Interaction of 3‧,4‧,6‧-trimyristoyl-uridine derivative as potential anticancer drug with phospholipids of tumorigenic and non-tumorigenic cells

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Salis, Luiz Fernando Grosso; Jaroque, Guilherme Nuñez; Escobar, Jhon Fernando Berrío; Giordani, Cristiano; Martinez, Alejandro Martinez; Fernández, Diana Margarita Márquez; Castelli, Francesco; Sarpietro, Maria Grazia; Caseli, Luciano

2017-12-01

Investigating the mechanism of action of drugs whose pharmaceutical activity is associated with cell membranes is fundamental to comprehending the biochemical and biophysical processes that occur on membrane surfaces. In this work, we investigated the interaction of an ester-type derivative of uridine, 3‧,4‧,6‧-trimyristoyl uridine, with models for cell membranes formed by lipid monolayers at the air-water interface. For that, selected lipids have been chosen in order to mimic tumorigenic and non-tumorigenic cells. For mixed monolayers with 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dihexadecanoyl-sn-glycero-3-phospho-L-serine (DPPS), the surface pressure-area isotherms exhibited a noticeable shift to lower areas in relation to the areas predicted for ideal mixtures, indicating a condensation of the monolayer structure. Changes in the viscoelastic properties of the interfacial film could be inferred by analyzing the compressibility modulus of the monolayer. Structural and morphological changes were also evidenced by using vibrational spectroscopy and Brewster angle microscopy, respectively, with distinctive effects on DPPC and DPPS. As conclusion we can state that the lipid composition of the monolayer modulates the interaction with this lipophilic drug, which may have important implications in understanding how this drug acts on specific sites of the cellular membrane.

Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

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Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

2001-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo

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Ginsburg Erika

2010-05-01

Full Text Available Abstract Background Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM derived from premenopausal African-American (AA or Caucasian-American (CAU breast tissue would affect the tumorigenicity of cancer cells in vitro and in vivo. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women. Methods The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested in vitro via soft agar and invasion assays, and in vivo via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry. Results Mouse mammary glands humanized with premenopausal CAU fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA humanized glands. Examination of 164 ECM molecules and cytokines from CAU-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast ECM was isolated; invasion and soft agar assays demonstrated that estrogen receptor (ER-, progesterone receptor (PR/PR- cells were significantly more aggressive when in contact with AA ECM, as were ER+/PR+ cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER+/PR+ cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1

Overexpression of YB1 C-terminal domain inhibits proliferation, angiogenesis and tumorigenicity in a SK-BR-3 breast cancer xenograft mouse model.

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Shi, Jian-Hong; Cui, Nai-Peng; Wang, Shuo; Zhao, Ming-Zhi; Wang, Bing; Wang, Ya-Nan; Chen, Bao-Ping

2016-01-01

Y-box-binding protein 1 (YB1) is a multifunctional transcription factor with vital roles in proliferation, differentiation and apoptosis. In this study, we have examined the role of its C-terminal domain (YB1 CTD) in proliferation, angiogenesis and tumorigenicity in breast cancer. Breast cancer cell line SK-BR-3 was infected with GFP-tagged YB1 CTD adenovirus expression vector. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) proliferation assay showed that YB1 CTD decreased SK-BR-3 cell proliferation, and down-regulated cyclin B1 and up-regulated p21 levels in SK-BR-3 cells. YB1 CTD overexpression changed the cytoskeletal organization and slightly inhibited the migration of SK-BR-3 cells. YB1 CTD also inhibited secreted VEGF expression in SK-BR-3 cells, which decreased SK-BR-3-induced EA.hy926 endothelial cell angiogenesis in vitro. YB1 CTD overexpression attenuated the ability of SK-BR-3 cells to form tumours in nude mice, and decreased in vivo VEGF levels and angiogenesis in the xenografts in SK-BR-3 tumour-bearing mice. Taken together, our findings demonstrate the vital role of YB1 CTD overexpression in inhibiting proliferation, angiogenesis and tumorigenicity of breast cancer cell line SK-BR-3.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

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Silvia Dotti

Full Text Available The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

Alterations in the extracellular matrix organization associated with the reexpression of tumorigenicity in human cell hybrids.

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Der, C J; Stanbridge, E J

1980-10-15

The expression of fibronectin on the cell surface was evaluated on a series of intraspecific human cell hybrids formed between HeLa and normal fibroblast strains. Although these hybrids continued to express many of the in vitro transformation properties of their corresponding tumorigenic HeLa parent, they were now unable to form tumors when inoculated into athymic nude mice. From these suppressed hybrid populations, rare tumorigenic segregant subpopulations arose which had regained their tumorigenic capacity. A comparison of the expression of fibronectin on the cell surface was made between these tumorigenic segregant cell lines and their corresponding non-tumorigenic HeLa/fibroblast hybrid. Following specific immunofluorescent staining for fibronectin, a striking alteration in the cell surface organization was observed to correspond with the reexpression of tumorigenicity in these hybrids. Tumorigenic HeLa/fibroblast hybrids were also significantly altered in both their cellular and colonial morphology. Double immunofluorescent staining to simultaneously visualize both surface fibronectin and collagen revealed that these two extracellular matrix proteins displayed an extensive degree of codistribution and expressed a coordinate shift in organization which correlated with the appearance of tumorigenic segregant hybrid populations. These observations are in agreement with the apparently close structural association between fibronectin and collagen and suggest that the organization of these two components in the extracellular matrix may be an important determinant for in vivo growth potential.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: Phenotypic characterization and tumorigenic potential

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Barbieri, Federica; Wurth, Roberto [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy); Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina [Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D' Aosta, National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila, 16129, Genova (Italy); Thellung, Stefano [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy); Daga, Antonio [Laboratory of Translational Oncology, IRCCS Azienda Ospedaliera Universitaria San Martino - IST- Istituto Nazionale Ricerca sul Cancro, L.go R. Benzi, 10, 16132 Genova Italy (Italy); Cilli, Michele [Animal Facility, IRCCS Azienda Ospedaliera Universitaria San Martino - IST- Istituto Nazionale Ricerca sul Cancro, L.go R. Benzi, 10, 16132 Genova Italy (Italy); Ferrari, Angelo [Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D' Aosta, National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila, 16129, Genova (Italy); Florio, Tullio, E-mail: tullio.florio@unige.it [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy)

2012-04-15

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-{alpha} and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: Black-Right-Pointing-Pointer Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 Black-Right-Pointing-Pointer These grow as spheres in serum-free medium and self-renew Black-Right-Pointing-Pointer Isolated stem-like cancer cells initiate tumor in immunodeficient mice Black-Right-Pointing-Pointer Xenografted tumors are phenotypically similar to the original tumor Black

Isolation of stem-like cells from spontaneous feline mammary carcinomas: Phenotypic characterization and tumorigenic potential

International Nuclear Information System (INIS)

Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

2012-01-01

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell–like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: ► Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 ► These grow as spheres in serum-free medium and self-renew ► Isolated stem-like cancer cells initiate tumor in immunodeficient mice ► Xenografted tumors are phenotypically similar to the original tumor ► Upon differentiation, cells grow as monolayers, loosing the tumorigenic potential

REST controls self-renewal and tumorigenic competence of human glioblastoma cells.

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Luciano Conti

Full Text Available The Repressor Element 1 Silencing Transcription factor (REST/NRSF is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

Caspase 8 and maspin are downregulated in breast cancer cells due to CpG site promoter methylation

International Nuclear Information System (INIS)

Wu, Yanyuan; Alvarez, Monica; Slamon, Dennis J; Koeffler, Phillip; Vadgama, Jaydutt V

2010-01-01

Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer. Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay. The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines. CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer

Molecular characterization of breast cancer cell lines through multiple omic approaches.

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Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated

Changes in chromatin state reveal ARNT2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness.

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Bogeas, Alexandra; Morvan-Dubois, Ghislaine; El-Habr, Elias A; Lejeune, François-Xavier; Defrance, Matthieu; Narayanan, Ashwin; Kuranda, Klaudia; Burel-Vandenbos, Fanny; Sayd, Salwa; Delaunay, Virgile; Dubois, Luiz G; Parrinello, Hugues; Rialle, Stéphanie; Fabrega, Sylvie; Idbaih, Ahmed; Haiech, Jacques; Bièche, Ivan; Virolle, Thierry; Goodhardt, Michele; Chneiweiss, Hervé; Junier, Marie-Pierre

2018-02-01

Although a growing body of evidence indicates that phenotypic plasticity exhibited by glioblastoma cells plays a central role in tumor development and post-therapy recurrence, the master drivers of their aggressiveness remain elusive. Here we mapped the changes in active (H3K4me3) and repressive (H3K27me3) histone modifications accompanying the repression of glioblastoma stem-like cells tumorigenicity. Genes with changing histone marks delineated a network of transcription factors related to cancerous behavior, stem state, and neural development, highlighting a previously unsuspected association between repression of ARNT2 and loss of cell tumorigenicity. Immunohistochemistry confirmed ARNT2 expression in cell sub-populations within proliferative zones of patients' glioblastoma. Decreased ARNT2 expression was consistently observed in non-tumorigenic glioblastoma cells, compared to tumorigenic cells. Moreover, ARNT2 expression correlated with a tumorigenic molecular signature at both the tissue level within the tumor core and at the single cell level in the patients' tumors. We found that ARNT2 knockdown decreased the expression of SOX9, POU3F2 and OLIG2, transcription factors implicated in glioblastoma cell tumorigenicity, and repressed glioblastoma stem-like cell tumorigenic properties in vivo. Our results reveal ARNT2 as a pivotal component of the glioblastoma cell tumorigenic signature, located at a node of a transcription factor network controlling glioblastoma cell aggressiveness.

TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells.

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Bartucci, M; Dattilo, R; Moriconi, C; Pagliuca, A; Mottolese, M; Federici, G; Benedetto, A Di; Todaro, M; Stassi, G; Sperati, F; Amabile, M I; Pilozzi, E; Patrizii, M; Biffoni, M; Maugeri-Saccà, M; Piccolo, S; De Maria, R

2015-02-05

Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

System-wide analysis reveals a complex network of tumor-fibroblast interactions involved in tumorigenicity.

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Megha Rajaram

Full Text Available Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8 or minimally (STC1 significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti

Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

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Reddy, Jay P; Atkinson, Rachel L; Larson, Richard; Burks, Jared K; Smith, Daniel; Debeb, Bisrat G; Ruffell, Brian; Creighton, Chad J; Bambhroliya, Arvind; Reuben, James M; Van Laere, Steven J; Krishnamurthy, Savitri; Symmans, William F; Brewster, Abenaa M; Woodward, Wendy A

2018-06-01

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44 + CD49f + CD133/2 + mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. 8 of 8 IBC samples expressed isolated CD44 + CD49f + CD133/2 + stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44 + CD49f + CD133/2 + cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

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Pham Phuc V

2011-12-01

Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

Induction of human breast cell carcinogenesis by triclocarban and intervention by curcumin

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Sood, Shilpa; Choudhary, Shambhunath; Wang, Hwa-Chain Robert, E-mail: hcrwang@utk.edu

2013-09-06

Highlights: •Triclocarban exposure induces breast epithelial cell carcinogenesis. •Triclocarban induces the Erk–Nox pathway, ROS elevation, and DNA damage. •Physiological doses of triclocarban induce cellular carcinogenesis. •Non-cytotoxic curcumin blocks triclocarban-induced carcinogenesis and pathways. -- Abstract: More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens and co-carcinogens. To identify co-carcinogens with abilities to induce cellular pre-malignancy, we studied the activity of triclocarban (TCC), an antimicrobial agent commonly used in household and personal care products. Here, we demonstrated, for the first time, that chronic exposure to TCC at physiologically-achievable nanomolar concentrations resulted in progressive carcinogenesis of human breast cells from non-cancerous to pre-malignant. Pre-malignant carcinogenesis was measured by increasingly-acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth and increased cell proliferation, without acquisition of cellular tumorigenicity. Long-term TCC exposure also induced constitutive activation of the Erk–Nox pathway and increases of reactive oxygen species (ROS) in cells. A single TCC exposure induced transient induction of the Erk–Nox pathway, ROS elevation, increased cell proliferation, and DNA damage in not only non-cancerous breast cells but also breast cancer cells. Using these constitutively- and transiently-induced changes as endpoints, we revealed that non-cytotoxic curcumin was effective in intervention of TCC-induced cellular pre-malignancy. Our results lead us to suggest that the co-carcinogenic potential of TCC should be seriously considered in epidemiological studies to reveal the significance of TCC in the development of sporadic breast cancer. Using TCC-induced transient and constitutive endpoints as targets will likely help identify non-cytotoxic preventive

Bioengineering Embryonic Stem Cell Microenvironments for the Study of Breast Cancer

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Yubing Xie

2011-11-01

Full Text Available Breast cancer is the most prevalent disease amongst women worldwide and metastasis is the main cause of death due to breast cancer. Metastatic breast cancer cells and embryonic stem (ES cells display similar characteristics. However, unlike metastatic breast cancer cells, ES cells are nonmalignant. Furthermore, embryonic microenvironments have the potential to convert metastatic breast cancer cells into a less invasive phenotype. The creation of in vitro embryonic microenvironments will enable better understanding of ES cell-breast cancer cell interactions, help elucidate tumorigenesis, and lead to the restriction of breast cancer metastasis. In this article, we will present the characteristics of breast cancer cells and ES cells as well as their microenvironments, importance of embryonic microenvironments in inhibiting tumorigenesis, convergence of tumorigenic and embryonic signaling pathways, and state of the art in bioengineering embryonic microenvironments for breast cancer research. Additionally, the potential application of bioengineered embryonic microenvironments for the prevention and treatment of invasive breast cancer will be discussed.

Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer.

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Antonis Kourtidis

Full Text Available Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120, which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.

Crucial role of carbonic anhydrase IX in tumorigenicity of xenotransplanted adult T-cell leukemia-derived cells.

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Nasu, Kentaro; Yamaguchi, Kazunori; Takanashi, Tomoka; Tamai, Keiichi; Sato, Ikuro; Ine, Shoji; Sasaki, Osamu; Satoh, Kennichi; Tanaka, Nobuyuki; Tanaka, Yuetsu; Fukushima, Takuya; Harigae, Hideo; Sugamura, Kazuo

2017-03-01

Carbonic anhydrase IX (CA9) is a membrane-associated carbonic anhydrase that regulates cellular pH, is upregulated in various solid tumors, and is considered to be a therapeutic target. Here, we describe the essential role of CA9 in the tumorigenicity of cells derived from human adult T-cell leukemia/lymphoma (ATL). We previously established the highly tumorigenic ST1-N6 subline from the ATL-derived ST1 cell line by serial xenotransplantation in NOG mice. In the present study, we first show that CA9 expression is strongly enhanced in ST1-N6 cells. We then sorted ST1 cells by high or low CA9 expression and established ST1-CA9 high and ST1-CA9 low sublines. ST1-CA9 high cells, like ST1-N6 cells, were more strongly tumorigenic than ST1-CA9 low or parental ST1 cells when injected into NOG mice. Knockdown of CA9 with shRNAs suppressed the ability of ST1-CA9 high cells to initiate tumors, and the tumorigenicity of ST1 cells was significantly enhanced by introducing wild-type CA9 or a CA9 mutant with deletion of an intracytoplasmic domain. However, a CA9 with point mutations in the catalytic site did not increase the tumorigenicity of ST1 cells. Furthermore, we detected a small population of CA9 + CD25 + cells in lymph nodes of ATL patients. These findings suggest that CA9, and particularly its carbonic anhydrase activity, promotes the tumorigenicity of ATL-derived cells and may be involved in malignant development of lymphoma-type ATL. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Peroxisome Proliferator Activated Receptor-α/Hypoxia Inducible Factor-1α Interplay Sustains Carbonic Anhydrase IX and Apoliprotein E Expression in Breast Cancer Stem Cells

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Papi, Alessio; Storci, Gianluca; Guarnieri, Tiziana; De Carolis, Sabrina; Bertoni, Sara; Avenia, Nicola; Sanguinetti, Alessandro; Sidoni, Angelo; Santini, Donatella; Ceccarelli, Claudio; Taffurelli, Mario; Orlandi, Marina; Bonafé, Massimiliano

2013-01-01

Aims Cancer stem cell biology is tightly connected to the regulation of the pro-inflammatory cytokine network. The concept of cancer stem cells “inflammatory addiction” leads to envisage the potential role of anti-inflammatory molecules as new anti-cancer targets. Here we report on the relationship between nuclear receptors activity and the modulation of the pro-inflammatory phenotype in breast cancer stem cells. Methods Breast cancer stem cells were expanded as mammospheres from normal and tumor human breast tissues and from tumorigenic (MCF7) and non tumorigenic (MCF10) human breast cell lines. Mammospheres were exposed to the supernatant of breast tumor and normal mammary gland tissue fibroblasts. Results In mammospheres exposed to the breast tumor fibroblasts supernatant, autocrine tumor necrosis factor-α signalling engenders the functional interplay between peroxisome proliferator activated receptor-α and hypoxia inducible factor-1α (PPARα/HIF1α). The two proteins promote mammospheres formation and enhance each other expression via miRNA130b/miRNA17-5p-dependent mechanism which is antagonized by PPARγ. Further, the PPARα/HIF1α interplay regulates the expression of the pro-inflammatory cytokine interleukin-6, the hypoxia survival factor carbonic anhydrase IX and the plasma lipid carrier apolipoprotein E. Conclusion Our data demonstrate the importance of exploring the role of nuclear receptors (PPARα/PPARγ) in the regulation of pro-inflammatory pathways, with the aim to thwart breast cancer stem cells functioning. PMID:23372804

Real-time motion analysis reveals cell directionality as an indicator of breast cancer progression.

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Michael C Weiger

Full Text Available Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF or lysophosphatitic acid (LPA are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas.

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Adams, April; Warner, Kristy; Pearson, Alexander T; Zhang, Zhaocheng; Kim, Hong Sun; Mochizuki, Daiki; Basura, Gregory; Helman, Joseph; Mantesso, Andrea; Castilho, Rogério M; Wicha, Max S; Nör, Jacques E

2015-09-29

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

Interplay of Stem Cell Characteristics, EMT, and Microtentacles in Circulating Breast Tumor Cells

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Charpentier, Monica; Martin, Stuart

2013-01-01

Metastasis, not the primary tumor, is responsible for the majority of breast cancer-related deaths. Emerging evidence indicates that breast cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis. CTCs with both CSC and EMT characteristics have recently been identified in the bloodstream of patients with metastatic disease. Breast CSCs have elevated tumorigenicity required for metastatic outgrowth, while EMT may promote CSC character and endows breast cancer cells with enhanced invasive and migratory potential. Both CSCs and EMT are associated with a more flexible cytoskeleton and with anoikis-resistance, which help breast carcinoma cells survive in circulation. Suspended breast carcinoma cells produce tubulin-based extensions of the plasma membrane, termed microtentacles (McTNs), which aid in reattachment. CSC and EMT-associated upregulation of intermediate filament vimentin and increased detyrosination of α-tubulin promote the formation of McTNs. The combined advantages of CSCs and EMT and their associated cytoskeletal alterations increase metastatic efficiency, but understanding the biology of these CTCs also presents new therapeutic targets to reduce metastasis

Interplay of Stem Cell Characteristics, EMT, and Microtentacles in Circulating Breast Tumor Cells

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Charpentier, Monica [Program in Molecular Medicine, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-20, Baltimore, MD 21201 (United States); Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-29, Baltimore, MD 21201 (United States); Martin, Stuart, E-mail: ssmartin@som.umaryland.edu [Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-29, Baltimore, MD 21201 (United States); Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Bressler Bldg., Rm 10-29, Baltimore, MD 21201 (United States)

2013-11-14

Metastasis, not the primary tumor, is responsible for the majority of breast cancer-related deaths. Emerging evidence indicates that breast cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis. CTCs with both CSC and EMT characteristics have recently been identified in the bloodstream of patients with metastatic disease. Breast CSCs have elevated tumorigenicity required for metastatic outgrowth, while EMT may promote CSC character and endows breast cancer cells with enhanced invasive and migratory potential. Both CSCs and EMT are associated with a more flexible cytoskeleton and with anoikis-resistance, which help breast carcinoma cells survive in circulation. Suspended breast carcinoma cells produce tubulin-based extensions of the plasma membrane, termed microtentacles (McTNs), which aid in reattachment. CSC and EMT-associated upregulation of intermediate filament vimentin and increased detyrosination of α-tubulin promote the formation of McTNs. The combined advantages of CSCs and EMT and their associated cytoskeletal alterations increase metastatic efficiency, but understanding the biology of these CTCs also presents new therapeutic targets to reduce metastasis.

Genome-wide binding of transcription factor ZEB1 in triple-negative breast cancer cells.

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Maturi, Varun; Enroth, Stefan; Heldin, Carl-Henrik; Moustakas, Aristidis

2018-05-10

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcriptional regulator involved in embryonic development and cancer progression. ZEB1 induces epithelial-mesenchymal transition (EMT). Triple-negative human breast cancers express high ZEB1 mRNA levels and exhibit features of EMT. In the human triple-negative breast cancer cell model Hs578T, ZEB1 associates with almost 2,000 genes, representing many cellular functions, including cell polarity regulation (DLG2 and FAT3). By introducing a CRISPR-Cas9-mediated 30 bp deletion into the ZEB1 second exon, we observed reduced migratory and anchorage-independent growth capacity of these tumor cells. Transcriptomic analysis of control and ZEB1 knockout cells, revealed 1,372 differentially expressed genes. The TIMP metallopeptidase inhibitor 3 and the teneurin transmembrane protein 2 genes showed increased expression upon loss of ZEB1, possibly mediating pro-tumorigenic actions of ZEB1. This work provides a resource for regulators of cancer progression that function under the transcriptional control of ZEB1. The data confirm that removing a single EMT transcription factor, such as ZEB1, is not sufficient for reverting the triple-negative mesenchymal breast cancer cells into more differentiated, epithelial-like clones, but can reduce tumorigenic potential, suggesting that not all pro-tumorigenic actions of ZEB1 are linked to the EMT. © 2018 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Long-term estrogen exposure promotes carcinogen bioactivation, induces persistent changes in gene expression, and enhances the tumorigenicity of MCF-7 human breast cancer cells

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Spink, Barbara C.; Bennett, James A.; Pentecost, Brian T.; Lostritto, Nicole; Englert, Neal A.; Benn, Geoffrey K.; Goodenough, Angela K.; Turesky, Robert J.; Spink, David C.

2009-01-01

The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor α (ERα) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERα and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERα- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17β-estradiol (E 2 ). With these LTEE cells and with parallel control cells cultured without E 2 supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E 2 -dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E 2 .

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

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M. Ryan Smith

2016-08-01

Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

AZU-1: A Candidate Breast Tumor Suppressor and Biomarker for Tumor Progression

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Chen, Huei-Mei; Schmeichel, Karen L; Mian, I. Saira; Lelie`vre, Sophie; Petersen, Ole W; Bissell, Mina J

2000-02-04

To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.

Label-Free Raman Imaging to Monitor Breast Tumor Signatures

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Ciubuc, John

Methods built on Raman spectroscopy have shown major potential in describing and discriminating between malignant and benign specimens. Accurate, real-time medical diagnosis benefits in substantial improvements through this vibrational optical method. Not only is acquisition of data possible in milliseconds and analysis in minutes, Raman allows concurrent detection and monitoring of all biological components. Besides validating a significant Raman signature distinction between non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) breast epithelial cells, this study reveals a label-free method to assess overexpression of epidermal growth factor receptors (EGFR) in tumor cells. EGFR overexpression sires Raman features associated with phosphorylated threonine and serine, and modifications of DNA/RNA characteristics. Investigations by gel electrophoresis reveal EGF induction of phosphorylated Akt, agreeing with the Raman results. The analysis presented is a vital step toward Raman-based evaluation of EGF receptors in breast cancer cells. With the goal of clinically applying Raman-guided methods for diagnosis of breast tumors, the current results lay the basis for proving label-free optical alternatives in making prognosis of the disease.

Human Breast Cancer Cells Are Redirected to Mammary Epithelial Cells upon Interaction with the Regenerating Mammary Gland Microenvironment In-Vivo

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Bussard, Karen M.; Smith, Gilbert H.

2012-01-01

Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display ‘normal’ behavior when placed into ‘normal’ ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for ‘normal’ gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo. PMID:23155468

Human breast cancer cells are redirected to mammary epithelial cells upon interaction with the regenerating mammary gland microenvironment in-vivo.

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Karen M Bussard

Full Text Available Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown; however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display 'normal' behavior when placed into 'normal' ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for 'normal' gland development in-vivo. Here, we show that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo.

Targeting the S1P Axis and Development of a Novel Therapy for Obesity Related Triple Negative Breast Cancer

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2017-09-01

with the options ‘intersection-nonempty’ mode, non- stranded , minimum quality 10, and ‘exon’ was used as the feature type, with ‘gene_id’ as the GTF...Keywords: sphingosine-1-phosphate, sphingosine kinase, estradiol, breast cancer, FTY720/fingolimod. Abbreviations: BCSCs, breast cancer stem cells; ,ER...pathway along with p38MAPK in breast cancer stem cells (BCSCs) leading to proliferation and tumorigenicity (Hirata et al., 2014). BCSCs can also be

Mutant p53 transfection of astrocytic cells results in altered cell cycle control, radiation sensitivity, and tumorigenicity

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Kanady, Kirk E.; Mei Su; Proulx, Gary; Malkin, David M.; Pardo, Francisco S.

1995-01-01

Introduction: Alterations in the p53 tumor suppressor gene are one of the most frequent genetic alterations in malignant gliomas. An understanding of the molecular genetic events leading to glial tumor progression would aid in designing therapeutic vectors for controlling these challenging tumor types. We investigated whether mutations in coding exons of the p53 gene result in functional changes altering cell cycle 'checkpoint' control and the intrinsic radiation sensitivity of glial cells. Methods: An astrocytic cell line was derived from a low grade astrocytoma and characterized to be of human karyotype and GFAP positivity. Additionally, the cellular population has never formed tumors in immune-deficient mice. At early passage ( 2 as parameters. Cell kinetic analyses after 2, 5, and 10 Gy of ionizing radiation were conducted using propidium iodide FACS analyses. Results: Overall levels of p53 expression were increased 5-10 fold in the transfected cellular populations. Astrocytic cellular populations transfected with mutant p53 revealed a statistically significant increase in levels of resistance to ionizing radiation in vitro (2-tailed test, SF2, MID). Astrocytic cellular populations transfected with mutant p53, unlike the parental cells, were tumorigenic in SCID mice. Cell kinetic analyses indicated that the untransfected cell line demonstrated dose dependent G1 and G2 arrests. Following transfection, however, the resultant cellular population demonstrated a predominant G2 arrest. Conclusions: Astrocytic cellular populations derived from low grade astrocytomas, are relatively radiation sensitive, non-tumorigenic, and have intact cell cycle ''checkpoints.'' Cellular populations resulting upon transfection of parental cells with a dominant negative p53 mutation, are relatively radiation resistant, when compared to both parental and mock-transfected cells. Transfected cells demonstrate abnormalities of cell cycle control at the G1/S checkpoint, increases in levels

Breast Cancer-Initiating Cells: Insights into Novel Treatment Strategies

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Santilli, Guido; Binda, Mara; Zaffaroni, Nadia; Daidone, Maria Grazia

2011-01-01

There is accumulating evidence that breast cancer may arise from mutated mammary stem/progenitor cells which have been termed breast cancer-initiating cells (BCIC). BCIC identified in clinical specimens based on membrane phenotype (CD44 + /CD24 −/low and/or CD133 + expression) or enzymatic activity of aldehyde dehydrogenase 1 (ALDH1 + ), have been demonstrated to have stem/progenitor cell properties, and are tumorigenic when injected in immunocompromized mice at very low concentrations. BCIC have also been isolated and in vitro propagated as non-adherent spheres of undifferentiated cells, and stem cell patterns have been recognized even in cancer cell lines. Recent findings indicate that aberrant regulation of self renewal is central to cancer stem cell biology. Alterations in genes involved in self-renewal pathways, such as Wnt, Notch, sonic hedgehog, PTEN and BMI, proved to play a role in breast cancer progression. Hence, targeting key elements mediating the self renewal of BCIC represents an attractive option, with a solid rationale, clearly identifiable molecular targets, and adequate knowledge of the involved pathways. Possible concerns are related to the poor knowledge of tolerance and efficacy of inhibiting self-renewal mechanisms, because the latter are key pathways for a variety of biological functions and it is unknown whether their interference would kill BCIC or simply temporarily stop them. Thus, efforts to develop BCIC-targeted therapies should not only be focused on interfering on self-renewal, but could seek to identify additional molecular targets, like those involved in regulating EMT-related pathways, in reversing the MDR phenotype, in inducing differentiation and controlling cell survival pathways

The PIKfyve–ArPIKfyve–Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines

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Ikonomov, Ognian C.; Filios, Catherine; Sbrissa, Diego; Chen, Xuequn; Shisheva, Assia

2013-01-01

Highlights: •We assess PAS complex proteins and phosphoinositide levels in breast cancer cells. •Sac3 and ArPIKfyve are markedly elevated in triple-negative breast cancer cells. •Sac3 silencing inhibits proliferation in triple-negative breast cancer cell lines. •Phosphoinositide profiles are altered in breast cancer cells. •This is the first evidence linking high Sac3 with breast cancer cell proliferation. -- Abstract: The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve–ArPIKfyve–Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P–PtdIns(3,5)P 2 synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P–PtdIns(3,5)P 2 conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P 2 in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or indirectly the

Berberine Suppresses Cell Motility Through Downregulation of TGF-β1 in Triple Negative Breast Cancer Cells

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Sangmin Kim

2018-01-01

Full Text Available Background/Aims: Transforming growth factor-beta proteins (TGF-βs are multifunctional growth factors and powerful modulators of the epithelial-mesenchymal transition (EMT in a variety of cancer types including breast and lung cancer cells. Here, we demonstrated the inhibitory effect of berberine (BBR on tumor growth and metastasis of triple negative breast cancer (TNBC cells via suppression of TGF-β1 expression. Methods: The levels of mRNA expression were analyzed by real-time PCR. The levels of MMP-2, MMP-9 and TGF-β1 protein expression were analyzed by zymography and confocal microscopy, respectively. Cell migration was analyzed by wound healing assay. Tumorigenicity of TNBC cells such as tumor growth and metastasis was analyzed using xenograft models. Results: In a clinical data set, aberrant TGF-β1 expression was associated with poor prognosis of breast cancer patients. Our in vitro results using TNBC cells showed that the expression levels of matrix metalloproteinase (MMP-2 and MMP-9 and the capacity for cell migration were increased by TGF-β1 treatment. In contrast, basal levels of MMP-2 and MMP-9 were suppressed by a specific TGF-β receptor I inhibitor, SB431542. In addition, TGF-β1–induced MMP-2 and MMP-9 expression and cell migration were decreased by SB431542. Interestingly, we showed for the first time that BBR decreased the level of TGF-β1, but not TGF-β2, in TNBC cells. Furthermore, BBR significantly decreased the level of MMP-2 expression as well as the capacity for cell migration in TNBC cells. Finally, we examined the effect of BBR on in vivo tumor growth and lung metastasis in MDA-MB231 and 4T1 breast cancer xenograft models and showed that both were significantly decreased following BBR treatment. Conclusion: BBR suppresses tumorigenicity of TNBC cells through inhibition of TGF-β1 expression. Therefore, we demonstrate that BBR could be a promising drug for treatment of TNBC.

Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

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Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

2006-09-29

Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

Molecular Imaging of Stem Cells: Tracking Survival, Biodistribution, Tumorigenicity, and Immunogenicity

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Eugene Gu, Wen-Yi Chen, Jay Gu, Paul Burridge, Joseph C. Wu

2012-01-01

Full Text Available Being able to self-renew and differentiate into virtually all cell types, both human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs have exciting therapeutic implications for myocardial infarction, neurodegenerative disease, diabetes, and other disorders involving irreversible cell loss. However, stem cell biology remains incompletely understood despite significant advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment all hamper the transition from laboratory animal studies to human clinical trials. Although traditional histopathological techniques have been the primary approach for ex vivo analysis of stem cell behavior, these postmortem examinations are unable to further elucidate the underlying mechanisms in real time and in vivo. Fortunately, the advent of molecular imaging has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest. This review summarizes various molecular imaging technologies and how they have advanced the current understanding of stem cell survival, biodistribution, immunogenicity, and tumorigenicity.

The effects and mechanisms of SLC34A2 on maintaining stem cell-like phenotypes in CD147+ breast cancer stem cells.

Science.gov (United States)

Lv, Yonggang; Wang, Ting; Fan, Jing; Zhang, Zhenzhen; Zhang, Juliang; Xu, Cheng; Li, Yongping; Zhao, Ge; He, Chenyang; Meng, Huimin; Yang, Hua; Wang, Zhen; Liu, Jiayun; Chen, Jianghao; Wang, Ling

2017-04-01

The cancer stem cell (CSC) hypothesis has gained significant recognition in describing tumorigenesis. Identification of the factors critical to development of breast cancer stem cells (BCSCs) may provide insight into the improvement of effective therapies against breast cancer. In this study, we aim to investigate the biological function of SLC34A2 in affecting the stem cell-like phenotypes in BCSCs and its underlying mechanisms. We demonstrated that CD147 + cells from breast cancer tissue samples and cell lines possessed BCSC-like features, including the ability of self-renewal in vitro, differentiation, and tumorigenic potential in vivo. Flow cytometry analysis showed the presence of a variable fraction of CD147 + cells in 9 of 10 tumor samples. Significantly, SLC34A2 expression in CD147 + BCSCs was enhanced compared with that in differentiated adherent progeny of CD147 + BCSCs and adherently cultured cell line cells. In breast cancer patient cohorts, SLC34A2 expression was found increased in 9 of 10 tumor samples. By using lentiviral-based approach, si-SLC34A2-transduced CD147 + BCSCs showed decreased ability of sphere formation, cell viability in vitro, and tumorigenicity in vivo, which suggested the essential role of SLC34A2 in CD147 + BCSCs. Furthermore, PI3K/AKT pathway and SOX2 were found necessary to maintain the stemness of CD147 + BCSCs by using LY294002 or lentiviral-si-SOX2. Finally, we indicated that SLC34A2 could regulate SOX2 to maintain the stem cell-like features in CD147 + BCSCs through PI3K/AKT pathway. Therefore, our report identifies a novel role of SLC34A2 in BCSCs' state regulation and establishes a rationale for targeting the SLC34A2/PI3K/AKT/SOX2 signaling pathway for breast cancer therapy.

Novel anticancer activity of phloroglucinol against breast cancer stem-like cells

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Kim, Rae-Kwon; Uddin, Nizam [Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Hyun, Jin-Won [College of Medicine and Applied Radiological Science Research Institute, Jeju National University, Jeju-si 690-756 (Korea, Republic of); Kim, Changil [Department of Biotechnology, Konkuk University, Chungju 380-701 (Korea, Republic of); Suh, Yongjoon, E-mail: hiswork@hanmail.net [Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Lee, Su-Jae, E-mail: sj0420@hanyang.ac.kr [Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of)

2015-08-01

Poor prognosis of breast cancer patients is closely associated with metastasis and relapse. There is substantial evidence supporting that cancer stem-like cells (CSCs) are primarily responsible for relapse in breast cancer after anticancer treatment. However, there is a lack of suitable drugs that target breast cancer stem-like cells (BCSCs). Here, we report that phloroglucinol (PG), a natural phlorotannin component of brown algae, suppresses sphere formation, anchorage-independent colony formation and in vivo tumorigenicity. In line with these observations, treatment with PG also decreased CD44{sup +} cancer cell population as well as expression of CSC regulators such as Sox2, CD44, Oct4, Notch2 and β-catenin. Also, treatment with PG sensitized breast cancer cells to anticancer drugs such as cisplatin, etoposide, and taxol as well as to ionizing radiation. Importantly, PG inhibited KRAS and its downstream PI3K/AKT and RAF-1/ERK signaling pathways that regulate the maintenance of CSCs. Taken together, our findings implicate PG as a good candidate to target BCSCs and to prevent the disease relapse. - Highlights: • Phloroglucinol suppresses in vivo tumor formation. • Phloroglucinol sensitizes breast cancer cells to anticancer agents. • Phloroglucinol inhibits breast cancer stem-like cells. • Phloroglucinol inhibits PI3K/AKT and KRAS/RAF/ERK signaling pathways.

A cytotoxic Petiveria alliacea dry extract induces ATP depletion and decreases β-F1-ATPase expression in breast cancer cells and promotes survival in tumor-bearing mice

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John F. Hernández

Full Text Available Abstract Metabolic plasticity in cancer cells assures cell survival and cell proliferation under variable levels of oxygen and nutrients. Therefore, new anticancer treatments endeavor to target such plasticity by modifying main metabolic pathways as glycolysis or oxidative phosphorylation. In American traditional medicine Petiveria alliacea L., Phytolaccacea, leaf extracts have been used for leukemia and breast cancer treatments. Herein, we study cytotoxicity and antitumoral effects of P. alliacea extract in tumor/non-tumorigenic cell lines and murine breast cancer model. Breast cancer cells treated with P. alliacea dry extract showed reduction in β-F1-ATPase expression, glycolytic flux triggering diminished intracellular ATP levels, mitochondrial basal respiration and oxygen consumption. Consequently, a decline in cell proliferation was observed in conventional and three-dimension spheres breast cancer cells culture. Additionally, in vivo treatment of BALB/c mice transplanted with the murine breast cancer TS/A tumor showed that P. alliacea extract via i.p. decreases the primary tumor growth and increases survival in the TS/A model.

PCDH10 is required for the tumorigenicity of glioblastoma cells

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Echizen, Kanae; Nakada, Mitsutoshi; Hayashi, Tomoatsu; Sabit, Hemragul; Furuta, Takuya; Nakai, Miyuki; Koyama-Nasu, Ryo; Nishimura, Yukiko; Taniue, Kenzui; Morishita, Yasuyuki; Hirano, Shinji; Terai, Kenta; Todo, Tomoki; Ino, Yasushi; Mukasa, Akitake; Takayanagi, Shunsaku; Ohtani, Ryohei; Saito, Nobuhito; Akiyama, Tetsu

2014-01-01

Highlights: • PCDH10 is required for the proliferation, survival and self-renewal of glioblastoma cells. • PCDH10 is required for glioblastoma cell migration and invasion. • PCDH10 is required for the tumorigenicity of glioblastoma cells. • PCDH10 may be a promising target for the therapy of glioblastoma. - Abstract: Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell–cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma

Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells

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Cynthia C.T. Sprenger

2008-12-01

Full Text Available Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer.

Upregulation of CPE promotes cell proliferation and tumorigenicity in colorectal cancer

International Nuclear Information System (INIS)

Liang, Xing-Hua; He, Wen-guang; Huang, Yan-Nian; Zeng, Xian-Cheng; Li, Ling-ling; Wu, Geng-Gang; Xie, Yi-Cheng; Zhang, Guang-Xian; Chen, Wei; Yang, Hai-Feng; Liu, Qi-Long; Li, Wen-Hong

2013-01-01

Colorectal cancer (CRC) is one of the most common cancers worldwide and a leading cause of cancer related death. Although the mortality rate of CRC is decreasing, finding novel targets for its therapy remains urgent. Carboxypeptidase E (CPE), a member of the pro-protein convertases, which are involved in the maturation of protein precursors, has recently been reported as elevated in many types of cancer. However, its role and mechanisms in tumor progression are poorly understood. In the present study, we investigated expression of CPE in CRC cell lines and tumor tissues using Western blot and real-time qRT-PCR. Plasmids for overexpression and depletion of CPE were constructed and analyzed by Western blot, MTT and colony formation assays and bromodeoxyuridine incorporation assays. The relative expression of p21, p27, and cyclin D1 were analyzed by Real-time qRT-PCR in the indicated cells. Our study showed that CPE was significantly upregulated in CRC cell lines and tumor tissues. MTT and colony formation assays indicated that overexpression of CPE enhanced cell growth rates. BrdU incorporation and flow-cytometry assays showed that ectopic expression of CPE increased the S-phase fraction cells. Soft agar assay proved enhanced tumorigenicity activity in CPE over-expressing CRC cells. Further studies of the molecular mechanisms of CPE indicated that is promoted cell proliferation and tumorigenicity through downregulation of p21 and p27, and upregulation of cyclin D1. Taken together, these data suggest that CPE plays an important role in cell cycle regulation and tumorigenicity, and may serve as a potential target for CRC therapeutics

Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

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Kee Hang Lee

Full Text Available Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs immortalized by the human telomerase reverse transcriptase (hTERT gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM cells were injected into adult (4-6-week-old Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL, they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases.

siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells

International Nuclear Information System (INIS)

Dong, Xuejun; Liu, Anding; Zer, Cindy; Feng, Jianguo; Zhen, Zhuan; Yang, Mingfeng; Zhong, Li

2009-01-01

Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells. siRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice. The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined in vitro by MTT assay, FACS and SA-β-galactosidase staining. The hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 μM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 μM doxorubicin killed twice as many

Senescence-Induced Alterations of Laminin Chain Expression Modulate Tumorigenicity of Prostate Cancer Cells1

Science.gov (United States)

Sprenger, Cynthia C T; Drivdahl, Rolf H; Woodke, Lillie B; Eyman, Daniel; Reed, May J; Carter, William G; Plymate, Stephen R

2008-01-01

Prostate cancer is an age-associated epithelial cancer, and as such, it contributes significantly to the mortality of the elderly. Senescence is one possible mechanism by which the body defends itself against various epithelial cancers. Senescent cells alter the microenvironment, in part, through changes to the extracellular matrix. Laminins (LMs) are extracellular proteins important to both the structure and function of the microenvironment. Overexpression of the senescence-associated gene mac25 in human prostate cancer cells resulted in increased mRNA levels of the LM α4 and β2 chains compared to empty vector control cells. The purpose of this study was to examine the effects of these senescence-induced LM chains on tumorigenicity of prostate cancer cells. We created stable M12 human prostate cancer lines overexpressing either the LM α4 or β2 chain or both chains. Increased expression of either the LM α4 or β2 chain resulted in increased in vitro migration and in vivo tumorigenicity of those cells, whereas high expression of both chains led to decreased in vitro proliferation and in vivo tumorigenicity compared to M12 control cells. This study demonstrates that senescent prostate epithelial cells can alter the microenvironment and that these changes modulate progression of prostate cancer. PMID:19048114

Changes of tumorigenicity and gene expressions of melanoma cells mutated in outer space

International Nuclear Information System (INIS)

Xiang Qing; Xu Mei; Xiao Cheng; Xu Bo; Li Hongyan; Geng Chuanying; Pan Lin; Fang Qing; Guo Yupeng; Tang Jingtian

2006-01-01

Objective: To screen the cell lines with decreased tumorigenicity, and identify those genes related to the development and metastasis of melanoma. Methods: The murine melanoma B16 cells were carried into the outer space by No. 20 retrievable satellite, and the survival cells were cloned after returned to earth. Five monoclonal cell lines(No.1, No.5, No.6, No.7, No.8) were utilized for further study. The cells were injected into C57BL/6J mice subcutaneously and abdominally respectively, then tumor-free time and survival time were recorded, tumor lumps were examined by routine pathological method. Gene chips were used to detect the gene expressions in 2 cell lines(No.1, No.8)with decreased tumorigenicity. Results: Compared with the control group, the tumor-free time was longer for No.1 cell lines (P<0.05). The survival time was significantly increased (P<0.01) and the weights of tumors were significantly decreased (P<0.01) for both No.1 and No.8 cell lines. The lymphocytes were infiltrated into tumors and adjacent tissues in those mice injected with No.1 and No.8 cell lines. Changes in 145 gene expressions were identified in No.1 cell lines, and 124 genes in No.8 cell lines (P<0.05), 9 genes of them were common to both cell lines. Furthermore, prostaglandin D2 synthase gene was markedly upregulated. Conclusion: The study implied that the decrease of tumorigenicity was related to the changes of carcinoma-associated gene expressions. (authors)

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction

DEFF Research Database (Denmark)

Burns, Jorge S; Kristiansen, Malthe; Kristensen, Lars P

2011-01-01

. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells...... of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization....

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

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Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

2017-11-15

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Sprouty2 enhances the tumorigenic potential of glioblastoma cells.

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Park, Jong-Whi; Wollmann, Guido; Urbiola, Carles; Fogli, Barbara; Florio, Tullio; Geley, Stephan; Klimaschewski, Lars

2018-02-23

Sprouty2 (SPRY2), a feedback regulator of receptor tyrosine kinase (RTK) signaling, has been shown to be associated with drug resistance and cell proliferation in glioblastoma (GBM), but the underlying mechanisms are still poorly defined. SPRY2 expression and survival patterns of patients with gliomas were analyzed using publicly available databases. Effects of RNA interference targeting SPRY2 on cellular proliferation in established GBM or patient-derived GBM stemlike cells were examined. Loss- or gain-of-function of SPRY2 to regulate the tumorigenic capacity was assessed in both intracranial and subcutaneous xenografts. SPRY2 was found to be upregulated in GBM, which correlated with reduced survival in GBM patients. SPRY2 knockdown significantly impaired proliferation of GBM cells but not of normal astrocytes. Silencing of SPRY2 increased epidermal growth factor-induced extracellular signal-regulated kinase (ERK) and Akt activation causing premature onset of DNA replication, increased DNA damage, and impaired proliferation, suggesting that SPRY2 suppresses DNA replication stress. Abrogating SPRY2 function strongly inhibited intracranial tumor growth and led to significantly prolonged survival of U87 xenograft-bearing mice. In contrast, SPRY2 overexpression promoted tumor propagation of low-tumorigenic U251 cells. The present study highlights an antitumoral effect of SPRY2 inhibition that is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in GBM patients.

New use of an old drug: inhibition of breast cancer stem cells by benztropine mesylate.

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Cui, Jihong; Hollmén, Maija; Li, Lina; Chen, Yong; Proulx, Steven T; Reker, Daniel; Schneider, Gisbert; Detmar, Michael

2017-01-03

Cancer stem cells (CSCs) play major roles in cancer initiation, metastasis, recurrence and therapeutic resistance. Targeting CSCs represents a promising strategy for cancer treatment. The purpose of this study was to identify selective inhibitors of breast CSCs (BCSCs). We carried out a cell-based phenotypic screening with cell viability as a primary endpoint, using a collection of 2,546 FDA-approved drugs and drug-like molecules in spheres formed by malignant human breast gland-derived cells (HMLER-shEcad cells, representing BCSCs) and control immortalized non-tumorigenic human mammary cells (HMLE cells, representing normal stem cells). 19 compounds were identified from screening. The chemically related molecules benztropine mesylate and deptropine citrate were selected for further validation and both potently inhibited sphere formation and self-renewal of BCSCs in vitro. Benztropine mesylate treatment decreased cell subpopulations with high ALDH activity and with a CD44+/CD24- phenotype. In vivo, benztropine mesylate inhibited tumor-initiating potential in a 4T1 mouse model. Functional studies indicated that benztropine mesylate inhibits functions of CSCs via the acetylcholine receptors, dopamine transporters/receptors, and/or histamine receptors. In summary, our findings identify benztropine mesylate as an inhibitor of BCSCs in vitro and in vivo. This study also provides a screening platform for identification of additional anti-CSC agents.

Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation.

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Yamaguchi, Kazunori; Takanashi, Tomoka; Nasu, Kentaro; Tamai, Keiichi; Mochizuki, Mai; Satoh, Ikuro; Ine, Shoji; Sasaki, Osamu; Satoh, Kennichi; Tanaka, Nobuyuki; Harigae, Hideo; Sugamura, Kazuo

2016-05-01

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

miR-150 suppresses the proliferation and tumorigenicity of leukemia stem cells by targeting the Nanog signaling pathway

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Dan-dan Xu

2016-11-01

Full Text Available Proliferation, a key feature of cancer cells, accounts for the majority of cancer-related diseases resulting in mortality. MicroRNAs (miRNAs plays important post-transcriptional modulation roles by acting on multiple signaling pathways, but the underlying mechanism in proliferation and tumorigenicity is unclear. Here, we identified the role of miR-150 in proliferation and tumorigenicity in leukemia stem cells (LSCs (CD34+CD38- cells. miR-150 expression was significantly down-regulated in LSCs from leukemia cell lines and clinical samples. Functional assays demonstrated that increased miR-150 expression inhibited proliferation and clonal and clonogenic growth, enhanced chemosensitivity, and attenuated tumorigenic activity of LSCs in vitro. Transplantation animal studies revealed that miR-150 overexpression progressively abrogates tumour growth. Immunohistochemistry assays demonstrated that miR-150 overexpression enhanced caspase-3 level and reduced Ki-67 level. Moreover, luciferase reporter assays indicated Nanog is a direct and functional target of miR-150. Nanog silencing using small interfering RNA recapitulated anti-proliferation and tumorigenicity inhibition effects. Furthermore, miR-150 directly down-regulated the expression of other cancer stem cell factors including Notch2 and CTNNB1. These results provide insights into the specific biological behaviour of miR-150 in regulating LSC proliferation and tumorigenicity. Targeting this miR-150/Nanog axis would be a helpful therapeutic strategy to treat acute myeloid leukemia.

Canonical and Non-Canonical NF-κB Signaling Promotes Breast Cancer Tumor-Initiating Cells

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Kendellen, Megan F.; Bradford, Jennifer W.; Lawrence, Cortney L.; Clark, Kelly S.; Baldwin, Albert S.

2014-01-01

Tumor-initiating cells (TICs) are a sub-population of cells that exhibit a robust ability to self-renew and contribute to the formation of primary tumors, the relapse of previously treated tumors, and the development of metastases. TICs have been identified in various tumors, including those of the breast, and are particularly enriched in the basal-like and claudin-low subtypes of breast cancer. The signaling pathways that contribute to the function and maintenance of TICs are under intense study. We explored the potential involvement of the NF-κB family of transcription factors in TICs in cell lines that are representative of basal-like and claudin-low breast cancer. NF-κB was found to be activated in breast cancer cells that form tumorspheres efficiently. Moreover, both canonical and non-canonical NF-κB signaling is required for these cells to self-renew in vitro and to form xenograft tumors efficiently in vivo using limiting dilutions of cells. Consistent with this, canonical and non-canonical NF-κB signaling is activated in TICs isolated from breast cancer cell lines. Experimental results indicate that NF-κB promotes the function of TICs by stimulating epithelial-to-mesenchymal transition (EMT) and by upregulating the expression of the inflammatory cytokines IL-1β and IL-6. The results suggest the use of NF-κB inhibitors for clinical therapy of certain breast cancers. PMID:23474754

In vitro properties and tumorigenicity of radiation-transformed clones of mouse 10T1/2 cells

International Nuclear Information System (INIS)

Otsu, Hiroshi; Yasukawa, Mieko; Terasima, Toyozo

1983-01-01

Nineteen radiation-induced and one spontaneously developed transformed foci were cloned from mouse 10T1/2 cells. Each clone was grown with normal 10T1/2 cells, and typing (types II and III) was carried out by making reference to the description of Reznikoff et al. Morphological characteristics of foci and their response to co-cultured normal counterparts are described. Some in vitro properties of the clones were examined and the relationship to each focus type is discussed. A reduced serum requirement of transformed clones was not recognized. Soft agar colonies were produced exclusively by type III clones. Tumorigenicity testing of the clones revealed that 93 % of type III clones were tumorigenic upon inoculation into syngeneic mice in an immunosuppressed condition. From these findings, it can be concluded that the tumorigenic potential of radiation-induced transformed cells can be predicted from the ability of the cells to form colonies in agar. (author)

Conditionally reprogrammed cells (CRC) methodology does not allow the in vitro expansion of patient-derived primary and metastatic lung cancer cells.

Science.gov (United States)

Sette, Giovanni; Salvati, Valentina; Giordani, Ilenia; Pilozzi, Emanuela; Quacquarini, Denise; Duranti, Enrico; De Nicola, Francesca; Pallocca, Matteo; Fanciulli, Maurizio; Falchi, Mario; Pallini, Roberto; De Maria, Ruggero; Eramo, Adriana

2018-07-01

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently, conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However, the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here, we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay, monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly, pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast, brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion, patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions, limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus, CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells, as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells. © 2018 UICC.

Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

DEFF Research Database (Denmark)

Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V

2009-01-01

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane...... purification and comparative quantitative LC-MS/MS proteomic analysis identified 13 membrane proteins that were expressed at higher levels and 3 that were under-expressed in the metastatic compared to the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5...

The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity

International Nuclear Information System (INIS)

Mendonca, M.S.; Redpath, J.L.; Stanbridge, E.J.

1991-01-01

The authors investigated γ-ray sensitivity of several activated c-H-ras (EJ) containing clones established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p 21 ); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p 21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p 21 ; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p 21 ras protein similar to clone I-7. Comparison of single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2Gy (S 2 ) indicate no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance. (author)

Epigenetic reprogramming of breast cancer cells with oocyte extracts

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Kumari Rajendra

2011-01-01

Full Text Available Abstract Background Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis. Results We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts. Conclusions This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

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Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

2012-01-01

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

Effect of specific silencing of EMMPRIN on the growth and cell cycle distribution of MCF-7 breast cancer cells.

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Yang, X Q; Yang, J; Wang, R; Zhang, S; Tan, Q W; Lv, Q; Meng, W T; Mo, X M; Li, H J

2015-12-02

The extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin family and shows increased expression in tumor cells. We examined the effect of RNAi-mediated EMMPRIN gene silencing induced by lentiviral on the growth and cycle distribution of MCF-7 breast cancer cells. Lentiviral expressing EMMPRIN-short hairpin RNA were packaged to infect MCF-7 cells. The inhibition efficiency of EMMPRIN was validated by real-time fluorescent quantitation polymerase chain reaction and western blotting. The effect of EMMPRIN on cell proliferation ability was detected using the MTT assay and clone formation experiments. Changes in cell cycle were detected by flow cytometry. EMMPRIN-short hairpin RNA-packaged lentiviral significantly down-regulated EMMPRIN mRNA and protein expression, significantly inhibited cell proliferation and in vitro tumorigenicity, and induced cell cycle abnormalities. Cells in the G0/G1 and G2/M phases were increased, while cells in the S phase were decreased after infection of MCF-7 cells for 3 days. The EMMPRIN gene facilitates breast cancer cell malignant proliferation by regulating cell cycle distribution and may be a molecular target for breast cancer gene therapy.

Lentivirus mediated RNA interference of EMMPRIN (CD147) gene inhibits the proliferation, matrigel invasion and tumor formation of breast cancer cells.

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Yang, Jing; Wang, Rong; Li, Hongjiang; Lv, Qing; Meng, Wentong; Yang, Xiaoqin

2016-07-08

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) or cluster of differentiation 147 (CD147), a glycoprotein enriched on the plasma membrane of tumor cells, promotes proliferation, invasion, metastasis, and survival of malignant tumor cells. In this study, we sought to examine the expression of EMMPRIN in breast tumors, and to identify the potential roles of EMMPRIN on breast cancer cells. EMMPRIN expression in breast cancer tissues was assessed by immunohistochemistry. We used a lentivirus vector-based RNA interference (RNAi) approach expressing short hairpin RNA (shRNA) to knockdown EMMPRIN gene in breast cancer cell lines MDA-MB-231 and MCF-7. In vitro, Cell proliferative, invasive potential were determined by Cell Counting Kit (CCK-8), cell cycle analysis and matrigel invasion assay, respectively. In vivo, tumorigenicity was monitored by inoculating tumor cells into breast fat pad of female nude mice. EMMPRIN was over-expressed in breast tumors and breast cancer cell lines. Down-regulation of EMMPRIN by lentivirus vector-based RNAi led to decreased cell proliferative, decreased matrigel invasion in vitro, and attenuated tumor formation in vivo. High expression of EMMPRIN plays a crucial role in breast cancer cell proliferation, matrigel invasion and tumor formation.

Drug development for breast, colorectal, and non-small cell lung cancers from 1979 to 2014.

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Nixon, Nancy A; Khan, Omar F; Imam, Hasiba; Tang, Patricia A; Monzon, Jose; Li, Haocheng; Sun, Gavin; Ezeife, Doreen; Parimi, Sunil; Dowden, Scot; Tam, Vincent C

2017-12-01

Understanding the drug development pathway is critical for streamlining the development of effective cancer treatments. The objective of the current study was to delineate the drug development timeline and attrition rate of different drug classes for common cancer disease sites. Drugs entering clinical trials for breast, colorectal, and non-small cell lung cancer were identified using a pharmaceutical business intelligence database. Data regarding drug characteristics, clinical trials, and approval dates were obtained from the database, clinical trial registries, PubMed, and regulatory Web sites. A total of 411 drugs met the inclusion criteria for breast cancer, 246 drugs met the inclusion criteria for colorectal cancer, and 315 drugs met the inclusion criteria for non-small cell lung cancer. Attrition rates were 83.9% for breast cancer, 87.0% for colorectal cancer, and 92.0% for non-small cell lung cancer drugs. In the case of non-small cell lung cancer, there was a trend toward higher attrition rates for targeted monoclonal antibodies compared with other agents. No tumor site-specific differences were noted with regard to cytotoxic chemotherapy, immunomodulatory, or small molecule kinase inhibitor drugs. Drugs classified as "others" in breast cancer had lower attrition rates, primarily due to the higher success of hormonal medications. Mean drug development times were 8.9 years for breast cancer, 6.7 years for colorectal cancer, and 6.6 years for non-small cell lung cancer. Overall oncologic drug attrition rates remain high, and drugs are more likely to fail in later-stage clinical trials. The refinement of early-phase trial design may permit the selection of drugs that are more likely to succeed in the phase 3 setting. Cancer 2017;123:4672-4679. © 2017 American Cancer Society. © 2017 American Cancer Society.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

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Rafael Zúñiga

2018-03-01

Full Text Available TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

'A novel in vivo model for the study of human breast cancer metastasis using primary breast tumor-initiating cells from patient biopsies'

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Marsden, Carolyn G; Wright, Mary Jo; Carrier, Latonya; Moroz, Krzysztof; Pochampally, Radhika; Rowan, Brian G

2012-01-01

The study of breast cancer metastasis depends on the use of established breast cancer cell lines that do not accurately represent the heterogeneity and complexity of human breast tumors. A tumor model was developed using primary breast tumor-initiating cells isolated from patient core biopsies that would more accurately reflect human breast cancer metastasis. Tumorspheres were isolated under serum-free culture conditions from core biopsies collected from five patients with clinical diagnosis of invasive ductal carcinoma (IDC). Isolated tumorspheres were transplanted into the mammary fat pad of NUDE mice to establish tumorigenicity in vivo. Tumors and metastatic lesions were analyzed by hematoxylin and eosin (H+E) staining and immunohistochemistry (IHC). Tumorspheres were successfully isolated from all patient core biopsies, independent of the estrogen receptor α (ERα)/progesterone receptor (PR)/Her2/neu status or tumor grade. Each tumorsphere was estimated to contain 50-100 cells. Transplantation of 50 tumorspheres (1-5 × 10 3 cells) in combination with Matrigel into the mammary fat pad of NUDE mice resulted in small, palpable tumors that were sustained up to 12 months post-injection. Tumors were serially transplanted three times by re-isolation of tumorspheres from the tumors and injection into the mammary fat pad of NUDE mice. At 3 months post-injection, micrometastases to the lung, liver, kidneys, brain and femur were detected by measuring content of human chromosome 17. Visible macrometastases were detected in the lung, liver and kidneys by 6 months post-injection. Primary tumors variably expressed cytokeratins, Her2/neu, cytoplasmic E-cadherin, nuclear β catenin and fibronectin but were negative for ERα and vimentin. In lung and liver metastases, variable redistribution of E-cadherin and β catenin to the membrane of tumor cells was observed. ERα was re-expressed in lung metastatic cells in two of five samples. Tumorspheres isolated under defined culture

Culture conditions tailored to the cell of origin are critical for maintaining native properties and tumorigenicity of glioma cells.

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Ledur, Pítia F; Liu, Chong; He, Hua; Harris, Alexandra R; Minussi, Darlan C; Zhou, Hai-Yan; Shaffrey, Mark E; Asthagiri, Ashok; Lopes, Maria Beatriz S; Schiff, David; Lu, Yi-Cheng; Mandell, James W; Lenz, Guido; Zong, Hui

2016-10-01

Cell culture plays a pivotal role in cancer research. However, culture-induced changes in biological properties of tumor cells profoundly affect research reproducibility and translational potential. Establishing culture conditions tailored to the cancer cell of origin could resolve this problem. For glioma research, it has been previously shown that replacing serum with defined growth factors for neural stem cells (NSCs) greatly improved the retention of gene expression profile and tumorigenicity. However, among all molecular subtypes of glioma, our laboratory and others have previously shown that the oligodendrocyte precursor cell (OPC) rather than the NSC serves as the cell of origin for the proneural subtype, raising questions regarding the suitability of NSC-tailored media for culturing proneural glioma cells. OPC-originated mouse glioma cells were cultured in conditions for normal OPCs or NSCs, respectively, for multiple passages. Gene expression profiles, morphologies, tumorigenicity, and drug responsiveness of cultured cells were examined in comparison with freshly isolated tumor cells. OPC media-cultured glioma cells maintained tumorigenicity, gene expression profiles, and morphologies similar to freshly isolated tumor cells. In contrast, NSC-media cultured glioma cells gradually lost their OPC features and most tumor-initiating ability and acquired heightened sensitivity to temozolomide. To improve experimental reproducibility and translational potential of glioma research, it is important to identify the cell of origin, and subsequently apply this knowledge to establish culture conditions that allow the retention of native properties of tumor cells. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice

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Wei Xin Cai

2016-01-01

Full Text Available Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (n=8. A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells’ tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo.

Efficacy of melatonin, IL-25 and siIL-17B in tumorigenesis-associated properties of breast cancer cell lines.

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Gelaleti, Gabriela Bottaro; Borin, Thaiz Ferraz; Maschio-Signorini, Larissa Bazela; Moschetta, Marina Gobbe; Jardim-Perassi, Bruna Victorasso; Calvinho, Guilherme Berto; Facchini, Mariana Castilho; Viloria-Petit, Alicia M; de Campos Zuccari, Debora Aparecida Pires

2017-08-15

Mammary tumorigenesis can be modulated by melatonin, which has oncostatic action mediated by multiple mechanisms, including the inhibition of the activity of transcription factors such as NF-κB and modulation of interleukins (ILs) expression. IL-25 is an active cytokine that induces apoptosis in tumor cells due to differential expression of its receptor (IL-17RB). IL-17B competes with IL-25 for binding to IL-17RB in tumor cells, promoting tumorigenesis. This study purpose is to address the possibility of engaging IL-25/IL-17RB signaling to enhance the effect of melatonin on breast cancer cells. Breast cancer cell lines were cultured monolayers and 3D structures and treated with melatonin, IL-25, siIL-17B, each alone or in combination. Cell viability, gene and protein expression of caspase-3, cleaved caspase-3 and VEGF-A were performed by qPCR and immunofluorescence. In addition, an apoptosis membrane array was performed in metastatic cells. Treatments with melatonin and IL-25 significantly reduced tumor cells viability at 1mM and 1ng/mL, respectively, but did not alter cell viability of a non-tumorigenic epithelial cell line (MCF-10A). All treatments, alone and combined, significantly increased cleaved caspase-3 in tumor cells grown as monolayers and 3D structures (pmelatonin treatment. All treatments reduced VEGF-A protein expression in tumor cells (pmelatonin and IL-25-driven signaling in breast cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential Cell Adhesion of Breast Cancer Stem Cells on Biomaterial Substrate with Nanotopographical Cues

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Kenneth K.B. Tan

2015-04-01

Full Text Available Cancer stem cells are speculated to have the capability of self-renewal and re-establishment of tumor heterogeneity, possibly involved in the potential relapse of cancer. CD44+CD24−/lowESA+ cells have been reported to possess tumorigenic properties, and these biomarkers are thought to be highly expressed in breast cancer stem cells. Cell behavior can be influenced by biomolecular and topographical cues in the natural microenvironment. We hypothesized that different cell populations in breast cancer tissue exhibit different adhesion characteristics on substrates with nanotopography. Adhesion characterizations were performed using human mammary epithelial cells (HMEC, breast cancer cell line MCF7 and primary invasive ductal carcinoma (IDC cells obtained from patients’ samples, on micro- and nano-patterned poly-L-lactic acid (PLLA films. Topography demonstrated a significant effect on cell adhesion, and the effect was cell type dependent. Cells showed elongation morphology on gratings. The CD44+CD24−/lowESA+ subpopulation in MCF7 and IDC cells showed preferential adhesion on 350-nm gratings. Flow cytometry analysis showed that 350-nm gratings captured a significantly higher percentage of CD44+CD24− in MCF7. A slightly higher percentage of CD44+CD24−/lowESA+ was captured on the 350-nm gratings, although no significant difference was observed in the CD44+CD24−ESA+ in IDC cells across patterns. Taken together, the study demonstrated that the cancer stem cell subpopulation could be enriched using different nanopatterns. The enriched population could subsequently aid in the isolation and characterization of cancer stem cells.

Long non-coding RNA TUG1 promotes cell proliferation and metastasis in human breast cancer.

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Li, Teng; Liu, Yun; Xiao, Haifeng; Xu, Guanghui

2017-07-01

Long non-coding RNAs (LncRNAs) utilize a wide variety of mechanisms to regulate RNAs or proteins on the transcriptional or post-transcriptional levels. Accumulating studies have identified numerous LncRNAs to exert critical effects on different physiological processes, genetic disorders, and human diseases. Both clinical tissues from breast cancer patients and cultured cells were used for the qRT-PCR analysis. Specific siRNAs were included to assess the roles of TUG1 with cell viability assay, transwell assay, and cell apoptosis assay, respectively. The expression of TUG1 was enhanced in breast cancerous tissues and in highly invasive breast cancer cell lines and was associated with clinical variables, including tumor size, distant metastasis and TNM staging. Knockdown of TUG1 significantly slowed down cell proliferation, cell migration, and invasion in breast cancer cell lines MDA-MB-231 and MDA-MB-436. In addition, cell apoptotic rate was shown to increase upon siTUG1 treatment as evidenced by increases of the activities of caspase-3 and caspase-9. The identification of TUG1 as a critical mediator of breast cancer progression implied that it might serve as a biomarker for the diagnosis and treatment of breast cancer in clinic.

Overexpression of p42.3 promotes cell growth and tumorigenicity in hepatocellular carcinoma

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Sun, Wei; Dong, Wei-Wei; Mao, Lin-Lin; Li, Wen-Mei; Cui, Jian-Tao; Xing, Rui; Lu, You-Yong

2013-01-01

AIM: To investigate the association of p42.3 expression with clinicopathological characteristics and the biological function of p42.3 in human hepatocellular carcinoma (HCC). METHODS: We used reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR and western blotting to detect p42.3 mRNA and protein expression in hepatic cell lines. We examined primary HCC samples and matched adjacent normal tissue by immunohistochemistry to investigate the correlation between p42.3 expression and clinicopathological features. HepG2 cells were transfected with a pIRES2-EGFP-p42.3 expression vector to examine the function of the p42.3 gene. Transfected cells were analyzed for their viability and malignant transformation abilities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and tumorigenicity assay in nude mice. RESULTS: p42.3 is differentially expressed in primary HCC tumors and cell lines. Approximately 69.6% (96/138) of cells were p42.3-positive in hepatic tumor tissues, while 30.7% (35/114) were p42.3-positive in tumor-adjacent normal tissues. Clinicopathological characteristics of the HCC specimens revealed a significant correlation between p42.3 expression and tumor differentiation (P = 0.031). However, p42.3 positivity was not related to tumor tumor-node-metastasis classification, hepatitis B virus status, or hepatoma type. Regarding p42.3 overexpression in stably transfected HepG2 cells, we discovered significant enhancement of cancer cell growth and colony formation in vitro, and significantly enhanced tumorigenicity in nude mice. Western blot analysis of cell cycle proteins revealed that enhanced p42.3 levels promote upregulation of proliferating cell nuclear antigen, cyclin B1 and mitotic arrest deficient 2. CONCLUSION: p42.3 promotes tumorigenicity and tumor growth in HCC and may be a potential target for future clinical cancer therapeutics. PMID:23704824

Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.

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Spina, Raffaella; Filocamo, Gessica; Iaccino, Enrico; Scicchitano, Stefania; Lupia, Michela; Chiarella, Emanuela; Mega, Tiziana; Bernaudo, Francesca; Pelaggi, Daniela; Mesuraca, Maria; Pazzaglia, Simonetta; Semenkow, Samantha; Bar, Eli E; Kool, Marcel; Pfister, Stefan; Bond, Heather M; Eberhart, Charles G; Steinkühler, Christian; Morrone, Giovanni

2013-08-01

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner.

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David, Diana; Jagadeeshan, Sankar; Hariharan, Ramkumar; Nair, Asha Sivakumari; Pillai, Radhakrishna Madhavan

2014-01-01

Smurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. The purpose of the present study is to examine the effect of Smurf2 knockdown on the tumorigenic potential of human breast cancer cells emphasizing more on proliferative signaling pathway. siRNAs targeting different regions of the Smurf2 mRNA were employed to knockdown the expression of Smurf2. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth, cell cycle arrest, and cell cycle and cell proliferation related protein expressions upon Smurf2 silencing. Smurf2 silencing in human breast cancer cells resulted in a decreased focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of Smurf2 suppressed cell proliferation. Cell cycle analysis showed that the anti-proliferative effect of Smurf2 siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by decreased expression of cyclin D1and cdk4, followed by upregulation p21 and p27. Furthermore, we demonstrated that silencing of Smurf2 downregulated the proliferation of breast cancer cells by modulating the PI3K- PTEN-AKT-FoxO3a pathway via the scaffold protein CNKSR2 which is involved in RAS-dependent signaling pathways. The present study provides the first evidence that silencing Smurf2 using synthetic siRNAs can regulate the tumorigenic properties of human breast cancer cells in a CNKSR2 dependent manner. Our results

HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution.

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Stepanenko, A A; Dmitrenko, V V

2015-09-15

293 cell line (widely known as the Human Embryonic Kidney 293 cells) and its derivatives were the most used cells after HeLa in cell biology studies and after CHO in biotechnology as a vehicle for the production of adenoviral vaccines and recombinant proteins, for analysis of the neuronal synapse formation, in electrophysiology and neuropharmacology. Despite the historically long-term productive exploitation, the origin, phenotype, karyotype, and tumorigenicity of 293 cells are still debated. 293 cells were considered the kidney epithelial cells or even fibroblasts. However, 293 cells demonstrate no evident tissue-specific gene expression signature and express the markers of renal progenitor cells, neuronal cells and adrenal gland. This complicates efforts to reveal the authentic cell type/tissue of origin. On the other hand, the potential to propagate the highly neurotropic viruses, inducible synaptogenesis, functionality of the endogenous neuron-specific voltage-gated channels, and response to the diverse agonists implicated in neuronal signaling give credibility to consider 293 cells of neuronal lineage phenotype. The compound phenotype of 293 cells can be due to heterogeneous, unstable karyotype. The mean chromosome number and chromosome aberrations differ between 293 cells and derivatives as well as between 293 cells from the different cell banks/labs. 293 cells are tumorigenic, whereas acute changes of expression of the cancer-associated genes aggravate tumorigenicity by promoting chromosome instability. Importantly, the procedure of a stable empty vector transfection can also impact karyotype and phenotype. The discussed issues caution against misinterpretations and pitfalls during the different experimental manipulations with 293 cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Selection of metastatic breast cancer cells based on adaptability of their metabolic state.

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Balraj Singh

Full Text Available A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis

Selection of Metastatic Breast Cancer Cells Based on Adaptability of Their Metabolic State

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Singh, Balraj; Tai, Karen; Madan, Simran; Raythatha, Milan R.; Cady, Amanda M.; Braunlin, Megan; Irving, LaTashia R.; Bajaj, Ankur; Lucci, Anthony

2012-01-01

A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR.

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Ingthorsson, S; Andersen, K; Hilmarsdottir, B; Maelandsmo, G M; Magnusson, M K; Gudjonsson, T

2016-08-11

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492(HER2)) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492(HER2) (D492(HER2/EGFR)) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492(HER2/EGFR) xenografts grow slower than the D492(HER2) tumors, while overexpression of EGFR alone (D492(EGFR)) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492(HER2) xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492

SOX9-mediated upregulation of LGR5 is important for glioblastoma tumorigenicity

International Nuclear Information System (INIS)

Hiraoka, Koji; Hayashi, Tomoatsu; Kaneko, Ryusuke; Nasu-Nishimura, Yukiko; Koyama-Nasu, Ryo; Kawasaki, Yoshihiro; Akiyama, Tetsu

2015-01-01

LGR5 plays an important role in the self-renewal of stem cells and is used as a marker identifying self-renewing stem cells in small intestine and hair follicles. Moreover, LGR5 has been reported to be overexpressed in several cancers. SOX9 is a transcription factor that plays a key role in development, differentiation and lineage commitment in various tissues. It has also been reported that SOX9 is overexpressed in a variety of cancers and contributes to their malignant phenotype. Here we show that LGR5 is required for the tumorigenicity of glioblastoma cells. We further show that SOX9 is upregulated in glioblastoma cells and directly enhances the expression of LGR5. We also demonstrate that knockdown of SOX9 suppresses the proliferation and tumorigenicity of glioblastoma cells. These results suggest that SOX9-mediated transcriptional regulation of LGR5 is critical for the tumorigenicity of glioblastoma cells. We speculate that the SOX9-LGR5 pathway could be a potentially promising target for the therapy of glioblastoma. - Highlights: • LGR5 is required for the tumorigenicity of glioblastoma cells. • SOX9 directly enhances the expression of LGR5. • SOX9 is required for the tumorigenicity of glioblastoma cells

SOX9-mediated upregulation of LGR5 is important for glioblastoma tumorigenicity

Energy Technology Data Exchange (ETDEWEB)

Hiraoka, Koji; Hayashi, Tomoatsu; Kaneko, Ryusuke; Nasu-Nishimura, Yukiko; Koyama-Nasu, Ryo; Kawasaki, Yoshihiro; Akiyama, Tetsu, E-mail: akiyama@iam.u-tokyo.ac.jp

2015-05-01

LGR5 plays an important role in the self-renewal of stem cells and is used as a marker identifying self-renewing stem cells in small intestine and hair follicles. Moreover, LGR5 has been reported to be overexpressed in several cancers. SOX9 is a transcription factor that plays a key role in development, differentiation and lineage commitment in various tissues. It has also been reported that SOX9 is overexpressed in a variety of cancers and contributes to their malignant phenotype. Here we show that LGR5 is required for the tumorigenicity of glioblastoma cells. We further show that SOX9 is upregulated in glioblastoma cells and directly enhances the expression of LGR5. We also demonstrate that knockdown of SOX9 suppresses the proliferation and tumorigenicity of glioblastoma cells. These results suggest that SOX9-mediated transcriptional regulation of LGR5 is critical for the tumorigenicity of glioblastoma cells. We speculate that the SOX9-LGR5 pathway could be a potentially promising target for the therapy of glioblastoma. - Highlights: • LGR5 is required for the tumorigenicity of glioblastoma cells. • SOX9 directly enhances the expression of LGR5. • SOX9 is required for the tumorigenicity of glioblastoma cells.

Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

Science.gov (United States)

Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

2012-01-01

Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and

Tumor cell expression of CD163 is associated to postoperative radiotherapy and poor prognosis in patients with breast cancer treated with breast-conserving surgery.

Science.gov (United States)

Garvin, Stina; Oda, Husam; Arnesson, Lars-Gunnar; Lindström, Annelie; Shabo, Ivan

2018-05-03

Cancer cell fusion with macrophages results in highly tumorigenic hybrids that acquire genetic and phenotypic characteristics from both maternal cells. Macrophage traits, exemplified by CD163 expression, in tumor cells are associated with advanced stages and poor prognosis in breast cancer (BC). In vitro data suggest that cancer cells expressing CD163 acquire radioresistance. Tissue microarray was constructed from primary BC obtained from 83 patients treated with breast-conserving surgery, 50% having received postoperative radiotherapy (RT) and none of the patients had lymph node or distant metastasis. Immunostaining of CD163 in cancer cells and macrophage infiltration (MI) in tumor stroma were evaluated. Macrophage:MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation (0, 2.5 and 5 Gy γ-radiation), both hybrids and their maternal MCF-7 cells were examined by clonogenic survival. CD163-expression by cancer cells was significantly associated with MI and clinicopathological data. Patients with CD163-positive tumors had significantly shorter disease-free survival (DFS) after RT. In vitro generated macrophage:MCF-7 hybrids developed radioresistance and exhibited better survival and colony forming ability after radiation compared to maternal MCF-7 cancer cells. Our results suggest that macrophage phenotype in tumor cells results in radioresistance in breast cancer and shorter DFS after radiotherapy.

Mechanism of c-Src Synergy with the EGFR In Breast Cancer

National Research Council Canada - National Science Library

Tice, David

1999-01-01

... on tumorigenicity and growth of breast tumor cells. Furthermore, we have discovered a mechanism of c-Src synergy with the EGFR and located specific points at which the pathway can be interdicted...

Effect of doxorubicin/pluronic SP1049C on tumorigenicity, aggressiveness, DNA methylation and stem cell markers in murine leukemia.

Directory of Open Access Journals (Sweden)

Daria Y Alakhova

Full Text Available Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC and decrease tumorigenicity of cancer cells in vivo.P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a saline, (b Pluronics alone, (c Dox or (d SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1 in vitro colony formation potential, 2 in vivo tumorigenicity and aggressiveness, 3 development of drug resistance and Wnt signaling activation 4 global DNA methylation profiles, and 5 expression of CSC markers.SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-β-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+ P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(- cells.SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

The effect of neighboring cells on the stiffness of cancerous and non-cancerous human mammary epithelial cells

Science.gov (United States)

Guo, Xinyi; Bonin, Keith; Scarpinato, Karin; Guthold, Martin

2014-10-01

Using an Atomic Force Microscope (AFM) with a 5.3 μm diameter spherical probe, we determined mechanical properties of individual human mammary epithelial cells. The cells were derived from a pair of cell lines that mimic cell progression through four phases of neoplastic transformation: normal (non-transformed), immortal, tumorigenic, and metastatic. Measurements on cells in all four phases were taken over both the cytoplasmic and nuclear regions. Moreover, the measurements were made for cells in different microenvironments as related to cell-cell contacts: isolated cells; cells residing on the periphery of a contiguous cell monolayer; and cells on the inside of a contiguous cell monolayer. By fitting the AFM force versus indentation curves to a Hertz model, we determined the pseudo-elastic Young’s modulus, E. Combining all data for the cellular subregions (over nucleus and cytoplasm) and the different cell microenvironments, we obtained stiffness values for normal, immortal, tumorigenic, and metastatic cells of 870 Pa, 870 Pa, 490 Pa, and 580 Pa, respectively. That is, cells become softer as they advance to the tumorigenic phase and then stiffen somewhat in the final step to metastatic cells. We also found a distinct contrast in the influence of a cell’s microenvironment on cell stiffness. Normal mammary epithelial cells inside a monolayer are stiffer than peripheral cells, which are stiffer than isolated cells. However, the microenvironment had a slight, opposite effect on tumorigenic and little effect on immortal and metastatic cell stiffness. Thus, the stiffness of cancer cells is less sensitive to the microenvironment than normal cells. Our results show that the mechanical properties of a cell can depend on cancer progression and microenvironment (cell-cell interactions).

Characterization of a naturally occurring breast cancer subset enriched in EMT and stem cell characteristics

Energy Technology Data Exchange (ETDEWEB)

Hennessy, Bryan T.; Gonzalez-Angulo, Ana-Maria; Stemke-Hale, Katherine; Gilcrease, Michael Z.; Krishnamurthy, Savitri; Lee, Ju-Seog; Fridlyand, Jane; Sahin, Aysegul; Agarwal, Roshan; Joy, Corwin; Liu, Wenbin; Stivers, David; Baggerly, Keith; Carey, Mark; Lluch, Ana; Monteagudo, Carlos; He, Xiaping; Weigman, Victor; Fan, Cheng; Palazzo, Juan; Hortobagyi, Gabriel N.; Nolden, Laura K.; Wang, Nicholas J.; Valero, Vicente; Gray, Joe W.; Perou, Charles M.; Mills, Gordon B.

2009-05-19

Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor-positive cancers (34.5%; P = 0.32), 17 of 75 HER-2-positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a 'tumorigenic' signature defined using CD44{sup +}/CD24{sup -} breast tumor-initiating stem cell-like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell-like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets.

The effect of neighboring cells on the stiffness of cancerous and non-cancerous human mammary epithelial cells

International Nuclear Information System (INIS)

Guo, Xinyi; Bonin, Keith; Guthold, Martin; Scarpinato, Karin

2014-01-01

Using an Atomic Force Microscope (AFM) with a 5.3 μm diameter spherical probe, we determined mechanical properties of individual human mammary epithelial cells. The cells were derived from a pair of cell lines that mimic cell progression through four phases of neoplastic transformation: normal (non-transformed), immortal, tumorigenic, and metastatic. Measurements on cells in all four phases were taken over both the cytoplasmic and nuclear regions. Moreover, the measurements were made for cells in different microenvironments as related to cell–cell contacts: isolated cells; cells residing on the periphery of a contiguous cell monolayer; and cells on the inside of a contiguous cell monolayer. By fitting the AFM force versus indentation curves to a Hertz model, we determined the pseudo-elastic Young’s modulus, E. Combining all data for the cellular subregions (over nucleus and cytoplasm) and the different cell microenvironments, we obtained stiffness values for normal, immortal, tumorigenic, and metastatic cells of 870 Pa, 870 Pa, 490 Pa, and 580 Pa, respectively. That is, cells become softer as they advance to the tumorigenic phase and then stiffen somewhat in the final step to metastatic cells. We also found a distinct contrast in the influence of a cell’s microenvironment on cell stiffness. Normal mammary epithelial cells inside a monolayer are stiffer than peripheral cells, which are stiffer than isolated cells. However, the microenvironment had a slight, opposite effect on tumorigenic and little effect on immortal and metastatic cell stiffness. Thus, the stiffness of cancer cells is less sensitive to the microenvironment than normal cells. Our results show that the mechanical properties of a cell can depend on cancer progression and microenvironment (cell–cell interactions). (paper)

Kinomic and phospho-proteomic analysis of breast cancer stem-like cells

DEFF Research Database (Denmark)

Leth-Larsen, Rikke; Christensen, Anne Geske Lindhard; Ehmsen, Sidse

Kinomic and phospho-proteomic analysis of breast cancer stem-like cells Rikke Leth-Larsen1, Anne G Christensen1, Sidse Ehmsen1, Mark Møller1, Giuseppe Palmisano2, Martin R Larsen2, Henrik J Ditzel1,3 1Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 2Institute...... of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark 3Dept. of Oncology, Odense University Hospital, Odense, Denmark Cancer stem cells are thought to be responsible for tumorigenic potential and to possess resistance mechanisms against chemotherapy- and radiation-induced cancer...... cell death, while the bulk of a tumor lacks these capacities. The resistance mechanisms may cause these cells to survive and become the source of later tumor recurrence, highlighting the need for therapeutic strategies that specifically target pathways central to these cancer stem cells. The CD44hi...

A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer

Directory of Open Access Journals (Sweden)

Paul Savage

2017-10-01

Full Text Available Summary: Therapies targeting epidermal growth factor receptor (EGFR have variable and unpredictable responses in breast cancer. Screening triple-negative breast cancer (TNBC patient-derived xenografts (PDXs, we identify a subset responsive to EGFR inhibition by gefitinib, which displays heterogeneous expression of wild-type EGFR. Deep single-cell RNA sequencing of 3,500 cells from an exceptional responder identified subpopulations displaying distinct biological features, where elevated EGFR expression was significantly enriched in a mesenchymal/stem-like cellular cluster. Sorted EGFRhi subpopulations exhibited enhanced stem-like features, including ALDH activity, sphere-forming efficiency, and tumorigenic and metastatic potential. EGFRhi cells gave rise to EGFRhi and EGFRlo cells in primary and metastatic tumors, demonstrating an EGFR-dependent expansion and hierarchical state transition. Similar tumorigenic EGFRhi subpopulations were identified in independent PDXs, where heterogeneous EGFR expression correlated with gefitinib sensitivity. This provides new understanding for an EGFR-dependent hierarchy in TNBC and for patient stratification for therapeutic intervention. : Savage et al. demonstrate that sensitivity to EGFR inhibitor, gefitinib, in triple-negative breast cancer is paradoxically associated with EGFR heterogeneity. Using single-cell RNA sequencing in conjunction with functional assays, they identify TNBC tumors in which EGFR expression identifies cells with tumor-initiating capacity whose proliferative expansion is sensitive to EGFR inhibition. Keywords: breast cancer, tumor heterogeneity, patient-derived xenograft, single-cell RNA sequencing, EGFR inhibition, therapeutic response, tumor-initiating cell, cell hierarchy, BRCA1 mutation

Targeting the Ron-Dek Signaling Axis in Breast Cancer

Science.gov (United States)

2015-09-01

Words 5 Accomplishments 5 Impact & Conclusion 8 Changes/Problems 9 Products 9 Participants and Other Collaborating Organizations 11 Special...may represent an important new therapeutic option for the treatment of breast cancer. 2. KEY WORDS Ron receptor, Dek, beta-catenin, breast cancer 3...Finally, our data showed that Dek overexpression promoted tumorigenic properties in immortalized human mammary epithelial MCF10A cells and in the context

X-ray induction of immortalization in primary rat embryo cells associated with and without tumorigenicity

International Nuclear Information System (INIS)

Sierra, E.; Oberley, L.W.; Guernsey, D.L.

1985-01-01

Cultures of primary rat embryo fibroblasts were irradiated with X-rays (3 Gy). After 14 days the majority of colonies in both irradiated and control plates had senesced. Surviving clones were ring isolated from irradiated and control plates and grown in culture. A phase of rapid proliferation after isolation was observed, followed by a decline (crisis) leading to senescence. Several clones from the irradiated plates were able to recover from this crisis and gave rise to continuous cell lines, while all colonies from control plates senesced. Three types of cells have been identified among the irradiated survivors: (1) immortal fully transformed, capable of growth in soft agar (Aga/sup +/) and tumor formation, (2) immortal normal, not able to grow in soft agar (Aga/sup -/) and nontumorigenic, and (3) immortal Aga/sup -/ cells which progressed to malignancy (Aga/sup +/, tumorigenicity) after further sub-culture. These data support the suggestion that X-rays can induce immortalization of mammalian cells in the absence of tumorigenicity, in addition to (and separate from) the fully tumorigenetic state

Tumorigenic Heterogeneity in Cancer Stem Cells Evolved from Long-term Cultures of Telomerase-Immortalized

DEFF Research Database (Denmark)

Burns, Jorge S; Abdallah, Basem M; Guldberg, Per

2005-01-01

Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation or if...

Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

International Nuclear Information System (INIS)

Sadlonova, Andrea; Novak, Zdenek; Johnson, Martin R; Bowe, Damon B; Gault, Sandra R; Page, Grier P; Thottassery, Jaideep V; Welch, Danny R; Frost, Andra R

2005-01-01

Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF

Long non-coding RNA Loc554202 regulates proliferation and migration in breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Shi, Yongguo, E-mail: 1138303166@qq.com [Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Lu, Jianwei, E-mail: jianwei2010077@163.com [Cancer Hospital of Jiangsu Province, Nanjing, Jiangsu (China); Zhou, Jing, E-mail: 2310848@163.com [Department of Oncology, Taizhou People’ Hospital, Taizhou, Jiangsu (China); Tan, Xueming, E-mail: 843039795@qq.com [Department of Gastroenterology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); He, Ye, E-mail: 2825636@qq.com [Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Ding, Jie, E-mail: 9111165@qq.com [Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Tian, Yun, E-mail: 1815857@qq.com [Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wang, Li, E-mail: 2376737@qq.com [Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wang, Keming, E-mail: wkmys@sohu.com [Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China)

2014-04-04

Highlights: • First, we have shown that upregulated of the Loc554202 in breast cancer tissues. • Second, we demonstrated the function of Loc554202 in breast cancer cell. • Finally, we demonstrated that LOC554202 knockdown could inhibit tumor growth in vivo. - Abstract: Data derived from massive cloning and traditional sequencing methods have revealed that long non-coding RNAs (lncRNA) play important roles in the development and progression of cancer. Although many studies suggest that the lncRNAs have different cellular functions, many of them are not yet to be identified and characterized for the mechanism of their functions. To address this question, we assay the expression level of lncRNAs–Loc554202 in breast cancer tissues and find that Loc554202 is significantly increased compared with normal control, and associated with advanced pathologic stage and tumor size. Moreover, knockdown of Loc554202 decreased breast cancer cell proliferation, induced apoptosis and inhibits migration/invasion in vitro and impeded tumorigenesis in vivo. These data suggest an important role of Loc554202 in breast tumorigenesis.

Selective expression of long non-coding RNAs in a breast cancer cell progression model.

Science.gov (United States)

Tracy, Kirsten M; Tye, Coralee E; Page, Natalie A; Fritz, Andrew J; Stein, Janet L; Lian, Jane B; Stein, Gary S

2018-02-01

Long non-coding RNAs (lncRNAs) are acknowledged as regulators of cancer biology and pathology. Our goal was to perform a stringent profiling of breast cancer cell lines that represent disease progression. We used the MCF-10 series, which includes the normal-like MCF-10A, HRAS-transformed MCF-10AT1 (pre-malignant), and MCF-10CA1a (malignant) cells, to perform transcriptome wide sequencing. From these data, we have identified 346 lncRNAs with dysregulated expression across the progression series. By comparing lncRNAs from these datasets to those from an additional set of cell lines that represent different disease stages and subtypes, MCF-7 (early stage, luminal), and MDA-MB-231 (late stage, basal), 61 lncRNAs that are associated with breast cancer progression were identified. Querying breast cancer patient data from The Cancer Genome Atlas, we selected a lncRNA, IGF-like family member 2 antisense RNA 1 (IGFL2-AS1), of potential clinical relevance for functional characterization. Among the 61 lncRNAs, IGFL2-AS1 was the most significantly decreased. Our results indicate that this lncRNA plays a role in downregulating its nearest neighbor, IGFL1, and affects migration of breast cancer cells. Furthermore, the lncRNAs we identified provide a valuable resource to mechanistically and clinically understand the contribution of lncRNAs in breast cancer progression. © 2017 Wiley Periodicals, Inc.

SHOX2 Is a Direct miR-375 Target and a Novel Epithelial-to-Mesenchymal Transition Inducer in Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Sungguan Hong

2014-04-01

Full Text Available MicroRNAs have added a new dimension to our understanding of tumorigenesis and associated processes like epithelial-to-mesenchymal transition (EMT. Here, we show that miR-375 is elevated in epithelial-like breast cancer cells, and ectopic miR-375 expression suppresses EMT in mesenchymal-like breast cancer cells. We identified short stature homeobox 2 (SHOX2 as a miR-375 target, and miR-375–mediated suppression in EMT was reversed by forced SHOX2 expression. Ectopic SHOX2 expression can induce EMT in epithelial-like breast cancer cells, whereas SHOX2 knockdown diminishes EMT traits in mesenchymal-like breast cancer cells, demonstrating SHOX2 as an EMT inducer. We show that SHOX2 acts as a transcription factor to upregulate transforming growth factor β receptor I (TβR-I expression, and TβR-I inhibitor LY364947 abolishes EMT elicited by ectopic SHOX2 expression, suggesting that transforming growth factor β signaling is essential for SHOX2-induced EMT. Manipulating SHOX2 abundance in breast cancer cells impact in vitro invasion and in vivo dissemination. Analysis of breast tumor microarray database revealed that high SHOX2 expression significantly correlates with poor patient survival. Our study supports a critical role of SHOX2 in breast tumorigenicity.

The Application of Non-Invasive Apoptosis Detection Sensor (NIADS on Histone Deacetylation Inhibitor (HDACi-Induced Breast Cancer Cell Death

Directory of Open Access Journals (Sweden)

Kai-Wen Hsu

2018-02-01

Full Text Available Breast cancer is the most common malignancy in women and the second leading cause of cancer death in women. Triple negative breast cancer (TNBC subtype is a breast cancer subset without ER (estrogen receptor, PR (progesterone receptor and HER2 (human epidermal growth factor receptor 2 expression, limiting treatment options and presenting a poorer survival rate. Thus, we investigated whether histone deacetylation inhibitor (HDACi could be used as potential anti-cancer therapy on breast cancer cells. In this study, we found TNBC and HER2-enriched breast cancers are extremely sensitive to Panobinostat, Belinostat of HDACi via experiments of cell viability assay, apoptotic marker identification and flow cytometry measurement. On the other hand, we developed a bioluminescence-based live cell non-invasive apoptosis detection sensor (NIADS detection system to evaluate the quantitative and kinetic analyses of apoptotic cell death by HDAC treatment on breast cancer cells. In addition, the use of HDACi may also contribute a synergic anti-cancer effect with co-treatment of chemotherapeutic agent such as doxorubicin on TNBC cells (MDA-MB-231, but not in breast normal epithelia cells (MCF-10A, providing therapeutic benefits against breast tumor in the clinic.

The Rab2A GTPase Promotes Breast Cancer Stem Cells and Tumorigenesis via Erk Signaling Activation

Directory of Open Access Journals (Sweden)

Man-Li Luo

2015-04-01

Full Text Available Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs. Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

Science.gov (United States)

Nandakumar, Vivek; Kelbauskas, Laimonas; Hernandez, Kathryn F; Lintecum, Kelly M; Senechal, Patti; Bussey, Kimberly J; Davies, Paul C W; Johnson, Roger H; Meldrum, Deirdre R

2012-01-01

Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria. We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure. We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At pfibrocystic from the metastatic cell populations. Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.

Scaffold-Free Coculture Spheroids of Human Colonic Adenocarcinoma Cells and Normal Colonic Fibroblasts Promote Tumorigenicity in Nude Mice

Directory of Open Access Journals (Sweden)

Jong-il Park

2016-02-01

Full Text Available The aim of this study was to form a scaffold-free coculture spheroid model of colonic adenocarcinoma cells (CACs and normal colonic fibroblasts (NCFs and to use the spheroids to investigate the role of NCFs in the tumorigenicity of CACs in nude mice. We analysed three-dimensional (3D scaffold-free coculture spheroids of CACs and NCFs. CAC Matrigel invasion assays and tumorigenicity assays in nude mice were performed to examine the effect of NCFs on CAC invasive behaviour and tumorigenicity in 3D spheroids. We investigated the expression pattern of fibroblast activation protein-α (FAP-α by immunohistochemical staining. CAC monocultures did not form densely-packed 3D spheroids, whereas cocultured CACs and NCFs formed 3D spheroids. The 3D coculture spheroids seeded on a Matrigel extracellular matrix showed higher CAC invasiveness compared to CACs alone or CACs and NCFs in suspension. 3D spheroids injected into nude mice generated more and faster-growing tumors compared to CACs alone or mixed suspensions consisting of CACs and NCFs. FAP-α was expressed in NCFs-CACs cocultures and xenograft tumors, whereas monocultures of NCFs or CACs were negative for FAP-α expression. Our findings provide evidence that the interaction between CACs and NCFs is essential for the tumorigenicity of cancer cells as well as for tumor propagation.

Smad7 induces tumorigenicity by blocking TGF-beta-induced growth inhibition and apoptosis.

Science.gov (United States)

Halder, Sunil K; Beauchamp, R Daniel; Datta, Pran K

2005-07-01

Smad proteins play a key role in the intracellular signaling of the transforming growth factor beta (TGF-beta) superfamily of extracellular polypeptides that initiate signaling to regulate a wide variety of biological processes. The inhibitory Smad, Smad7, has been shown to function as intracellular antagonists of TGF-beta family signaling and is upregulated in several cancers. To determine the effect of Smad7-mediated blockade of TGF-beta signaling, we have stably expressed Smad7 in a TGF-beta-sensitive, well-differentiated, and non-tumorigenic cell line, FET, that was derived from human colon adenocarcinoma. Smad7 inhibits TGF-beta-induced transcriptional responses by blocking complex formation between Smad 2/3 and Smad4. While Smad7 has no effect on TGF-beta-induced activation of p38 MAPK and ERK, it blocks the phosphorylation of Akt by TGF-beta and enhances TGF-beta-induced phosphorylation of c-Jun. FET cells expressing Smad7 show anchorage-independent growth and enhance tumorigenicity in athymic nude mice. Smad7 blocks TGF-beta-induced growth inhibition by preventing TGF-beta-induced G1 arrest. Smad7 inhibits TGF-beta-mediated downregulation of c-Myc, CDK4, and Cyclin D1, and suppresses the expression of p21(Cip1). As a result, Smad7 inhibits TGF-beta-mediated downregulation of Rb phosphorylation. Furthermore, Smad7 inhibits the apoptosis of these cells. Together, Smad7 may increase the tumorigenicity of FET cells by blocking TGF-beta-induced growth inhibition and by inhibiting apoptosis. Thus, this study provides a mechanism by which a portion of human colorectal tumors may become refractory to tumor-suppressive actions of TGF-beta that might result in increased tumorigenicity.

RECK is not an independent prognostic marker for breast cancer

International Nuclear Information System (INIS)

Gomes, Luciana R.; Fujita, André; Mott, Joni D.; Soares, Fernando A.; Labriola, Leticia; Sogayar, Mari C.

2015-01-01

The REversion-inducing Cysteine-rich protein with Kazal motif (RECK) is a well-known inhibitor of matrix metalloproteinases (MMPs) and cellular invasion. Although high expression levels of RECK have already been correlated with a better clinical outcome for several tumor types, its main function, as well as its potential prognostic value for breast cancer patients, remain unclear. The RECK expression profile was investigated in a panel of human breast cell lines with distinct aggressiveness potential. RECK functional analysis was undertaken using RNA interference methodology. RECK protein levels were also analyzed in 1040 cases of breast cancer using immunohistochemistry and tissue microarrays (TMAs). The association between RECK expression and different clinico-pathological parameters, as well as the overall (OS) and disease-free (DFS) survival rates, were evaluated. Higher RECK protein expression levels were detected in more aggressive breast cancer cell lines (T4-2, MDA-MB-231 and Hs578T) than in non-invasive (MCF-7 and T47D) and non-tumorigenic (S1) cell lines. Indeed, silencing RECK in MDA-MB-231 cells resulted in elevated levels of pro-MMP-9 and increased invasion compared with scrambled (control) cells, without any effect on cell proliferation. Surprisingly, by RECK immunoreactivity analysis on TMAs, we found no association between RECK positivity and survival (OS and DFS) in breast cancer patients. Even considering the different tumor subtypes (luminal A, luminal B, Her2 type and basal-like) or lymph node status, RECK remained ineffective for predicting the disease outcome. Moreover, by multivariate Cox regression analysis, we found that RECK has no prognostic impact for OS and DFS, relative to standard clinical variables. Although it continues to serve as an invasion and MMP inhibitor in breast cancer, RECK expression analysis is not useful for prognosis of these patients

Comparative effects of 4-phenyl-3-butenoic acid and vorinostat on cell growth and signaling.

Science.gov (United States)

Burns, Timothy J; Ali, Amna; Matesic, Diane F

2015-02-01

4-phenyl-3-butenoic acid (PBA) is a small-molecule anti-inflammatory agent, which has been shown to inhibit growth, increase gap junction intercellular communication and modulate activation of p38 mitogen-activated protein kinase (p38 MAPK) and c-jun n-terminal kinase (JNK) in tumorigenic cells at concentrations that do not similarly affect non-tumorigenic cells. Vorinostat is an anticancer agent structurally similar to PBA. The purpose of this study was to compare the effects of these two agents on JNK and p38 activation, cell growth and gap junction intercellular communication (GJIC). Cell growth, GJIC and western blot analyses were performed utilizing tumorigenic WBras1 and H2009 human carcinoma cells, and non-tumorigenic WBneo3 and human bronchial epithelial (HBE) cells. Both compounds significantly inhibited WBras1 and H2009 tumorigenic cell growth and increased GJIC in WBras1 cells, as previously reported for PBA. Under similar conditions, both compounds increased phosphorylation of p38 MAPK in tumorigenic but not in non-tumorigenic cells and decreased phosphorylation of JNK in tumorigenic cells. However, a decrease in phosphorylation of JNK occurred in non-tumorigenic WBras1 cells following vorinostat treatment but not PBA treatment. Both compounds showed a selective growth inhibition of H2009 human carcinoma over normal HBE lung cells but, unlike PBA, vorinostat significantly decreased cell growth in WBneo3 cells. Overall, PBA exhibited similar effects to vorinostat in tumorigenic cells, while also showing reduced effects on JNK phosphorylation and growth in non-tumorigenic cells compared to ras-transformed cells. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Anti-fibrotic and anti-tumorigenic effects of rhein, a natural anthraquinone derivative, in mammalian stellate and carcinoma cells.

Science.gov (United States)

Tsang, Siu Wai; Bian, Zhao-Xiang

2015-03-01

Anthraquinone compounds have been recognized to possess antiinflammatory, anti-fibrotic and anti-tumour properties and thus applied in human and veterinary therapeutics as active substances of medicinal products. Amongst the anthraquinones isolated from Rheum palmatum, also known as da-huang, rhein was detected as one of the highest metabolite contents in the bloodstream of mammals. The biological activities of rhein therefore deserve detailed investigation. In this study, we aimed to delineate the mechanism of inhibitory actions of rhein on fibrotic and tumorigenic processes by means of various biochemical assays, such as immunofluorescent staining, real-time polymerase chain reaction (PCR) and western blotting analyses in rat pancreatic stellate cells (LTC-14), human pancreatic ductal adenocarcinoma cells (PANC-1) and human colon carcinoma cells (SW480 and SW620). Our results demonstrated that the application of rhein notably suppressed the mRNA and protein levels of various fibrotic and tumorigenic mediators including alpha-smooth muscle actin, type I collagen, fibronectin, N-cadherin and matrix metalloproteinases in the testing mammalian cells. The mechanism of the suppressive actions of rhein was associated with the modulation of the sonic hedgehog and serine-threonine kinase signalling pathways. In conclusion, we suggest that rhein may serve as a therapeutic or an adjuvant agent in anti-fibrotic and anti-tumorigenic approaches. Copyright © 2014 John Wiley & Sons, Ltd.

Simultaneous Expression of Cancer Stem Cell-Like Properties and Cancer-Associated Fibroblast-Like Properties in a Primary Culture of Breast Cancer Cells

Energy Technology Data Exchange (ETDEWEB)

Ishikawa, Mami; Inoue, Takahiro; Shirai, Takuma; Takamatsu, Kazuhiko; Kunihiro, Shiori; Ishii, Hirokazu [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Nishikata, Takahito, E-mail: nisikata@konan-u.ac.jp [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe 650-0047 (Japan)

2014-07-31

The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.

Protein Kinase C-ε Promotes EMT in Breast Cancer

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Jain, Kirti; Basu, Alakananda

2014-01-01

Protein kinase C (PKC), a family of serine/threonine kinases, plays critical roles in signal transduction and cell regulation. PKCε, a member of the novel PKC family, is known to be a transforming oncogene and a tumor biomarker for aggressive breast cancers. In this study, we examined the involvement of PKCε in epithelial to mesenchymal transition (EMT), the process that leads the way to metastasis. Overexpression of PKCε was sufficient to induce a mesenchymal phenotype in non-tumorigenic mammary epithelial MCF-10 A cells. This was accompanied by a decrease in the epithelial markers, such as E-cadherin, zonula occludens (ZO)-1, and claudin-1, and an increase in mesenchymal marker vimentin. Transforming growth factor β (TGFβ) induced Snail expression and mesenchymal morphology in MCF-10 A cells, and these effects were partially reversed by the PKCε knockdown. PKCε also mediated cell migration and anoikis resistance, which are hallmarks of EMT. Thus, our study demonstrates that PKCε is an important mediator of EMT in breast cancer. PMID:24701121

Nitric oxide-releasing nanoparticles: synthesis, characterization, and cytotoxicity to tumorigenic cells

Energy Technology Data Exchange (ETDEWEB)

Pelegrino, Milena T. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Silva, Letícia C.; Watashi, Carolina M. [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil); Haddad, Paula S. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Rodrigues, Tiago; Seabra, Amedea B., E-mail: amedea.seabra@ufabc.edu.br [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil)

2017-02-15

Nitric oxide (NO) is involved in several biological processes, including toxicity against tumor cells. The aim of this study was to synthesize, characterize, and evaluate the cytotoxicity of NO-releasing chitosan nanoparticles. A thiol-containing molecule, mercaptosuccinic acid (MSA), was encapsulated (encapsulation efficiency of 99%) in chitosan/sodium tripolyphosphate nanoparticles (CS NPs). The obtained nanoparticles showed an average hydrodynamic size of 108.40 ± 0.96 nm and polydispersity index of 0.26 ± 0.01. MSA-CS NPs were nitrosated leading to S-nitroso-MSA-CS NPs, which act as NO donor. The cytotoxicity of CS NPs, MSA-CS NPs, and S-nitroso-MSA-CS NPs were evaluated in several tumor cells, including human hepatocellular carcinoma (HepG2), mouse melanoma (B16F10), and human chronic myeloid leukemia (K562) cell lines and Lucena-1, a vincristine-resistant K562 cell line. Both CS NPs and MSA-CS NPs did not cause toxic effects in these cells, whereas S-nitroso-MSA-CS NPs caused potent cytotoxic effects in all the tested tumor cell lines. The half-maximal inhibitory concentration values of S-nitroso-MSA-CS NPs were 19.7, 10.5, 22.8, and 27.8 μg·mL{sup −1} for HepG2, B16F10, K562, and Lucena-1 cells, respectively. In contrast, S-nitroso-MSA-CS NPs exhibited lower cytotoxic to non-tumorigenic melanocytes (Melan-A) when compared with melanoma B16F10. Therefore, the results highlight the potential use of NO-releasing CS NPs in antitumor chemotherapy.

Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells.

Science.gov (United States)

Scarlatti, F; Maffei, R; Beau, I; Codogno, P; Ghidoni, R

2008-08-01

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

Science.gov (United States)

Melo, Sonia A.; Sugimoto, Hikaru; O’Connell, Joyce T.; Kato, Noritoshi; Villanueva, Alberto; Vidal, August; Qiu, Le; Vitkin, Edward; Perelman, Lev T.; Melo, Carlos A.; Lucci, Anthony; Ivan, Cristina; Calin, George A.; Kalluri, Raghu

2014-01-01

SUMMARY Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. PMID:25446899

Tumorigenic Effects of Tamoxifen on the Female Genital Tract

Directory of Open Access Journals (Sweden)

Kaei Nasu M.D., Ph.D.

2008-01-01

Full Text Available Tamoxifen is widely used for endocrine treatment and breast cancer prevention. It acts as both an estrogen antagonist in breast tissue and an estrogen agonist in the female lower genital tract. Tamoxifen causes severe gynecologic side effects, such as endometrial cancer. This review focuses on the effects of prolonged tamoxifen treatment on the human female genital tract and considers its tumorigenicity in the gynecologic organs through clinical data analysis. Tamoxifen is associated with an increased incidence of benign endometrial lesions such as polyps and hyperplasia and a two- to four-fold increased risk of endometrial cancer in postmenopausal patients. Moreover, the incidence of functional ovarian cysts is significantly high in premenopausal tamoxifen users. To prevent tamoxifen from having severe side effects in gynecologic organs, frequent gynecological examination should be performed for both premenopausal and postmenopausal patients with breast cancer who are treated with this drug.

Loss of Mel-18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway.

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Won, Hee-Young; Lee, Jeong-Yeon; Shin, Dong-Hui; Park, Ji-Hye; Nam, Jeong-Seok; Kim, Hyoung-Chin; Kong, Gu

2012-12-01

Mel-18 has been proposed as a negative regulator of Bmi-1, a cancer stem cell (CSC) marker, but it is still unclear whether Mel-18 is involved in CSC regulation. Here, we examined the effect of Mel-18 on the stemness of human breast CSCs. In Mel-18 small hairpin RNA (shRNA)-transduced MCF-7 cells, side population (SP) cells and breast CSC surface marker (CD44(+)/CD24(-)/ESA(+))-expressing cells, which imply a CSC population, were enriched. Moreover, the self-renewal of CSCs was enhanced by Mel-18 knockdown, as measured by the ability for tumorsphere formation in vitro and tumor-initiating capacity in vivo. Similarly, Mel-18 overexpression inhibited the number and self-renewal activity of breast CSCs in SK-BR-3 cells. Furthermore, our data showed that Mel-18 blockade up-regulated the expression of the Wnt/TCF target Jagged-1, a Notch ligand, and consequently activated the Notch pathway. Pharmacologic inhibition of the Notch and Wnt pathways abrogated Mel-18 knockdown-mediated tumorsphere formation ability. Taken together, our findings suggest that Mel-18 is a novel negative regulator of breast CSCs that inhibits the stem cell population and in vitro and in vivo self-renewal through the inactivation of Wnt-mediated Notch signaling.

Oxidases as Breast Cancer Oncogens

National Research Council Canada - National Science Library

Yeldandi, Anjana

2000-01-01

...) in a non-tumorigenic human mammary epithelial cell line to ascertain whether oxidase overexpressing cells undergo transformation when exposed to substrate xanthine for XOX and uric acid for UOX...

Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts

International Nuclear Information System (INIS)

Tsang, Chi Man; Cheung, Yuk Chun; Lui, Vivian Wai-Yan; Yip, Yim Ling; Zhang, Guitao; Lin, Victor Weitao; Cheung, Kenneth Chat-Pan; Feng, Yibin; Tsao, Sai Wah

2013-01-01

Cortidis rhizoma (Huanglian) and its major therapeutic component, berberine, have drawn extensive attention in recent years for their anti-cancer properties. Growth inhibitory effects of berberine on multiple types of human cancer cells have been reported. Berberine inhibits invasion, induces cell cycle arrest and apoptosis in human cancer cells. The anti-inflammatory property of berberine, involving inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) activation, has also been documented. In this study, we have examined the effects of berberine on tumorigenicity and growth of nasopharyngeal carcinoma (NPC) cells and their relationship to STAT3 signaling using both in vivo and in vitro models. Berberine effectively inhibited the tumorigenicity and growth of an EBV-positive NPC cell line (C666-1) in athymic nude mice. Inhibition of tumorigenic growth of NPC cells in vivo was correlated with effective inhibition of STAT3 activation in NPC cells inside the tumor xenografts grown in nude mice. In vitro, berberine inhibited both constitutive and IL-6-induced STAT3 activation in NPC cells. Inhibition of STAT3 activation by berberine induced growth inhibition and apoptotic response in NPC cells. Tumor-associated fibroblasts were found to secret IL-6 and the conditioned medium harvested from the fibroblasts also induced STAT3 activation in NPC cells. Furthermore, STAT3 activation by conditioned medium of tumor-associated fibroblasts could be blocked by berberine or antibodies against IL-6 and IL-6R. Our observation that berberine effectively inhibited activation of STAT3 induced by tumor-associated fibroblasts suggests a role of berberine in modulating the effects of tumor stroma on the growth of NPC cells. The effective inhibition of STAT3 activation in NPC cells by berberine supports its potential use in the treatment of NPC

Isotropic 3D nuclear morphometry of normal, fibrocystic and malignant breast epithelial cells reveals new structural alterations.

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Vivek Nandakumar

Full Text Available Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the

Breast cancer stem cell-like cells are more sensitive to ionizing radiation than non-stem cells: role of ATM.

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Seog-Young Kim

Full Text Available There are contradictory observations about the different radiosensitivities of cancer stem cells and cancer non-stem cells. To resolve these contradictory observations, we studied radiosensitivities by employing breast cancer stem cell (CSC-like MDA-MB231 and MDA-MB453 cells as well as their corresponding non-stem cells. CSC-like cells proliferate without differentiating and have characteristics of tumor-initiating cells [1]. These cells were exposed to γ-rays (1.25-8.75 Gy and survival curves were determined by colony formation. A final slope, D(0, of the survival curve for each cell line was determined to measure radiosensitivity. The D(0 of CSC-like and non-stem MDA-MB-453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB-231 cells (0.94 Gy vs. 1.56 Gy. After determination of radiosensitivity, we investigated intrinsic cellular determinants which influence radiosensitivity including cell cycle distribution, free-radical scavengers and DNA repair. We observed that even though cell cycle status and antioxidant content may contribute to differential radiosensitivity, differential DNA repair capacity may be a greater determinant of radiosensitivity. Unlike non-stem cells, CSC-like cells have little/no sublethal damage repair, a low intracellular level of ataxia telangiectasia mutated (ATM and delay of γ-H2AX foci removal (DNA strand break repair. These results suggest that low DNA repair capacity is responsible for the high radiosensitivity of these CSC-like cells.

Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

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Flores Juana M

2010-07-01

Full Text Available Abstract Background ErbB2-positive breast cancer is characterized by highly aggressive phenotypes and reduced responsiveness to standard therapies. Although specific ErbB2-targeted therapies have been designed, only a small percentage of patients respond to these treatments and most of them eventually relapse. The existence of this population of particularly aggressive and non-responding or relapsing patients urges the search for novel therapies. The purpose of this study was to determine whether cannabinoids might constitute a new therapeutic tool for the treatment of ErbB2-positive breast tumors. We analyzed their antitumor potential in a well established and clinically relevant model of ErbB2-driven metastatic breast cancer: the MMTV-neu mouse. We also analyzed the expression of cannabinoid targets in a series of 87 human breast tumors. Results Our results show that both Δ9-tetrahydrocannabinol, the most abundant and potent cannabinoid in marijuana, and JWH-133, a non-psychotropic CB2 receptor-selective agonist, reduce tumor growth, tumor number, and the amount/severity of lung metastases in MMTV-neu mice. Histological analyses of the tumors revealed that cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis. Cannabinoid antitumoral action relies, at least partially, on the inhibition of the pro-tumorigenic Akt pathway. We also found that 91% of ErbB2-positive tumors express the non-psychotropic cannabinoid receptor CB2. Conclusions Taken together, these results provide a strong preclinical evidence for the use of cannabinoid-based therapies for the management of ErbB2-positive breast cancer.

Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

Science.gov (United States)

Sayar, Nilufer; Karahan, Gurbet; Konu, Ozlen; Bozkurt, Betul; Bozdogan, Onder; Yulug, Isik G

2015-01-01

CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.

Science.gov (United States)

Coumans, Joëlle V F; Gau, David; Poljak, Anne; Wasinger, Valerie; Roy, Partha; Moens, Pierre D J

2014-12-01

Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer.

Breast cancer stem-like cells are inhibited by a non-toxic aryl hydrocarbon receptor agonist.

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Gérald J Prud'homme

2010-11-01

Full Text Available Cancer stem cells (CSCs have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH. CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231 mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation. It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of

Molecular mechanisms of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced inverted U-shaped dose responsiveness in anchorage independent growth and cell proliferation of human breast epithelial cells with stem cell characteristics

International Nuclear Information System (INIS)

Ahn, Nam-Shik; Hu, Hongbo; Park, Jin-Sung; Park, Joon-Suk; Kim, Jong-Sik; An, Sungwhan; Kong, Gu; Aruoma, Okezie I.; Lee, Yong-Soon; Kang, Kyung-Sun

2005-01-01

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a variety of carcinogenic and noncarcinogenic effects in experimental animals, its role in human carcinogenicity remain controversial. A simian virus 40-immortalized cell line from normal human breast epithelial cells with stem cells and luminal characteristics (M13SV1) was used to study whether TCDD can induce AIG positive colony formation and cause increased cell numbers in a inverted U-shaped dose-response manner. TCDD activated Akt, ERK2, and increased the expression of CYP1A1, PAI-2, IL-lb mRNA, and ERK2 protein levels. TCDD was able to increased phosphorylation and expression of ERK2 in same dose-response manner as AIG positive colony formation. Thus, TCDD induced tumorigenicity in M13SV1, possibly through the phosphorylation of ERK2 and/or Akt. Further, cDNA microarray with 7448 sequence-verified clones was used to profile various gene expression patterns after treatment of TCDD. Three clear patterns could be delineated: genes that were dose-dependently up-regulated, genes expressed in either U-shape and/or inverted U-shape. The fact that these genes are intrinsically related to breast epithelial cell proliferation and survival clearly suggests that they may be involved in the TCDD-induced breast tumorigenesis

Centchroman regulates breast cancer angiogenesis via inhibition of HIF-1α/VEGFR2 signalling axis.

Science.gov (United States)

Dewangan, Jayant; Kaushik, Shweta; Rath, Srikanta Kumar; Balapure, Anil K

2018-01-15

Angiogenesis is a recognized hallmark of cancer which promotes cancer cell progression and metastasis. Inhibition of angiogenesis to attenuate cancer growth is becoming desirable strategy for breast cancer management. The present study is aimed to investigate the antiangiogenic efficacy of a novel selective estrogen receptor modulator Centchroman (CC) on human breast cancer cells. Effect of CC on cell viability was evaluated using Sulforhodamine B assay. Endothelial cell proliferation, wound healing, Boyden chamber cell invasion, tube formation and chorioallantoic membrane (CAM) assays were performed to assess the effect of CC on migration, invasion and angiogenesis. Apoptosis, reactive oxygen species generation, caspase-3/7 and intracellular calcium ion level were measured through flow cytometry. Expression levels of HIF-1α, VEGF, VEGFR2, AKT and ERK were assessed by western blot analysis. CC selectively induces apoptosis in human breast cancer cells without affecting non-tumorigenic breast epithelial cells MCF-10A. Moreover, it inhibits migratory, invasive and mammosphere forming potential of breast cancer. Furthermore, CC also inhibited VEGF-induced migration, invasion and tube formation of HUVECs in vitro. CC effectively inhibited neovasculature formation in chicken CAM. Western blot analysis demonstrated that CC inhibited expression of HIF-1α and its downstream target VEGF. Interestingly, CC also suppressed VEGFR2 phosphorylation and consequently attenuated AKT and ERK phosphorylation. Our findings suggest that CC downregulates VEGF-induced angiogenesis by modulating HIF-1α/VEGFR2 pathway and recommend it (CC) as a potential therapeutic drug for breast cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

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Ivanov, Sergey V., E-mail: Sergey.Ivanov@med.nyu.edu [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States); Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I. [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States)

2009-05-08

Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

Polyploidization of murine mesenchymal cells is associated with suppression of the long noncoding RNA H19 and reduced tumorigenicity.

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Shoshani, Ofer; Massalha, Hassan; Shani, Nir; Kagan, Sivan; Ravid, Orly; Madar, Shalom; Trakhtenbrot, Luba; Leshkowitz, Dena; Rechavi, Gideon; Zipori, Dov

2012-12-15

Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumor-forming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA.

Lithium inhibits tumorigenic potential of PDA cells through targeting hedgehog-GLI signaling pathway.

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Zhonglu Peng

Full Text Available Hedgehog signaling pathway plays a critical role in the initiation and development of pancreatic ductal adenocarcinoma (PDA and represents an attractive target for PDA treatment. Lithium, a clinical mood stabilizer for mental disorders, potently inhibits the activity of glycogen synthase kinase 3β (GSK3β that promotes the ubiquitin-dependent proteasome degradation of GLI1, an important downstream component of hedgehog signaling. Herein, we report that lithium inhibits cell proliferation, blocks G1/S cell-cycle progression, induces cell apoptosis and suppresses tumorigenic potential of PDA cells through down-regulation of the expression and activity of GLI1. Moreover, lithium synergistically enhances the anti-cancer effect of gemcitabine. These findings further our knowledge of mechanisms of action for lithium and provide a potentially new therapeutic strategy for PDA through targeting GLI1.

Evaluating the immortal strand hypothesis in cancer stem cells: symmetric/self-renewal as the relevant surrogate marker of tumorigenicity.

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Winquist, Raymond J; Hall, Amy B; Eustace, Brenda K; Furey, Brinley F

2014-09-15

Stem cells subserve repair functions for the lifetime of the organism but, as a consequence of this responsibility, are candidate cells for accumulating numerous genetic and/or epigenetic aberrations leading to malignant transformation. However, given the importance of this guardian role, stem cells likely harbor some process for maintaining their precious genetic code such as non-random segregation of chromatid strands as predicted by the Immortal Strand Hypothesis (ISH). Discerning such non-random chromosomal segregation and asymmetric cell division in normal or cancer stem cells has been complicated by methodological shortcomings but also by differing division kinetics amongst tissues and the likelihood that both asymmetric and symmetric cell divisions, dictated by local extrinsic factors, are operant in these cells. Recent data suggest that cancer stem cells demonstrate a higher incidence of symmetric versus asymmetric cell division with both daughter cells retaining self-renewal characteristics, a profile which may underlie poorly differentiated morphology and marked clonal diversity in tumors. Pathways and targets are beginning to emerge which may provide opportunities for preventing such a predilection in cancer stem cells and that will hopefully translate into new classes of chemotherapeutics in oncology. Thus, although the existence of the ISH remains controversial, the shift of cell division dynamics to symmetric random chromosome segregation/self-renewal, which would negate any likelihood of template strand retention, appears to be a surrogate marker for the presence of highly malignant tumorigenic cell populations. Copyright © 2014 Elsevier Inc. All rights reserved.

Downregulation of the long non-coding RNA TUG1 is associated with cell proliferation, migration, and invasion in breast cancer.

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Fan, Shulin; Yang, Zhaoying; Ke, Zirui; Huang, Keke; Liu, Ning; Fang, Xuedong; Wang, Keren

2017-11-01

Recent studies have identified many long non-coding RNAs (lncRNAs) with critical roles in various biological processes including tumorigenesis. Taurine-upregulated gene 1 (TUG1), is an lncRNA recently reported to be involved in the progression of several human cancers. This study aimed to investigate the clinical significance and biological functions of TUG1 in breast cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure TUG1 expression in cells from breast cancer cell lines and in 58 matched pairs of breast cancer and normal tissue samples from patients with clinicopathological comparisons. Gain-and loss-of-function experiments were performed in vitro to investigate the biological role of TUG1. TUG1 expression was significantly downregulated in both breast cancer tissues and cell lines compared to controls, and low TUG1 expression was significantly correlated with mutant p53 expression (p=0.037) and lymph node metastasis (p=0.044). In vitro experiments revealed that TUG1 overexpression significantly suppressed cell proliferation by causing cell cycle arrest and inducing apoptosis in breast cancer cells, while TUG1 knockdown caused increased cell growth via promoting cell cycle progression and regulating the expression of cyclinD1 and CDK4. Further functional assays indicated that TUG1 overexpression significantly promoted cell migration and invasion while TUG1 knockdown had the opposite effects. Our findings indicate that the lncRNA TUG1 is a tumor suppressor in breast cancer, and may serve as a novel prognostic biomarker and potential therapeutic target for patients with breast cancer. Copyright © 2017. Published by Elsevier Masson SAS.

FoxD3 deficiency promotes breast cancer progression by induction of epithelial–mesenchymal transition

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Chu, Tian-Li [Department of General Surgery, The People’s Hospital of Wuqing, Tianjin (China); Zhao, Hong-Meng [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Li, Yue [Department of Respiration, Affiliated Hospital of Medical College of Chinese People’s Armed Police Force, Tianjin (China); Chen, Ao-Xiang; Sun, Xuan [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Ge, Jie, E-mail: gejie198003@163.com [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China)

2014-04-04

Highlights: • FOXD3 is down-regulated in breast cancer tissues. • FOXD3 inhibits breast cancer cell proliferation and invasion. • FoxD3 deficiency induces epithelial–mesenchymal transition. - Abstract: The transcription factor forkhead box D3 (FOXD3) plays an important role in the development of neural crest and gastric cancer cells. However, the function and mechanisms of FOXD3 in the breast tumorigenesis and progression is still limited. Here, we report that FOXD3 is a tumor suppressor of breast cancer tumorigenicity and aggressiveness. We found that FOXD3 is down-regulated in breast cancer tissues. Patients with low FOXD3 expression have a poor outcome. Depletion of FOXD3 expression promotes breast cancer cell proliferation and invasion in vitro, whereas overexpression of FOXD3 inhibits breast cancer cell proliferation and invasion both in vitro and in vivo. In addition, depletion of FOXD3 is linked to epithelial–mesenchymal transition (EMT)-like phenotype. Our results indicate FOXD3 exhibits tumor suppressive activity and may be useful for breast therapy.

FoxD3 deficiency promotes breast cancer progression by induction of epithelial–mesenchymal transition

International Nuclear Information System (INIS)

Chu, Tian-Li; Zhao, Hong-Meng; Li, Yue; Chen, Ao-Xiang; Sun, Xuan; Ge, Jie

2014-01-01

Highlights: • FOXD3 is down-regulated in breast cancer tissues. • FOXD3 inhibits breast cancer cell proliferation and invasion. • FoxD3 deficiency induces epithelial–mesenchymal transition. - Abstract: The transcription factor forkhead box D3 (FOXD3) plays an important role in the development of neural crest and gastric cancer cells. However, the function and mechanisms of FOXD3 in the breast tumorigenesis and progression is still limited. Here, we report that FOXD3 is a tumor suppressor of breast cancer tumorigenicity and aggressiveness. We found that FOXD3 is down-regulated in breast cancer tissues. Patients with low FOXD3 expression have a poor outcome. Depletion of FOXD3 expression promotes breast cancer cell proliferation and invasion in vitro, whereas overexpression of FOXD3 inhibits breast cancer cell proliferation and invasion both in vitro and in vivo. In addition, depletion of FOXD3 is linked to epithelial–mesenchymal transition (EMT)-like phenotype. Our results indicate FOXD3 exhibits tumor suppressive activity and may be useful for breast therapy

Primary non-Hodgkin's lymphoma of breast – A rare cause of breast lump

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Veena Gupta

2017-03-01

Full Text Available We, here, report a case of primary breast lymphoma in a 59 years old female. The diagnosis was suspected on fine needle aspiration cytology and confirmed on excision biopsy of the tumor. Histology and immunophenotyping were in accordance with non-Hodgkin's diffuse large B-cell lymphoma. The patient has been planned for adjuvant chemoradiation. The management and outcome of primary breast lymphoma and carcinoma are totally different. Early and prompt diagnosis of primary breast lymphoma is of utmost importance to avoid unnecessary mastectomies. Fine needle aspiration cytology supplemented by immuno-cytochemistry can be applied as a reliable and cost-effective tool in the early diagnosis of primary breast lymphomas, while histopathology and immunohistochemistry are conclusive.

Effect of curcumin on the cell surface markers CD44 and CD24 in breast cancer.

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Calaf, Gloria M; Ponce-Cusi, Richard; Abarca-Quinones, Jorge

2018-04-20

Human breast cell lines are often characterized based on the expression of the cell surface markers CD44 and CD24. CD44 is a type I transmembrane glycoprotein that regulates cell adhesion and cell-cell, as well as cell-extracellular matrix interactions. CD24 is expressed in benign and malignant solid tumors and is also involved in cell adhesion and metastasis. The aim of the present study was to investigate the effects of curcumin on the surface expression of CD44 and CD24 in breast epithelial cell lines. An established breast cancer model derived from the MCF-10F cell line was used. The results revealed that curcumin decreased CD44 and CD24 gene and protein expression levels in MCF-10F (normal), Alpha5 (premalignant) and Tumor2 (malignant) cell lines compared with the levels in their counterpart control cells. Flow cytometry revealed that the CD44+/CD24+ cell subpopulation was greater than the CD44+/CD24- subpopulation in these three cell lines. Curcumin increased CD44+/CD24+ to a greater extent and decreased CD44+/CD24- subpopulations in the normal MCF-10F and the pre-tumorigenic Alpha5 cells, but had no significant effect on Tumor2 cells compared with the corresponding control cells. Conversely, curcumin increased CD44 and decreased CD24 gene expression in MCF-7 breast cancer cells, and decreased CD44 gene expression in MDA-MB-231 cell line, while CD24 was not present in these cells. Curcumin did not alter the CD44+/CD24+ or CD44+/CD24- subpopulations in the MCF-7 cell line. However, it increased CD44+/CD24+ and decreased CD44+/CD24- subpopulations in MDA-MB-231 cells. In breast cancer specimens from patients, normal tissues were negative for CD44 and CD24 expression, while benign lesions were positive for both markers, and malignant tissues were found to be negative for CD44 and positive for CD24 in most cases. In conclusion, these results indicated that curcumin may be used to improve the proportion of CD44+/CD24+ cells and decrease the proportion of CD44

Disseminated breast cancer cells acquire a highly malignant and aggressive metastatic phenotype during metastatic latency in the bone.

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Carolyn G Marsden

Full Text Available BACKGROUND: Disseminated tumor cells (DTCs in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation in the bone marrow. METHODOLOGY/PRINCIPAL FINDINGS: Total bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype. CONCLUSIONS/SIGNIFICANCE: Dormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during

Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells.

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Burns, Jorge S; Harkness, Linda; Aldahmash, Abdullah; Gautier, Laurent; Kassem, Moustapha

2017-12-01

Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures. Copyright © 2017. Published by Elsevier B.V.

Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: A historic prospective study

International Nuclear Information System (INIS)

Baltzell, Kimberly A; Moghadassi, Michelle; Rice, Terri; Sison, Jennette D; Wrensch, Margaret

2008-01-01

Past studies have shown that women with abnormal cytology or epithelial cells in nipple aspirate fluid (NAF) have an increased relative risk (RR) of breast cancer when compared to women from whom NAF was attempted but not obtained (non-yielders). This study analyzed NAF results from a group of women seen in a breast clinic between 1970–1991 (N = 2480). Our analysis presented here is an aggregate of two sub-groups: women with questionnaire data (n = 712) and those with NAF visits beginning in 1988 (n = 238), the year in which cancer case information was uniformly collected in California. Cytological classification was determined for a group of 946 women using the most abnormal epithelial cytology observed in fluid specimens. Breast cancer incidence and mortality status was determined through June 2006 using data from the California Cancer Registry, California Vital Statistics and self-report. We estimated odd ratios (ORs) for breast cancer using logistic regression analysis, adjusting for age. We analyzed breast cancer risk related to abnormality of NAF cytology using non-yielders as the referent group and breast cancer risk related to the presence or absence of epithelial cells in NAF, using non-yielders/fluid without epithelial cells as the referent group. Overall, 10% (93) of the 946 women developed breast cancer during the follow-up period. Age-adjusted ORs and 95% confidence intervals (C.I.) compared to non-yielders were 1.4 (0.3 to 6.4), 1.7 (0.9 to 3.5), and 2.0 (1.1 to 3.6) for women with fluid without epithelial cells, normal epithelial cells and hyperplasia/atypia, respectively. Comparing the presence or absence of epithelial cells in NAF, women with epithelial cells present in NAF were more likely to develop breast cancer than non-yielders or women with fluid without epithelial cells (RR = 1.9, 1.2 to 3.1). These results support previous findings that 1) women with abnormal epithelial cells in NAF have an increased risk of breast cancer when compared to

EMT-induced stemness and tumorigenicity are fueled by the EGFR/Ras pathway.

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Dominic Chih-Cheng Voon

Full Text Available Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT. However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 (-/- p53 (-/- murine gastric epithelial (GIF-14 cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-β1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-β1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.

Tafazzin (TAZ promotes the tumorigenicity of cervical cancer cells and inhibits apoptosis.

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Mei Chen

Full Text Available Tafazzin (TAZ is often aberrantly expressed in some cancers, including rectal cancer and thyroid neoplasms. However, the function of TAZ in cervical cancer cells remains unknown. This study aims to explore the expression and function of TAZ in cervical cancer cells. Here, we determined the expression of TAZ protein in normal cervical tissue (NC, n = 27, high-grade squamous intraepithelial lesions (HSIL, n = 26 and squamous cervical carcinoma (SCC, n = 41 by immunohistochemistry, the expression of TAZ protein gradually increased from NC to HSIL to SCC. TAZ was overexpressed or down-regulated in cervical cancer cells by stably transfecting a TAZ-expressing plasmid or a shRNA plasmid targeting TAZ. In vitro, the cell growth curves and MTT assays showed that TAZ may promote the growth and viability of cervical cancer cells. In vivo, xenografts experiment showed that TAZ may increase tumor-forming ability. The percentage of apoptosis cells analyzed by FACS and TUNEL assays consistently showed that TAZ inhibits apoptosis in cervical cancer cells. Furthermore, the Cleaved Caspase 9 and Cleaved Caspase 3 were down-regulated by TAZ in cervical cancer cells. Taken together, this study demonstrated that TAZ is overexpressed in cervical cancer and may promote tumorigenicity of cervical cancer cells and inhibit apoptosis.

Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells

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Vaapil, Marica; Helczynska, Karolina; Villadsen, René

2012-01-01

Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less...... is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis....

Effects of DCK knockdown on proliferation, apoptosis and tumorigenicity in vivo of cervical cancer HeLa cells.

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Shang, Q-Y; Wu, C-S; Gao, H-R

2017-09-01

The present study explored the effect that deoxycytidine kinase (DCK) knockdown had on proliferation, apoptosis and tumorigenicity in vivo of cervical cancer HeLa cells. Human cervical cancer HeLa cells that had received no prior treatment were selected from the HeLa group. The HeLa-negative control (NC) group consisted of cells that had undergone an empty vector treatment, and finally the HeLa-short hairpin RNA (shRNA) group included cells that were treated by means of shRNA-DCK expression. DCK expressions were evaluated by quantitative real-time polymerase chain reaction in addition to western blotting assays. Cell proliferation was estimated using the Cell Counting Kit-8 (CCK-8) assay and cell cycle progression. Cell apoptosis was determined by flow cytometry. BALB/c nude mice (n=24) were selected to establish transplanted tumor models, with gross tumor volume measured every 3 days. The results in vitro were as follows: compared with the HeLa group, the HeLa-shRNA group exhibited downregulation of DCK expression and inhibition of cell proliferation at 48, 72 and 96 h. Additionally, more cells in the HeLa-shRNA group were arrested in G0/G1 stage and less in S and G2/M stages, as well as in promotion of cell apoptosis. In vivo results are as follows: when comparing the HeLa and HeLa-NC groups, the gross tumor volume of the transplanted tumor in nude mice in the HeLa-shRNA group was found to have decreased in 13, 16, 19 and 22 days. Based on these findings, our study suggests that DCK knockdown facilitates apoptosis while inhibiting proliferation and tumorigenicity in vivo of cervical cancer HeLa cells.

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

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Jorge S Burns

Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

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Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

Death receptor pathways mediate targeted and non-targeted effects of ionizing radiations in breast cancer cells

International Nuclear Information System (INIS)

Luce, A.; Courtin, A.; Levalois, C.; Altmeyer-Morel, S.; Chevillard, S.; Lebeau, J.; Romeo, P.H.

2009-01-01

Delayed cell death by mitotic catastrophe is a frequent mode of solid tumor cell death after γ-irradiation, a widely used treatment of cancer. Whereas the mechanisms that underlie the early γ-irradiation-induced cell death are well documented, those that drive the delayed cell death are largely unknown. Here we show that the Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor (TNF)-α death receptor pathways mediate the delayed cell death observed after γ-irradiation of breast cancer cells. Early after irradiation, we observe the increased expression of Fas, TRAIL-R and TNF-R that first sensitizes cells to apoptosis. Later, the increased expression of FasL, TRAIL and TNF-α permit the apoptosis engagement linked to mitotic catastrophe. Treatments with TNF-α, TRAIL or anti-Fas antibody, early after radiation exposure, induce apoptosis, whereas the neutralization of the three death receptors pathways impairs the delayed cell death. We also show for the first time that irradiated breast cancer cells excrete soluble forms of the three ligands that can induce the death of sensitive bystander cells. Overall, these results define the molecular basis of the delayed cell death of irradiated cancer cells and identify the death receptors pathways as crucial actors in apoptosis induced by targeted as well as non-targeted effects of ionizing radiation. (authors)

Breast Metastasis from Renal Cell Carcinoma: A Case Report

International Nuclear Information System (INIS)

Kim, Seon Jeong; Kim, Ji Young; Jeong, Myeong Ja; Kim, Jae Hyung; Kim, Soung Hee; Kim, Soo Hyun; Jun, Woo Sun; Kim, Hyun Jung; Han, Se Hwan

2010-01-01

Metastatic breast cancer from renal cell carcinoma is extremely rare and has non-specific findings that include a well circumscribed lesion without calcification on mammography and a well circumscribed hypoechoic lesion without posterior acoustic shadowing on sonography. We report a case of metastatic breast cancer from renal cell carcinoma and describe the radiologic findings in a 63-year-old woman who has no history of primary neoplasm

Breast Metastasis from Renal Cell Carcinoma: A Case Report

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Kim, Seon Jeong; Kim, Ji Young; Jeong, Myeong Ja; Kim, Jae Hyung; Kim, Soung Hee; Kim, Soo Hyun; Jun, Woo Sun; Kim, Hyun Jung; Han, Se Hwan [Sanggye Paik Hospital, Seoul (Korea, Republic of)

2010-01-15

Metastatic breast cancer from renal cell carcinoma is extremely rare and has non-specific findings that include a well circumscribed lesion without calcification on mammography and a well circumscribed hypoechoic lesion without posterior acoustic shadowing on sonography. We report a case of metastatic breast cancer from renal cell carcinoma and describe the radiologic findings in a 63-year-old woman who has no history of primary neoplasm.

The Apoptotic Effects of the P300 Activator on Breast Cancer and Lung Fibroblast Cell Lines

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Mohammad Reza Salahshoor

2013-10-01

Full Text Available Background: P300 is an enzyme that acetylates histones during stress. It alsoacetylates several non-histone proteins, including P53 which is the most important tumorsuppressor gene. P53 plays an important role in the apoptosis of tumor cells. Hereby,this study describes the potency of cholera toxin B subunit as a P300 activator to induceapoptosis in a breast cancer cell line (MCF-7 and a lung fibroblast cell line (MRC-5as a non-tumorigenic control sample. Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with orwithout cholera toxin B subunit at the concentration of 85.43 μmol/L, based on the half-maximal inhibitory concentration index at different times (24, 48 and 72 h. Thepercentage of apoptotic cells was measured by flow cytometry. Real-time quantitativeRT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with cholera toxin B subunit at different times. We used the ELISA and Bradford proteintechniques to detect levels of total and acetylated P53 protein generated in MCF-7 andMRC-5. Results: Our findings indicated that the cholera toxin B subunit effectively andsignificantly induced more apoptosis in MCF-7 compared to MRC-5. We showed thatexpression of P300 up-regulated by increasing the time of the cholera toxin B subunittreatment in MCF-7 but not in MRC-5. In addition, the acetylated and total P53protein levels increased more in MCF-7 cells than in MRC-5 cells.Conclusion: Cholera toxin B subunit induced significant cell death in MCF-7, butit could be well tolerated in MRC-5. Therefore, cholera toxin B subunit can besuggested as an anti-cancer agent.

Age-related modifications of type I collagen impair DDR1-induced apoptosis in non-invasive breast carcinoma cells.

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Charles, Saby; Hassan, Rammal; Kevin, Magnien; Emilie, Buache; Sylvie, Brassart-Pasco; Laurence, Van-Gulick; Pierre, Jeannesson; Erik, Maquoi; Hamid, Morjani

2018-05-07

Type I collagen and DDR1 axis has been described to decrease cell proliferation and to initiate apoptosis in non-invasive breast carcinoma in three-dimensional cell culture matrices. Moreover, MT1-MMP down-regulates these effects. Here, we address the effect of type I collagen aging and MT1-MMP expression on cell proliferation suppression and induced-apoptosis in non-invasive MCF-7 and ZR-75-1 breast carcinoma. We provide evidence for a decrease in cell growth and an increase in apoptosis in the presence of adult collagen when compared to old collagen. This effect involves a differential activation of DDR1, as evidenced by a higher DDR1 phosphorylation level in adult collagen. In adult collagen, inhibition of DDR1 expression and kinase function induced an increase in cell growth to a level similar to that observed in old collagen. The impact of aging on the sensitivity of collagen to MT1-MMP has been reported recently. We used the MT1-MMP expression strategy to verify whether, by degrading adult type I collagen, it could lead to the same phenotype observed in old collagen 3D matrix. MT1-MMP overexpression abrogated the proliferation suppression and induced-apoptosis effects only in the presence of adult collagen. This suggests that differential collagen degradation by MT1-MMP induced a structural disorganization of adult collagen and inhibits DDR1 activation. This could in turn impair DDR1-induced cell growth suppression and apoptosis. Taken together, our data suggest that modifications of collagen structural organization, due to aging, contribute to the loss of the growth suppression and induced apoptosis effect of collagen in luminal breast carcinoma. MT1-MMP-dependent degradation and aging of collagen have no additive effects on these processes.

Marrow-derived mesenchymal stem cells: role in epithelial tumor cell determination.

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Fierro, Fernando A; Sierralta, Walter D; Epuñan, Maria J; Minguell, José J

2004-01-01

Marrow stroma represents an advantageous environment for development of micrometastatic cells. Within the cellular structure of marrow stroma, mesenchymal stem cells (MSC) have been postulated as an interacting target for disseminated cancer cells. The studies reported here were performed to gain more information on the interaction of the human breast cancer cell line MCF-7 with human bone marrow-derived MSC cells and to investigate whether this interaction affects tumor cell properties. The results showed that after co-culture with MSC, changes were detected in the morphology, proliferative capacity and aggregation pattern of MCF-7 cells, but these parameters were not affected after the co-culture of MSC cells with a non-tumorigenic breast epithelial cell line, MCF-10. Since the indirect culture of MCF-7 with MSC or its products also resulted in functional changes in the tumor cells, we evaluated whether these effects could be attributed to growth factors produced by MSC cells. It was found that VEGF and IL-6 mimic the effects produced by MSC or its products on the proliferation and aggregation properties of MCF-7, cells, respectively. Thus, it seems that after entry of disseminated tumor cells into the marrow space, their proliferative and morphogenetic organization patterns are modified after interaction with distinct stromal cells and/or with specific signals from the marrow microenvironment.

Coxsackie-adenovirus receptor as a novel marker of stem cells in treatment-resistant non-small cell lung cancer

International Nuclear Information System (INIS)

Zhang, Xiaochun; Fang, Bingliang; Mohan, Radhe; Chang, Joe Y.

2012-01-01

Background: Treatment resistance resulting from the presence of cancer stem cells (CSCs) remains a challenge in cancer treatment. Little is known about possible markers of CSCs in treatment-resistant non-small cell lung cancer (NSCLC). We explored the coxsackie-adenovirus receptor (CAR) as one such marker of CSCs in models of treatment-resistant NSCLC. Materials and methods: Resistant H460 and A549 cell lines were established by repeated exposure to paclitaxel or fractionated radiation. CSC markers were measured by Western blotting and flow cytometry. We also established stable CAR-overexpressing and stable shRNA-CAR-knockdown cell lines and assessed their survival, invasiveness, and tumorigenic capabilities with clonogenic, telomerase, Matrigel, and tumor formation assays. Results: CAR expression was associated with CSC phenotype both in vitro and in vivo. CAR-overexpressing cells were more treatment-resistant, self-renewing, and tumorigenic than were parental cells, and shRNA-mediated knockdown of CAR expression was sufficient to inhibit these functions. CAR expression also correlated with the epithelial–mesenchymal transition. Conclusions: We showed for the first time that CAR is a marker of CSCs and may affect the activities of CSCs in treatment-resistant NSCLC. CAR may prove to be a target for CSC treatment and a predictor of treatment response in patients with NSCLC.

Effects of non-thermal mobile phone radiation on breast adenocarcinoma cells

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Zen Fourie

2011-09-01

Full Text Available Mobile phone usage currently exceeds landline communication in Africa. The extent of this usage has raised concerns about the long-term health effects of the ongoing use of mobile phones. To assess the physiological effects of radiation from mobile phones in vitro, MCF-7 breast adenocarcinoma cells were exposed to 2W/kg non-thermal 900-MHz mobile phone radiation. The effects investigated were those on metabolic activity, cell morphology, cell cycle progression, phosphatidylserine (PS externalisation and the generation of reactive oxygen species and nitrogen species. Statistically insignificant increases in mitochondrial dehydrogenase activity were observed in irradiated cells when compared to controls. Fluorescent detection of F-actin demonstrated an increase in F-actin stress fibre formation in irradiated MCF-7 cells. Cell cycle progression revealed no statistically significant variation. A small increase in early and late apoptotic events in irradiated MCF-7 cells was observed. No statistically significant changes were observed in reactive oxygen and reactive nitrogen species generation. In addition, quantitative and qualitative analyses of cell cycle activity and nuclear and cytosolic changes, respectively, revealed no significant changes. In conclusion, exposure to 1 h of 900-MHz irradiation induced an increase in PS externalisation and an increase in the formation of F-actin stress fibres in MCF-7 cells. Data obtained from this study, and their correlation with other studies, provides intriguing links between radio frequency radiation and cellular events and warrant further investigation.

Cell membrane softening in human breast and cervical cancer cells

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Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

2015-08-01

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

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José J. Gaforio

2011-10-01

Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

Cytogenetic Evolution of Human Ovarian Cell Lines Associated with Chemoresistance and Loss of Tumorigenicity

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Stéphanie Struski

2003-01-01

Full Text Available In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex‐FISH and Spectral Karyotype and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug‐resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8t(8;13(p22;q? and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8 or the loss of tumorigenicity properties (del(11p. Colour figure can be viewed on http://www.esacp.org/acp/2003/25‐3/struski.htm.

Androgen receptor expression on circulating tumor cells in metastatic breast cancer.

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Takeo Fujii

Full Text Available Androgen receptor (AR is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+ breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer.Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK, and biomarkers of interest (AR, estrogen receptor [ER], and HER2 on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms.Of 68 patients, 51 (75% had at least 1 CTC, and 49 of these 51 (96% had hormone-receptor-positive (HR+/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells.CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

The non-protein coding breast cancer susceptibility locus Mcs5a acts in a non-mammary cell-autonomous fashion through the immune system and modulates T-cell homeostasis and functions.

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Smits, Bart M G; Sharma, Deepak; Samuelson, David J; Woditschka, Stephan; Mau, Bob; Haag, Jill D; Gould, Michael N

2011-08-16

Mechanisms underlying low-penetrance, common, non-protein coding variants in breast cancer risk loci are largely undefined. We showed previously that the non-protein coding mammary carcinoma susceptibility locus Mcs5a/MCS5A modulates breast cancer risk in rats and women. The Mcs5a allele from the Wistar-Kyoto (WKy) rat strain consists of two genetically interacting elements that have to be present on the same chromosome to confer mammary carcinoma resistance. We also found that the two interacting elements of the resistant allele are required for the downregulation of transcript levels of the Fbxo10 gene specifically in T-cells. Here we describe mechanisms through which Mcs5a may reduce mammary carcinoma susceptibility. We performed mammary carcinoma multiplicity studies with three mammary carcinoma-inducing treatments, namely 7,12-dimethylbenz(a)anthracene (DMBA) and N-nitroso-N-methylurea (NMU) carcinogenesis, and mammary ductal infusion of retrovirus expressing the activated HER2/neu oncogene. We used mammary gland and bone marrow transplantation assays to assess the target tissue of Mcs5a activity. We used immunophenotyping assays on well-defined congenic rat lines carrying susceptible and resistant Mcs5a alleles to identify changes in T-cell homeostasis and function associated with resistance. We show that Mcs5a acts beyond the initial step of mammary epithelial cell transformation, during early cancer progression. We show that Mcs5a controls susceptibility in a non-mammary cell-autonomous manner through the immune system. The resistant Mcs5a allele was found to be associated with an overabundance of gd T-cell receptor (TCR)+ T-cells as well as a CD62L (L-selectin)-high population of all T-cell classes. In contrast to in mammary carcinoma, gdTCR+ T-cells are the predominant T-cell type in the mammary gland and were found to be overabundant in the mammary epithelium of Mcs5a resistant congenic rats. Most of them simultaneously expressed the CD4, CD8, and CD161�

International study on inter-reader variability for circulating tumor cells in breast cancer

NARCIS (Netherlands)

Ignatiadis, Michail; Riethdorf, Sabine; Bidard, François-Clement; Vaucher, Isabelle; Khazour, Mustapha; Rothe, Francoise; Metallo, Jessica; Rouas, Ghizlane; Payne, Rachel E.; Coombes, Raoul Charles; Teufel, Ingrid; Andergassen, Ulrich; Apostolaki, Stella; Politaki, Eleni; Mavroudis, Dimitris; Bessi, Silvia; Pestrin, Martta; di Leo, Angelo; Campion, Michael; Reinholz, Monica; Perez, Edith; Piccart, Martine; Borgen, Elin; Naume, Bjorn; Jimenez, Jose; Aura, Claudia Monica; Zorzino, Laura; Cassatella, Maria Cristina; Sandri, Maria Teresa; Mostert, Bianca; Sleijfer, Stefan; Kraan, Jaco; Janni, Wolfgang; Fehm, Tanja; Rack, Brigitte; Terstappen, Leonardus Wendelinus Mathias Marie; Repollet, Madeline; Pierga, Jean-Yves; Miller, Craig; Sotiriou, Christos; Michiels, Stefan; Pantel, Klaus

2014-01-01

IntroductionCirculating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. MethodsCellSearch® images (N = 272) of either CTCs or white blood cells or

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP).

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Zscharnack, Matthias; Krause, Christoph; Aust, Gabriela; Thümmler, Christian; Peinemann, Frank; Keller, Thomas; Smink, Jeske J; Holland, Heidrun; Somerson, Jeremy S; Knauer, Jens; Schulz, Ronny M; Lehmann, Jörg

2015-05-20

The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair. The testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC). No human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin. In summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.

Heterogeneity of functional properties of Clone 66 murine breast cancer cells expressing various stem cell phenotypes.

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Mukhopadhyay, Partha; Farrell, Tracy; Sharma, Gayatri; McGuire, Timothy R; O'Kane, Barbara; Sharp, J Graham

2013-01-01

Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. The proportion of cells expressing CD44(high)CD24(low/neg), side population (SP) cells, ALDH1(+), CD49f(high), CD133(high), and CD34(high) differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1(+), CD34(low), and CD49f(high) suggested properties of transit amplifying cells. Colony formation was higher from ALDH1(-) and non-SP cells than ALDH1(+) and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than "non-stem" cells. Fewer SP cells were needed to form tumors than ALDH1(+) cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

Anandamide inhibits adhesion and migration of breast cancer cells

International Nuclear Information System (INIS)

Grimaldi, Claudia; Pisanti, Simona; Laezza, Chiara; Malfitano, Anna Maria; Santoro, Antonietta; Vitale, Mario; Caruso, Maria Gabriella; Notarnicola, Maria; Iacuzzo, Irma; Portella, Giuseppe; Di Marzo, Vincenzo; Bifulco, Maurizio

2006-01-01

The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB 1 receptors could induce a non-invasive phenotype in breast mtastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB 1 antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB 1 receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB 1 receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo

Epigenetic modulation of cancer-germline antigen gene expression in tumorigenic human mesenchymal stem cells: implications for cancer therapy

DEFF Research Database (Denmark)

Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole

2009-01-01

Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found...... of cancer-germline antigens in hMSC-TERT20 cells, while their expression levels in primary human mesenchymal stem cells remained unaffected. The expression pattern of cancer-germline antigens in tumorigenic mesenchymal stem cells and sarcomas, plus their susceptibility to enhancement by epigenetic...

CDDO-Me protects normal lung and breast epithelial cells but not cancer cells from radiation.

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Mariam El-Ashmawy

Full Text Available Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs. In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF = 1.3, and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients.

Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.

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Pai, Vaibhav P; Marshall, Aaron M; Hernandez, Laura L; Buckley, Arthur R; Horseman, Nelson D

2009-01-01

The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs

Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

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Li J

2017-09-01

Full Text Available Jun Li,1,* Wenqing Xu,2,* Xiaoli Yuan,3,* Huaiwen Chen,3 Hao Song,1,4 Bingquan Wang,5 Jun Han5 1College of Pharmacy, Liaocheng University, Liaocheng, Shandong, 2Railway Police College, Zhengzhou, 3Department of Cadre Health Care, Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu, 4Centre for Stem Cell & Regenerative Medicine, Liaocheng People’s Hospital, 5Institute of Biopharmaceutical Research, Liaocheng University, Liaocheng, Shandong, China *These authors contributed equally to this work Purpose: Breast cancer stem cells (CSCs are responsible for the initiation, recurrence, and metastasis of breast cancer. Sufficient evidence has established that breast cancer cells can spontaneously turn into breast CSCs. Thus, it is essential to simultaneously target breast CSCs and cancer cells to maximize the efficacy of breast cancer therapy. HER2 has been found to be overexpressed in both breast CSCs and cancer cells. We developed salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles (Sali-NP-HER2 to target both HER2-positive breast CSCs and cancer cells.Methods: The antitumor activity of Sali-NP-HER2 constructed by conjugating anti-HER2 antibodies to polymer–lipid salinomycin nanoparticles was evaluated in vitro and in vivo.Results: Sali-NP-HER2 efficiently bound to HER2-positive breast CSCs and cancer cells, resulting in enhanced cytotoxic effects compared with non-targeted nanoparticles or salinomycin. In mice bearing breast cancer xenografts, administration of Sali-NP-HER2 exhibited superior efficacy in inhibiting tumor growth. Sali-NP-HER2 reduced the breast tumorsphere formation rate and the proportion of breast CSCs more effectively than non-targeted nanoparticles or salinomycin alone.Conclusion: Sali-NP-HER2 represents a promising approach in treating HER2-positive breast cancer by targeting both breast CSCs and cancer cells. Keywords: nanoparticles, breast cancer, cancer stem cells, salinomycin, HER2

Optical imaging of metabolic adaptability in metastatic and non-metastatic breast cancer

Science.gov (United States)

Rebello, Lisa; Rajaram, Narasimhan

2018-02-01

Accurate methods for determining metastatic risk from the primary tumor are crucial for patient survival. Cell metabolism could potentially be used as a marker of metastatic risk. Optical imaging of the endogenous fluorescent molecules nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) provides a non-destructive and label-free method for determining cell metabolism. The optical redox ratio (FAD/FAD+NADH) is sensitive to the balance between glycolysis and oxidative phosphorylation (OXPHOS). We have previously established that hypoxia-reoxygenation stress leads to metastatic potential-dependent changes in optical redox ratio. The objective of this study was to monitor the changes in optical redox ratio in breast cancer cells in response to different periods of hypoxic stress as well various levels of hypoxia to establish an optimal protocol. We measured the optical redox ratio of highly metastatic 4T1 murine breast cancer cells under normoxic conditions and after exposure to 30, 60, and 120 minutes of 0.5% O2. This was followed by an hour of reoxygenation. We found an increase in the optical redox ratio following reoxygenation from hypoxia for all durations. Statistically significant differences were observed at 60 and 120 minutes (p˂0.01) compared with normoxia, implying an ability to adapt to OXPHOS after reoxygenation. The switch to OXPHOS has been shown to be a key promoter of cell invasion. We will present our results from these investigations in human breast cancer cells as well as non-metastatic breast cancer cells exposed to various levels of hypoxia.

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

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Matthew J Billard

Full Text Available Triple negative breast cancer (TNBC is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3 is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

Science.gov (United States)

Billard, Matthew J.; Fitzhugh, David J.; Parker, Joel S.; Brozowski, Jaime M.; McGinnis, Marcus W.; Timoshchenko, Roman G.; Serafin, D. Stephen; Lininger, Ruth; Klauber-Demore, Nancy; Sahagian, Gary; Truong, Young K.; Sassano, Maria F.; Serody, Jonathan S.; Tarrant, Teresa K.

2016-01-01

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis. PMID:27049755

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

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McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

2011-04-01

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

Squamous cell carcinoma of the trachea in a non-smoking woman after radiation therapy for breast cancer

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Ishiwa, Naoki; Yamada, Kouzou; Nakayama, Haruhiko; Noda, Kazumasa [Kanagawa Cancer Center, Yokohama (Japan); Kameda, Youichi; Maehara, Takamitsu

2000-04-01

Few cases of squamous cell carcinoma of the trachea have been reported in non-smoking women. We report one case which was probably due to radiation therapy. A 50-year-old non-smoking woman was referred to our hospital because wheezing and dyspnea had been aggravated for 3 months. She had received radiation therapy ({sup 60}Cobalt) after standard radical mastectomy for right breast cancer 13 years previously. Bronchoscopic findings and chest CT scan showed tracheal stenosis due to the tumor. Sleeve resection of the trachea was performed, and the tumor was histologically diagnosed as poorly differentiated squamous cell carcinoma. We may consider this case to be radiation-induced cancer compatible with the most strict criteria of radiation induced malignancy. (author)

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

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2014-01-01

Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

Primary breast lymphoma presenting as non-healing axillary abscess.

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Anele, Chukwuemeka; Phan, Yih Chyn; Wong, Suanne; Poddar, Anil

2015-10-07

A 67-year-old woman with non-insulin dependent diabetes mellitus with a history consistent with a right axillary abscess, presented to her general practitioner (GP). A diagnosis of folliculitis was made and the GP started a course of flucloxacillin. Despite antibiotics, the patient's symptoms worsened and the abscess increased in size. This prompted her GP to perform an incision and drainage procedure of the abscess. The practice nurse subsequently oversaw the follow-up care of the wound. Two months after the incision and drainage, and after regular wound dressing, the patient was referred to the acute surgical team with a complicated, non-healing right axillary abscess cavity and associated generalised right breast cellulitis. There was no history of breast symptoms prior to the onset of the axillary abscess. The patient underwent wound debridement, washout and application of negative pressure vacuum therapy. Biopsies revealed primary breast lymphoma (B-cell). She underwent radical chemotherapy and is currently in remission. 2015 BMJ Publishing Group Ltd.

HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

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Yolanda Stypula-Cyrus

Full Text Available Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC. However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

Role of IGF1R in breast cancer subtypes, stemness, and lineage differentiation

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Susan M Farabaugh

2015-04-01

Full Text Available Insulin-like growth factor (IGF signaling is fundamental for growth and survival. A large body of evidence (laboratory, epidemiological, and clinical implicates the exploitation of this pathway in cancer. Up to 50% of breast tumors express the activated form of the IGF1 receptor (IGF1R. Breast cancers are categorized into subtypes based upon hormone and ERRB2 receptor expression and/or gene expression profiling. Even though IGF1R influences tumorigenic phenotypes and drug resistance across all breast cancer subtypes, it has specific expression and function in each. In some subtypes, IGF1R levels correlate with a favorable prognosis, while in others it is associated with recurrence and poor prognosis, suggesting different actions based upon cellular and molecular contexts. In this review, we examine IGF1R expression and function as it relates to breast cancer subtype and therapy-acquired resistance. Additionally, we discuss the role of IGF1R in stem cell maintenance and lineage differentiation and how these cell fate influences may alter the differentiation potential and cellular composition of breast tumors.

Molecular Mechanisms of Metastasis Suppression in Human Breast Cancer

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1997-07-01

immune system? Ann N Y Acad Sci, JR, 1986, The role of NK cells in tumour growth and 741, 212-15. metastasis in beige mice. Nature, 284, 622-4. 89. Stone ...77. Simmons ML and Brick JO, 1969, The Laboratory 96. Senger DR, Brown LF, Claffey KP and Dvorak HF, Mouse. Hollaender A, ed. Englewood Cliffs, NJ...ranfe of huan tumo sme I I su ding the human chromosome 11 into the highly metastatic MDA-MB-435 breast tumorigenic phenotype of several tumor lines

BAD overexpression inhibits cell growth and induces apoptosis via mitochondrial-dependent pathway in non-small cell lung cancer.

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Jiang, Li; Luo, Man; Liu, Dan; Chen, Bojiang; Zhang, Wen; Mai, Lin; Zeng, Jing; Huang, Na; Huang, Yi; Mo, Xianming; Li, Weimin

2013-06-01

The pro-apoptotic Bcl-2 protein BAD initiated apoptosis in human cells and has been identified as a prognostic marker in non-small cell lung cancer (NSCLC). In this study, we aimed to explore the functions of BAD in NSCLC. Overexpression of BAD was performed by transfecting different NSCLC cell lines with wild-type BAD. Cell proliferation, cell cycle, apoptosis, and invasion were characterized in vitro. Tumorigenicity was analyzed in vivo. Western blot was performed to determine the effects of BAD overexpression on the Bcl-2 family proteins and apoptosis-related proteins. Overexpression of BAD significantly inhibited cell proliferation in H1299, H292, and SPC-A1 but not in SK-MES-1 and H460 cell lines in vitro. BAD overexpression also reduced the tumorigenicity of H1299/SPC-A1 cell in vivo. However, no appreciable effects on cell cycle distribution and invasion were observed in all these cell lines. BAD overexpression also induced apoptosis in all cell types, in which process expression of mitochondrial cytochrom c (cyto-c) and caspase 3 were increased, whereas Bcl-xl, Bcl-2, Bax and caspase 8 expressions did not changed. These findings indicated that a mitochondrial pathway, in which process cyto-c was released from mitochondrial to activate caspase 3, was involved in BAD overexpression-mediated apoptosis. Our data suggested that increased expression of BAD enhance apoptosis and has negative influence on cell proliferation and tumor growth in NSCLC. Bad is a new potential target for tumor interventions.

Investigating the expression, effect and tumorigenic pathway of PADI2 in tumors

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Guo W

2017-03-01

Full Text Available Wei Guo,1,2,* Yabing Zheng,2,* Bing Xu,1 Fang Ma,1 Chang Li,3 Xiaoqian Zhang,4 Yao Wang,1 Xiaotian Chang1 1Medical Research Center, Shandong Provincial Qianfoshan Hospital, 2Obstetrical Department, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, Shandong, 3Pathology Department, Tengzhou Central People’s Hospital, Tengzhou, 4Clinical Laboratory, PKU Care Luzhong Hospital, Zibo, Shandong, People’s Republic of China *These authors contributed equally to this work Background: Peptidylarginine deiminase (PAD catalyzes the conversion of arginine residues to citrulline residues, termed citrullination. Recent studies have suggested that PAD isoform 2 (PADI2 plays an important role in tumors, although its tumorigenic effect and mechanism are largely unknown. Materials and methods: Immunohistochemistry and enzyme-linked immunosorbent assay (ELISA were used to investigate the expression level of PADI2 in various tumor tissues and patient blood samples, respectively. MNK-45 and Bel-7402 tumor cell lines originating from gastric and liver tumors, respectively, were treated with anti-PADI2 siRNA, and the subsequent cell proliferation, apoptosis and migration were observed. Polymerase chain reaction (PCR arrays, including Cancer PathwayFinder, Oncogenes and Tumor Suppressor Genes, p53 Signaling Pathway, Signal Transduction Pathway and Tumor Metastasis PCR arrays, were used to investigate the tumorigenic pathway of PADI2 in the siRNA-treated tumor cells. This analysis was verified by real-time PCR. Results: Immunohistochemistry detected significantly increased expression of PADI2 in invasive breast ductal carcinoma, cervical squamous cell carcinoma, colon adenocarcinoma, liver hepatocellular carcinoma, lung cancer, ovarian serous papillary adenocarcinoma and papillary thyroid carcinoma samples. ELISA detected a twofold increase in PADI2 expression in the blood of 48.3% of patients with liver cancer, 38% of patients with cervical

Molecular Mechanisms of Breast Cancer Metastasis and Potential Anti-metastatic Compounds.

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Tungsukruthai, Sucharat; Petpiroon, Nalinrat; Chanvorachote, Pithi

2018-05-01

Throughout the world, breast cancer is among the major causes of cancer-related death and is the most common cancer found in women. The development of cancer molecular knowledge has surpassed the novel concept of cancer biology and unraveled principle targets for anticancer drug developments and treatment strategies. Metastatic breast cancer cells acquire their aggressive features through several mechanisms, including augmentation of survival, proliferation, tumorigenicity, and motility-related cellular pathways. Clearly, natural product-derived compounds have since long been recognized as an important source for anticancer drugs, several of which have been shown to have promising anti-metastasis activities by suppressing key molecular features supporting such cell aggressiveness. This review provides the essential details of breast cancer, the molecular-based insights into metastasis, as well as the effects and mechanisms of potential compounds for breast cancer therapeutic approaches. As the abilities of cancer cells to invade and metastasize are addressed as the hallmarks of cancer, compounds possessing anti-metastatic effects, together with their defined molecular drug action could benefit the development of new drugs as well as treatment strategies. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Clinicopathologic and prognostic implications of progranulin in breast carcinoma.

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Li, Li-qin; Huang, Hui-lian; Ping, Jin-liang; Wang, Xiao-hong; Zhong, Jing; Dai, Li-cheng

2011-07-05

Progranulin is a newly discovered 88-kDa glycoprotein originally purified from the highly tumorigenic mouse teratoma-derived cell line PC. Its expression is closely correlated with the development and metastasis of several cancers. However, no immunohistochemical evidence currently exists to correlate progranulin expression with clinicopathologic features in breast carcinoma biopsies, and the role of progranulin as a new marker of metastatic risk and prognosis in breast cancer has not yet been studied. The aim of this study was to investigate the clinicopathologic and prognostic implications of progranulin expression in breast carcinoma and its correlation with tumor angiogenesis. Progranulin expression was determined immunohistochemically in 183 surgical specimens from patients with breast cancer and 20 tissue samples from breast fibroadenomas. The tumor angiogenesis-related biomarker, vascular endothelial growth factor was assayed and microvessel density was assessed by counting vascular endothelial cells in tumor tissues labeled with endoglin antibody. The relationship between progranulin expression and the clinicopathologic data were analyzed. Progranulin proteins were overexpressed in breast cancer. The level of progranulin expression was significantly correlated with tumor size (P = 0.004), lymph node metastasis (P progranulin expression was associated with higher tumor angiogenesis, reflected by increased vascular endothelial growth factor expression (P Progranulin may be a valuable marker for assessing the metastasis and prognosis of breast cancer, and could provide the basis for new combination regimens with antiangiogenic activity.

Degradation of endothelial basement membrane by human breast cancer cell lines

International Nuclear Information System (INIS)

Yee, C.; Shiu, R.P.

1986-01-01

During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of [35S]methionine-labeled and [3H]proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer

Transcriptomic profiling of curcumin-treated human breast stem cells identifies a role for stearoyl-coa desaturase in breast cancer prevention.

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Colacino, Justin A; McDermott, Sean P; Sartor, Maureen A; Wicha, Max S; Rozek, Laura S

2016-07-01

Curcumin is a potential agent for both the prevention and treatment of cancers. Curcumin treatment alone, or in combination with piperine, limits breast stem cell self-renewal, while remaining non-toxic to normal differentiated cells. We paired fluorescence-activated cell sorting with RNA sequencing to characterize the genome-wide changes induced specifically in normal breast stem cells following treatment with these compounds. We generated genome-wide maps of the transcriptional changes that occur in epithelial-like (ALDH+) and mesenchymal-like (ALDH-/CD44+/CD24-) normal breast stem/progenitor cells following treatment with curcumin and piperine. We show that curcumin targets both stem cell populations by down-regulating expression of breast stem cell genes including ALDH1A3, CD49f, PROM1, and TP63. We also identified novel genes and pathways targeted by curcumin, including downregulation of SCD. Transient siRNA knockdown of SCD in MCF10A cells significantly inhibited mammosphere formation and the mean proportion of CD44+/CD24- cells, suggesting that SCD is a regulator of breast stemness and a target of curcumin in breast stem cells. These findings extend previous reports of curcumin targeting stem cells, here in two phenotypically distinct stem/progenitor populations isolated from normal human breast tissue. We identified novel mechanisms by which curcumin and piperine target breast stem cell self-renewal, such as by targeting lipid metabolism, providing a mechanistic link between curcumin treatment and stem cell self-renewal. These results elucidate the mechanisms by which curcumin may act as a cancer-preventive compound and provide novel targets for cancer prevention and treatment.

First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells.

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Engel, Nadja; Adamus, Anna; Frank, Marcus; Kraft, Karin; Kühn, Juliane; Müller, Petra; Nebe, Barbara; Kasten, Annika; Seitz, Guido

2018-01-01

The bioactive lipid sphingosine-1-phosphate (S1P) is a main regulator of cell survival, proliferation, motility, and platelet aggregation, and it is essential for angiogenesis and lymphocyte trafficking. In that S1P acts as a second messenger intra- and extracellularly, it might promote cancer progression. The main cause is found in the high S1P concentration in the blood, which encourage cancer cells to migrate through the endothelial barrier into the blood vessels. The irreversible degradation of S1P is solely caused by the sphingosine-1-phosphate lyase (SGPL1). SGPL1 overexpression reduces cancer cell migration and therefore silences the endogenous S1P siren, which promotes cancer cell attraction-the main reason for metastasis. Since our previous metabolomics studies revealed an increased SGPL1 activity in association with successful breast cancer cell treatment in vitro, we further investigated expression and localization of SGPL1. Expression analyses confirmed a very low SGPL1 expression in all breast cancer samples, regardless of their subtype. Additionally, we were able to prove a novel SGPL expression in the cytoplasm membrane of non-tumorigenic breast cells by fusing three independent methods. The general SGPL1 downregulation and the loss of the plasma membrane expression resulted in S1P dependent stimulation of migration in the breast cancer cell lines MCF-7 and BT-20. Not only S1P stimulated migration could be repressed by overexpressing the natural SGPL1 variant not but also more general migratory activity was significantly reduced. Here, for the first time, we report on the SGPL1 plasma membrane location in human, non-malignant breast epithelial cell lines silencing the extracellular S1P siren in vitro, and thereby regulating pivotal cellular functions. Loss of this plasma membrane distribution as well as low SGPL1 expression levels could be a potential prognostic marker and a viable target for therapy. Therefore, the precise role of SGPL1 for cancer

First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells.

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Nadja Engel

Full Text Available The bioactive lipid sphingosine-1-phosphate (S1P is a main regulator of cell survival, proliferation, motility, and platelet aggregation, and it is essential for angiogenesis and lymphocyte trafficking. In that S1P acts as a second messenger intra- and extracellularly, it might promote cancer progression. The main cause is found in the high S1P concentration in the blood, which encourage cancer cells to migrate through the endothelial barrier into the blood vessels. The irreversible degradation of S1P is solely caused by the sphingosine-1-phosphate lyase (SGPL1. SGPL1 overexpression reduces cancer cell migration and therefore silences the endogenous S1P siren, which promotes cancer cell attraction-the main reason for metastasis. Since our previous metabolomics studies revealed an increased SGPL1 activity in association with successful breast cancer cell treatment in vitro, we further investigated expression and localization of SGPL1. Expression analyses confirmed a very low SGPL1 expression in all breast cancer samples, regardless of their subtype. Additionally, we were able to prove a novel SGPL expression in the cytoplasm membrane of non-tumorigenic breast cells by fusing three independent methods. The general SGPL1 downregulation and the loss of the plasma membrane expression resulted in S1P dependent stimulation of migration in the breast cancer cell lines MCF-7 and BT-20. Not only S1P stimulated migration could be repressed by overexpressing the natural SGPL1 variant not but also more general migratory activity was significantly reduced. Here, for the first time, we report on the SGPL1 plasma membrane location in human, non-malignant breast epithelial cell lines silencing the extracellular S1P siren in vitro, and thereby regulating pivotal cellular functions. Loss of this plasma membrane distribution as well as low SGPL1 expression levels could be a potential prognostic marker and a viable target for therapy. Therefore, the precise role of

AFM indentation study of breast cancer cells

International Nuclear Information System (INIS)

Li, Q.S.; Lee, G.Y.H.; Ong, C.N.; Lim, C.T.

2008-01-01

Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 deg. C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis

Tumorigenic hybrids between mesenchymal stem cells and gastric cancer cells enhanced cancer proliferation, migration and stemness

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Xue, Jianguo; Zhu, Yuan; Sun, Zixuan; Ji, Runbi; Zhang, Xu; Xu, Wenrong; Yuan, Xiao; Zhang, Bin; Yan, Yongmin; Yin, Lei; Xu, Huijuan; Zhang, Leilei; Zhu, Wei; Qian, Hui

2015-01-01

Emerging evidence indicates that inappropriate cell-cell fusion might contribute to cancer progression. Similarly, mesenchymal stem cells (MSCs) can also fuse with other cells spontaneously and capable of adopting the phenotype of other cells. The aim of our study was to investigate the role of MSCs participated cell fusion in the tumorigenesis of gastric cancer. We fused human umbilical cord mesenchymal stem cells (hucMSCs) with gastric cancer cells in vitro by polyethylene glycol (PEG), the hybrid cells were sorted by flow cytometer. The growth and migration of hybrids were assessed by cell counting, cell colony formation and transwell assays. The proteins and genes related to epithelial-mesenchymal transition and stemness were tested by western blot, immunocytochemistry and real-time RT-PCR. The expression of CD44 and CD133 was examined by immunocytochemistry and flow cytometry. The xenograft assay was used to evaluation the tumorigenesis of the hybrids. The obtained hybrids exhibited epithelial- mesenchymal transition (EMT) change with down-regulation of E-cadherin and up-regulation of Vimentin, N-cadherin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The hybrids also increased expression of stemness factors Oct4, Nanog, Sox2 and Lin28. The expression of CD44 and CD133 on hybrid cells was stronger than parental gastric cancer cells. Moreover, the migration and proliferation of heterotypic hybrids were enhanced. In addition, the heterotypic hybrids promoted the growth abilities of gastric xenograft tumor in vivo. Taken together, our results suggest that cell fusion between hucMSCs and gastric cancer cells could contribute to tumorigenic hybrids with EMT and stem cell-like properties, which may provide a flexible tool for investigating the roles of MSCs in gastric cancer. The online version of this article (doi:10.1186/s12885-015-1780-1) contains supplementary material, which is available to authorized users

Cell plasticity and heterogeneity in cancer.

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Marjanovic, Nemanja D; Weinberg, Robert A; Chaffer, Christine L

2013-01-01

Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models. The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. However, evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity. Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies. © 2012 American Association for Clinical Chemistry

A human breast cell model of pre-invasive to invasive transition

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Bissell, Mina J; Rizki, Aylin; Weaver, Valerie M.; Lee, Sun-Young; Rozenberg, Gabriela I.; Chin, Koei; Myers, Connie A.; Bascom, Jamie L.; Mott, Joni D.; Semeiks, Jeremy R.; Grate, Leslie R.; Mian, I. Saira; Borowsky, Alexander D.; Jensen, Roy A.; Idowu, Michael O.; Chen, Fanqing; Chen, David J.; Petersen, Ole W.; Gray, Joe W.; Bissell, Mina J.

2008-03-10

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from pre-invasive to invasive phenotype as it may occur 'spontaneously' in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted-basement membrane cultures. These cells remained non-invasive; however, unlike their non-malignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher grade tumors. To find functionally significant changes in transition from pre-invasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between pre-invasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP-9,-13,-15,-17 was up regulated in the invasive cells. Using siRNA based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which pre-invasive breast cells could acquire invasiveness in a metaplastic context.

Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy

International Nuclear Information System (INIS)

Gioanni, J.; Courdi, A.; Lalanne, C.M.; Fischel, J.L.; Zanghellini, E.; Lambert, J.C.; Ettore, F.; Namer, M.

1985-01-01

In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro

Monocytopenia; Induction by Vinorelbine, Cisplatin and Doxorubicin in Breast, Non-Small Cell Lung and Cervix Cancer Patients

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Nazir, Taha; Taha, Nida; Islam, Azahrul; Abraham, Suraj; Mahmood, Adeel; Mustafa, Mazhar

2016-01-01

Background The neoplasm is still a potential threat for breast, Non-Small Cell Lung (NSCL) and cervix cancer patients. Those gradually invade into other body organs, inducing complex pathological complications. Whereas, the anticancer drugs suppress the bone marrow, resulting serious hematological toxicities. Thus, the monocytic toxicity may the chance of infections, particularly in AID’s patients. Objective We aimed this retrospective study to investigate the monocytopenia induced by vinorelbine following chemotherapy in cancer patients. Patients and method A total 60 adult cancer patients were divided into two groups; Group-1 patients received the treatment of Vinorelbine alone while group 2 patients received Vinorelbine based combination chemotherapy. Result The overall comparison of mean monocyte count (×103 per μl) with time showed a significant statistical difference (p value <0.001) for G-I and no significant difference for G-II (p value <0.08). The independent comparison of mean values for two groups at every week confirms the non-significant statistical difference during all of the five weeks (p values 0.551, 0.112, 0.559, 0.372, 0.468 respectively). In addition of that, the comparison of mean values observed before therapy with that of week 4 (after therapy) showed significant difference in G-I (p value <0.001) and non-significant in G-II (p value 0.053). Conclusion Monocytopenia is induced in both of the chemotherapy protocols allows the clinical oncologists and consultant physicians to select either of the chemotherapy protocol. The therapeutic efficacy should constitute the intervening consideration to treat the breast, cervix and NSCL (Non-Small Cell Lung’s) cancers. PMID:27833519

Promotion of breast cancer by β-Hexachlorocyclohexane in MCF10AT1 cells and MMTV-neu mice

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Matsumura Fumio

2007-07-01

Full Text Available Abstract Background Exposure to β-Hexachlorocyclohexane (β-HCH, a contaminant of the hexachlorohexane pesticide lindane, has been implicated as a risk factor in the development of breast cancers in epidemiological studies. Previous studies in our laboratory have demonstrated the ability of β-HCH to elicit its actions via a ligand-independent activation of the estrogen receptor through increased c-Neu (= erbB2 or HER-2 expression and kinase activation in both the BG-1 and MCF-7 cell lines. In addition, long term exposure (33 passages to β-HCH was shown to promote the selection of MCF-7 cells which exhibit a more metastatic phenotype. Methods In this current study, we decided to investigate the long-term effects of β-HCH in both the MCF10AT1 cell line which was derived from a normal epithelial cell line by stably transfecting a mutated c-Ha-ras and a MMTV-Neu mouse model for mammary cancer in vivo. MCF10AT1 cells were exposed for 20 passages with β-HCH, 4-OH-Tamoxifen (Tam, or 17-β-estradiol (E2 after which cells were analyzed for proliferation rates and mRNA expression by RT-PCR. In our in vivo studies, MMTV-Neu mice were injected with β-HCH and observed for tumor formation over a 70 week period. Results β-HCH and Tam selected MCF10AT1 cells demonstrated increased mRNA expression of MMP-13 (collagenase-3 a marker of increased invasiveness. β-HCH treatment was also seen to increase the expression in a number of proto-oncogenes (c-Neu, Cyclin D1, p27, cell status markers (Met-1, CK19, and the inflammatory marker NFκB. Previous studies, have demonstrated the role of these markers as evidence of malignant transformations, and further illustrate the ability of β-HCH to be carcinogenic. To demonstrate β-HCH's tumorigenic properties in an in vivo system, we used an MMTV-Neu mouse model. MMTV-Neu is a c-Neu overexpressing strain which has been shown to spontaneously develop mammary tumors at later stages of aging. In this experiment, �

Promotion of breast cancer by β-Hexachlorocyclohexane in MCF10AT1 cells and MMTV-neu mice

International Nuclear Information System (INIS)

Wong, Patrick S; Matsumura, Fumio

2007-01-01

Exposure to β-Hexachlorocyclohexane (β-HCH), a contaminant of the hexachlorohexane pesticide lindane, has been implicated as a risk factor in the development of breast cancers in epidemiological studies. Previous studies in our laboratory have demonstrated the ability of β-HCH to elicit its actions via a ligand-independent activation of the estrogen receptor through increased c-Neu (= erbB 2 or HER-2) expression and kinase activation in both the BG-1 and MCF-7 cell lines. In addition, long term exposure (33 passages) to β-HCH was shown to promote the selection of MCF-7 cells which exhibit a more metastatic phenotype. In this current study, we decided to investigate the long-term effects of β-HCH in both the MCF10AT1 cell line which was derived from a normal epithelial cell line by stably transfecting a mutated c-Ha-ras and a MMTV-Neu mouse model for mammary cancer in vivo. MCF10AT1 cells were exposed for 20 passages with β-HCH, 4-OH-Tamoxifen (Tam), or 17-β-estradiol (E 2 ) after which cells were analyzed for proliferation rates and mRNA expression by RT-PCR. In our in vivo studies, MMTV-Neu mice were injected with β-HCH and observed for tumor formation over a 70 week period. β-HCH and Tam selected MCF10AT1 cells demonstrated increased mRNA expression of MMP-13 (collagenase-3) a marker of increased invasiveness. β-HCH treatment was also seen to increase the expression in a number of proto-oncogenes (c-Neu, Cyclin D1, p27), cell status markers (Met-1, CK19), and the inflammatory marker NFκB. Previous studies, have demonstrated the role of these markers as evidence of malignant transformations, and further illustrate the ability of β-HCH to be carcinogenic. To demonstrate β-HCH's tumorigenic properties in an in vivo system, we used an MMTV-Neu mouse model. MMTV-Neu is a c-Neu overexpressing strain which has been shown to spontaneously develop mammary tumors at later stages of aging. In this experiment, β-HCH exposure was shown to both accelerate

H19 lncRNA mediates 17β-estradiol-induced cell proliferation in MCF-7 breast cancer cells.

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Sun, Hong; Wang, Guo; Peng, Yan; Zeng, Ying; Zhu, Qiong-Ni; Li, Tai-Lin; Cai, Jia-Qin; Zhou, Hong-Hao; Zhu, Yuan-Shan

2015-06-01

Estrogen plays a critical role in breast cancer development and progression. However, the mechanism involved in the promotion of breast cancer development and progression by estrogen remains unclear although it has been intensively studied. In the present study, we investigated the estrogen inducibility and functional significance of H19 lncRNA in breast cancer cells and tumor tissues. The screening of 83 disease-related long non-coding RNAs (lncRNAs) revealed that H19 lncRNA was much higher in estrogen receptor (ER)-positive MCF-7 breast cancer cells than in ER-negative MDA-MB-231 cells. 17β-estradiol produced a dose- and time-dependent induction of H19 expression in MCF-7 cells, which was mediated via ERα as evident by the blockade of this 17β-estradiol effect with ICI 182780, a specific ER antagonist and knockdown of ERα using specific RNAi. Moreover, knockdown of H19 lncRNA decreased cell survival and blocked estrogen-induced cell growth while overexpression of H19 lncRNA stimulated cell proliferation. Quantitation of H19 lncRNA in human breast cancer tissues showed that the level of H19 lncRNA was >10-fold higher in ER-positive than in ER-negative tumor tissues. These results suggest that H19 is an estrogen-inducible gene and plays a key role in cell survival and in estrogen-induced cell proliferation in MCF-7 cells, indicating that H19 lncRNA may serve as a biomarker for breast cancer diagnosis and progression, and as a valuable target for breast cancer therapy.

Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

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Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

2014-10-24

Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

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Zhang, Lei; Shamaladevi, Nagarajarao; Jayaprakasha, Guddadarangavvanahally K.; Patil, Bhimu S.; Lokeshwar, Bal L.

2015-01-01

Bioactive compounds from edible plants have limited efficacy in treating advanced cancers, but they have potential to increase the efficacy of chemotherapy drugs in a combined treatment. An aqueous extract of berries of Pimenta dioica (Allspice) shows promise as one such candidate for combination therapy or chemoprevention. An aqueous extract of Allspice (AAE) was tested against human breast cancer (BrCa) cells in vitro and in vivo. AAE reduced the viability and clonogenic growth of several types of BrCa cells (IC50 ≤ 100 μg/ml) with limited toxicity in non-tumorigenic, quiescent cells (IC50 >200 μg/ml). AAE induced cytotoxicity in BrCa was inconsistent with apoptosis, but was associated with increased levels of autophagy markers LC3B and LC3B-positive puncta. Silencing the expression of autophagy related genes (ATGs) prevented AAE-induced cell death. Further, AAE caused inhibition of Akt/mTOR signaling, and showed enhanced cytotoxicity when combined with rapamycin, a chemotherapy drug and an inhibitor of mTOR signaling. Oral administration (gavage) of AAE into athymic mice implanted with MDA-MB231 tumors inhibited tumor growth slightly but not significantly (mean decrease ~ 14%, p ≥ 0.20) if mice were gavaged post-tumor implant. Tumor growth showed a significant delay (38%) in tumor palpability and growth rate (time to reach tumor volume ≥ 1,000 mm3) when mice were pre-dosed with AAE for two weeks. Analysis of tumor tissues showed increased levels of LC3B in AAE treated tumors, indicating elevated autophagic tumor cell death in vivo in treated mice. These results demonstrate antitumor and chemo-preventive activity of AAE against BrCa and potential for adjuvant to mTOR inhibition. PMID:25945840

METCAM/MUC18 promoted tumorigenesis of human breast cancer SK-BR-3 cells in a dosage-specific manner.

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Huang, Chang-Yu; Wu, Guang-Jer

2016-04-01

Overexpression of METCAM/MUC18, an immunoglobulin-like cell-adhesion molecule, promotes tumorigenesis and progression of human breast cancer cells. We also observed an intriguing phenomenon that a high-expressing SK-BR-3 clone manifested a transient tumor suppression effect in vivo. The purpose of this study was to understand if this was caused by clonal variation, METCAM/MUC18-dosage effect, or the number of cells injected. Several G418-resistant clones of SK-BR-3, expressing different levels of METCAM/MUC18, were obtained for testing effects of human METCAM/MUC18 on in vitro motility, invasiveness, and anchorage-independent colony formation (in vitro tumorigenicity) and in vivo tumorigenesis in female Balb/C athymic nude mice. Tumor sections were made for histology and immunohistochemistry analyses, and tumor lysates for Western blot analysis to determine the effects of human METCAM/MUC18 expression on levels of various downstream effectors. METCAM/MUC18 promoted in vitro motility, invasiveness, and in vitro tumorigenicity of SK-BR-3 cells in a dosage-specific manner. Overexpression of METCAM/MUC18 could promote in vivo tumorigenesis of SK-BR-3 cells even when one tenth of the previously used cell number (5 × 10(5)) was injected and in vivo tumorigenesis of SK-BR-3 cells was directly proportional to the dosage of the protein. The previously observed transient tumor suppression effect from the same clone was no longer observed. The downstream effector, such as phospho-AKT/AKT ratio, was elevated in the tumors. Transient suppression observed previously in the clone was caused by injection of a high cell number (2 × 10(6)-5 × 10(6)). METCAM/MUC18 positively promotes tumorigenesis of SK-BR-3 cells by increasing the survival and proliferation pathway. Copyright © 2016. Published by Elsevier B.V.

ERK/CANP rapid signaling mediates 17β-estradiol-induced proliferation of human breast cancer cell line MCF-7 cells.

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Wang, Guo-Sheng; Huang, Yan-Gang; Li, Huan; Bi, Shi-Jie; Zhao, Jin-Long

2014-01-01

17β-estradiol (E2) exerts its functions through both genomic and non-genomic signaling pathways. Because E2 is important in breast cancer development, we investigated whether its actions in promoting breast cancer cell proliferation occur through the non-genomic signaling pathway via extracellular signal-regulated kinase 1/2 (ERK1/2)/calcium-activated neutral protease (CANP). MCF-7 breast cancer cells were treated with ERKl/2 inhibitor (PD98059) or CANP inhibitor (calpeptin) before exposure to 1×10(-8) M E2. MTT colorimetry and flow cytometry were used to analyze effects on cell proliferation and cell cycle progression, respectively. Expression of phosphorylated-ERK (p-ERK), total ERK, and Capn4 proteins were assessed by Western blotting. Cell proliferation increased in cells treated with E2 for 24 h (P<0.05), and the proportion of cells in G0/G1 was decreased, accompanied by accelerated G1/S. Calpeptin pre-treatment significantly inhibited the E2-induced proliferation of MCF-7 cells (P<0.05), while also ameliorating the effects of E2 on cell cycle progression. Further, expression of p-ERK was rapidly up-regulated (after 10 min) by E2 (P<0.05), an effect that persisted 16 h after E2 exposure but which was significantly inhibited by PD98059 (P<0.05). Finally, expression of Capn4 protein was rapidly up-regulated in E2-exposed cells (P<0.05), but this change was significantly inhibited by PD98059 or calpeptin (P<0.05) pre-treatment. Thus, the rapid, non-genomic ERK/CANP signaling pathway mediates E2-induced proliferation of human breast cancer cells.

Non-Hodgkin's lymphoma of the breast presenting as breast abscess during pregnancy.

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Sultan, Naheed; Khalid, Mahvesh; Khan, Sarah Rafi; Khan, Fahadullah

2012-10-01

Primary non-Hodgkin's lymphoma of the breast is an uncommon disease. In all patients with breast lump, primary lymphoma of breast should be considered as it is one of the most easily missed pathology. We report a case of a 22 years old lactating mother who presented with the complaint of a painful swelling in the right breast, noticed during the last trimester of her pregnancy, mimicking breast abscess.

Non-hodgkin's lymphoma of the breast presenting as breast abscess during pregnancy

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Sultan, N.; Khalid, M.; Khan, S.R.; Khan, F.

2012-01-01

Primary non-Hodgkin's lymphoma of the breast is an uncommon disease. In all patients with breast lump, primary lymphoma of breast should be considered as it is one of the most easily missed pathology. We report a case of a 22 years old lactating mother who presented with the complaint of a painful swelling in the right breast, noticed during the last trimester of her pregnancy, mimicking breast abscess. (author)

Aldehyde dehydrogenase 1 (ALDH1) expression is an independent prognostic factor in triple negative breast cancer (TNBC).

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Ma, Fei; Li, Huihui; Li, Yiqun; Ding, Xiaoyan; Wang, Haijuan; Fan, Ying; Lin, Chen; Qian, Haili; Xu, Binghe

2017-04-01

Triple negative breast cancer (TNBC) is a subset of breast cancer that is highly aggressive and has a poor prognosis. Meanwhile, cancer stem cells (CSCs) are also characterized by a strong tumorigenic potential, which might be partly responsible for the aggressive behavior of TNBC. We previously showed that CSCs are enriched in TNBC cell lines and tissues. Further experiments in animal models revealed higher tumorigenicity of CSCs sorted from TNBC cell lines. In this study, we aimed to determine the clinical relationship between CSCs and TNBC by exploring the expression of aldehyde dehydrogenase 1 (ALDH1), which is a putative marker of breast CSCs, in TNBC tissues.ALDH1 levels in paraffin-embedded tumor tissues from 158 TNBC patients were evaluated by immunohistochemistry staining using an ALDH1A1 primary antibody. Staining evaluation was performed independently by two pathologists, and the expression level of ALDH1 was evaluated in terms of the percentage and intensity of positive cells. The association of immunohistochemistry staining of ALDH1 expression with clinical parameters was also analyzed.ALDH1 expression in tumor cells was observed in 88 out of 158 cases (55.7%). Analysis of clinicopathological parameters showed that the immunohistochemistry staining of ALDH1 was significantly correlated with tumor size (P = 0.02) and stage (P = 0.04). Survival analysis in patients with ALDH1 expression demonstrated shorter relapse-free survival (RFS) and overall survival (OS) times (P = 0.01; P = 0.001). Moreover, Cox multivariate analysis revealed that ALDH1 expression was an independent prognostic indicator of RFS and OS (P = 0.04; P = 0.04).Immunohistochemistry staining of ALDH1 in tumor cells is an independent prognostic indicator of RFS and OS in TNBC patients.

Use of Ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains

International Nuclear Information System (INIS)

Burr, T.J.; Norelli, J.L.; Katz, B.H.; Bishop, A.L.

1990-01-01

Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two 32 P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10 6 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains

mTOR in breast cancer: differential expression in triple-negative and non-triple-negative tumors.

LENUS (Irish Health Repository)

Walsh, S

2012-04-01

Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptors (ER), progesterone receptors (PR) and overexpression of HER2. Targeted therapy is currently unavailable for this subgroup of breast cancer patients. mTOR controls cancer cell growth, survival and invasion and is thus a potential target for the treatment of patients with TNBC. Using immunohistochemistry, mTOR and p-mTOR were measured in 89 TNBCs and 99 non-TNBCs. While mTOR expression was confined to tumor cell cytoplasm, p-mTOR staining was located in the nucleus, perinuclear area and in the cytoplasm. Potentially important, was our finding that nuclear p-mTOR was found more frequently in triple-negative than non triple-negative cancers (p < 0.001). These results suggest that mTOR may play a more important role in the progression of TNBC compared to non-TNBC. Based on these findings, we conclude that mTOR may be a new target for the treatment of triple-negative breast cancer.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

International Nuclear Information System (INIS)

Cheng, Gang; Zielonka, Jacek; McAllister, Donna M; Mackinnon, A Craig Jr; Joseph, Joy; Dwinell, Michael B; Kalyanaraman, Balaraman

2013-01-01

Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

The Composition of Cigarette Smoke: Problems with Lists of Tumorigens

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Rodgman A

2014-12-01

Full Text Available Since the mid-1960s, various investigators, agencies, and institutions have disseminated lists of cigarette mainstream smoke (MSS components reported to be tumorigenic on the basis of laboratory bioassays conducted under conditions significantly different from those encountered by the smoker during exposure to the components in the cigarette MSS aerosol. Since 1990, numerous lists of cigarette MSS components, defined as significant tumorigens, have been compiled by American Health Foundation personnel, Occupational Safety and Health Administration (OSHA, Fowles and Bates, and R.J. Reynolds R&D personnel. The purpose of most of the reports was to define human risk assessment and to dissuade smokers from smoking. Various investigators and agencies have frequently cited the earlier and/or the more recent lists of tumorigenic entities. The recent compilations, involving nearly 80 MSS components, suffer from serious deficiencies including: a Use of per cigarette delivery ranges for specified components which often include analytical data from cigarettes manufactured in the 1950s and 1960s which are not comparable to lower-'tar’ yield cigarettes manufactured since the mid-1970s. b Absence of standard analytical procedures for most of the listed components. c Methodological considerations regarding bioassays used to determine tumorigenicity of the listed MSS components. d Difficulty in extrapolating in vivo bioassay data obtained by non-inhalation modes of administration of a single compound to the human smoking situation involving inhalation of a complex aerosol containing that compound. e Inhalation data inadequacies regarding the tumorigenicity of many of the components. f Several tobacco smoke components are listed despite the fact their presence has not been confirmed, their MSS level has not been defined, or their MSS level is no longer relevant. g Insufficient consideration of inhibitors of tumorigenesis and mutagenesis found in MSS. h

Increased Levels of Erythropoietin in Nipple Aspirate Fluid and in Ductal Cells from Breast Cancer Patients

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Ferdinando Mannello

2008-01-01

Full Text Available Background: Erythropoietin (Epo is an important regulator of erythropoiesis, and controls proliferation and differentiation of both erythroid and non-erythroid tissues. Epo is actively synthesized by breast cells during lactation, and also plays a role in breast tissues promoting hypoxia-induced cancer initiation. Our aims are to perform an exploratory investigation on the Epo accumulation in breast secretions from healthy and cancer patients and its localization in breast cancer cells.

ATM suppresses SATB1-induced malignant progression in breast epithelial cells.

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Ellen Ordinario

Full Text Available SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2, but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.

Elasticity and tumorigenic characteristics of cells in a monolayer after nanosecond pulsed electric field exposure.

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Steuer, A; Wende, K; Babica, P; Kolb, J F

2017-09-01

Nanosecond pulsed electric fields (nsPEFs) applied to cells can induce different biological effects depending on pulse duration and field strength. One known process is the induction of apoptosis whereby nsPEFs are currently investigated as a novel cancer therapy. Another and probably related change is the breakdown of the cytoskeleton. We investigated the elasticity of rat liver epithelial cells WB-F344 in a monolayer using atomic force microscopy (AFM) with respect to the potential of cells to undergo malignant transformation or to develop a potential to metastasize. We found that the elastic modulus of the cells decreased significantly within the first 8 min after treatment with 20 pulses of 100 ns and with a field strength of 20 kV/cm but was still higher than the elasticity of their tumorigenic counterpart WB-ras. AFM measurements and immunofluorescent staining showed that the cellular actin cytoskeleton became reorganized within 5 min. However, both a colony formation assay and a cell migration assay revealed no significant changes after nsPEF treatment, implying that cells seem not to adopt malignant characteristics associated with metastasis formation despite the induced transient changes to elasticity and cytoskeleton that can be observed for up to 1 h.

Loss of Endocan tumorigenic properties after alternative splicing of exon 2

International Nuclear Information System (INIS)

Depontieu, Florence; Grigoriu, Bogdan-Dragos; Scherpereel, Arnaud; Adam, Estelle; Delehedde, Maryse; Gosset, Philippe; Lassalle, Philippe

2008-01-01

Endocan was originally described as a dermatan sulfate proteoglycan found freely circulating in the blood. Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells. This molecule is the product of a single gene composed of 3 exons. Previous data showed that endocan mRNA is subject to alternative splicing with possible generation of two protein products. In the present study we identified, and functionally characterized, the alternative spliced product of the endocan gene: the exon 2-deleted endocan, called endocanΔ2. Stable, endocanΔ2-overexpressing cell lines were generated to investigate the biological activities of this new alternatively spliced product of endocan gene. Tumorigenesis was studied by inoculating endocan and endocanΔ2 expressing cell lines subcutaneously in SCID mice. Biochemical properties of endocan and endocanΔ2 were studied after production of recombinant proteins in various cell lines of human and murine origin. Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan. EndocanΔ2 did not promote tumor formation by 293 cells implanted in the skin of severe combined immunodeficient (SCID) mice. Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR

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Ingthorsson, Saevar; Andersen, K; Hilmarsdóttir, Bylgja; Mælandsmo, Gunhild M; Magnusson, Magnus Karl; Gudjonsson, Thorarinn

2015-01-01

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal ...

Expression of the pluripotency transcription factor OCT4 in the normal and aberrant mammary gland

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Foteini eHassiotou

2013-04-01

Full Text Available Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell (ESC self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs. This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast (from non-pregnant, non-lactating women showed minimal OCT4 nuclear expression (0.9%. However, this increased in the normal lactating breast (11.4%, with further increase in lactating adenomas, lactating carcinomas and pregnancy-associated breast cancer (30.7-48.3%. OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs demonstrated upregulation of OCT4, SOX2 and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both in vitro and in vivo. Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the

Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells

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Wernet Peter

2010-08-01

Full Text Available Abstract Background The miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors. Findings We show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus. Conclusions The present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.

Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells.

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Neves, Rui; Scheel, Christina; Weinhold, Sandra; Honisch, Ellen; Iwaniuk, Katharina M; Trompeter, Hans-Ingo; Niederacher, Dieter; Wernet, Peter; Santourlidis, Simeon; Uhrberg, Markus

2010-08-03

The miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors. We show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus. The present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.

UWB based low-cost and non-invasive practical breast cancer early detection

Science.gov (United States)

Vijayasarveswari, V.; Khatun, S.; Fakir, M. M.; Jusoh, M.; Ali, S.

2017-03-01

Breast cancer is one of the main causes of women death worldwide. Breast tumor is an early stage of cancer that locates in cells of a human breast. As there is no remedy, early detection is crucial. Towards this, Ultra-Wideband (UWB) is a prominent candidate. It is a wireless communication technology which can achieve high bandwidth with low power utilization. UWB is suitable to be used for short range communication systems including breast cancer detection since it is secure, non-invasive and human health friendly. This paper presents the low-cost and non-invasive early breast cancer detection strategy using UWB sensor (or antenna). Emphasis is given here to detect breast tumor in 2D and 3D environments. The developed system consisted of hardware and software. Hardware included UWB transceiver and a pair of home-made directional sensor/antenna. The software included feed-forward back propagation Neural Network (NN) module to detect the tumor existence, size and location along with soft interface between software and hardware. Forward scattering technique was used by placing two sensors diagonally opposite sides of a breast phantom. UWB pulses were transmitted from one side of phantom and received from other side, controlled by the software interface in PC environment. Collected received signals were then fed into the NN module for training, testing and validation. The system exhibited detection efficiency on tumor existence, location (x, y, z), and size were approximately 100%, (78.17%, 70.66%, 92.46%), 85.86% respectively. The proposed UWB based early breast cancer detection system could be more practical with low-cost, user friendly and non-harmful features. This project may help users to monitor their breast health regularly at their home.

Myeloid cells in circulation and tumor microenvironment of breast cancer patients.

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Toor, Salman M; Syed Khaja, Azharuddin Sajid; El Salhat, Haytham; Faour, Issam; Kanbar, Jihad; Quadri, Asif A; Albashir, Mohamed; Elkord, Eyad

2017-06-01

Pathological conditions including cancers lead to accumulation of a morphological mixture of highly immunosuppressive cells termed as myeloid-derived suppressor cells (MDSC). The lack of conclusive markers to identify human MDSC, due to their heterogeneous nature and close phenotypical and functional proximity with other cell subsets, made it challenging to identify these cells. Nevertheless, expansion of MDSC has been reported in periphery and tumor microenvironment of various cancers. The majority of studies on breast cancers were performed on murine models and hence limited literature is available on the relation of MDSC accumulation with clinical settings in breast cancer patients. The aim of this study was to investigate levels and phenotypes of myeloid cells in peripheral blood (n = 23) and tumor microenvironment of primary breast cancer patients (n = 7), compared with blood from healthy donors (n = 21) and paired non-tumor normal breast tissues from the same patients (n = 7). Using multicolor flow cytometric assays, we found that breast cancer patients had significantly higher levels of tumor-infiltrating myeloid cells, which comprised of granulocytes (P = 0.022) and immature cells that lack the expression of markers for fully differentiated monocytes or granulocytes (P = 0.016). Importantly, this expansion was not reflected in the peripheral blood. The immunosuppressive potential of these cells was confirmed by expression of Arginase 1 (ARG1), which is pivotal for T-cell suppression. These findings are important for developing therapeutic modalities to target mechanisms employed by immunosuppressive cells that generate an immune-permissive environment for the progression of cancer.

Endogenous reverse transcriptase (RT) activity and Chromatin remodeling in normal and transformed cells and early embryos

International Nuclear Information System (INIS)

Spadafora, C.; Sciamanna, I.; Misteli, T.

2009-01-01

Endogenous Reverse Transcriptase (RT) is an enzyme encoded by two classes of genomic retro-elements: retro-transposons and endogenous retroviruses. Basal levels of RT are expressed in all non pathological, differentiated tissues while high RT expression levels characterize tumorigenic cells, germ cells and embryonic tissues. Preliminary studies carried out in our laboratory have shown that RT inhibition using pharmacological inhibitors (nevirapine and efavirenz, two drugs currently used in AIDS therapy) drastically reduces cell proliferation, promotes differentiation of tumorigenic cells in vitro, induces a reprogrammed gene expression and antagonizes tumor progression in nude mice inoculated with tumorigenic human cell lines, including melanoma, prostate and colon carcinoma and microcitoma

Multistep carcinogenesis of normal human fibroblasts. Human fibroblasts immortalized by repeated treatment with Co-60 gamma rays were transformed into tumorigenic cells with Ha-ras oncogenes.

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Namba, M; Nishitani, K; Fukushima, F; Kimoto, T

1988-01-01

Two normal mortal human fibroblast cell strains were transformed into immortal cell lines, SUSM-1 and KMST-6, by treatment with 4-nitroquinoline 1-oxide (4NQO) and Co-60 gamma rays, respectively. These immortalized cell lines showed morphological changes of cells and remarkable chromosome aberrations, but neither of them grew in soft agar or formed tumors in nude mice. The immortal cell line, KMST-6, was then converted into neoplastic cells by treatment with Harvey murine sarcoma virus (Ha-MSV) or the c-Ha-ras oncogene derived from a human lung carcinoma. These neoplastically transformed cells acquired anchorage-independent growth potential and developed tumors when transplanted into nude mice. All the tumors grew progressively without regression until the animals died of tumors. In addition, the tumors were transplantable into other nude mice. Normal human fibroblasts, on the other hand, were not transformed into either immortal or tumorigenic cells by treatment with Ha-MSV or c-Ha-ras alone. Our present data indicate that (1) the chemical carcinogen, 4NQO, or gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to immortality, and (2) the ras gene played a role in the progression of the immortally transformed cells to more malignant cells showing anchorage-independent growth and tumorigenicity. In other words, the immortalization process of human cells seems to be a pivotal or rate-limiting step in the carcinogenesis of human cells.

Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

International Nuclear Information System (INIS)

Foster, James S; Fish, Lindsay M; Phipps, Jonathan E; Bruker, Charles T; Lewis, James M; Bell, John L; Solomon, Alan; Kestler, Daniel P

2013-01-01

The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior

GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

International Nuclear Information System (INIS)

Lin, Tao; Meng, Lingjun; Tsai, Robert Y.L.

2011-01-01

Highlights: → Strong synergy between mycophenolic acid (MPA) and 5-FU in MDA-MB-231 cells. → Cell type-dependent synergy between MPA and anti-proliferative agents. → The synergy of MPA on 5-FU is recapitulated by RNA polymerase-I inhibition. → The synergy of MPA on 5-FU requires the expression of nucleostemin. -- Abstract: Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.

Cadmium promotes the proliferation of triple-negative breast cancer cells through EGFR-mediated cell cycle regulation

International Nuclear Information System (INIS)

Wei, Zhengxi; Song, Xiulong; Shaikh, Zahir A.

2015-01-01

Cadmium (Cd) is a carcinogenic metal which is implicated in breast cancer by epidemiological studies. It is reported to promote breast cancer cell growth in vitro through membrane receptors. The study described here examined Cd-mediated growth of non-metastatic human breast cancer derived cells that lack receptors for estrogen, progesterone, and HER2. Treatment of triple-negative HCC 1937 cells with 0.1–0.5 μM Cd increased cell growth by activation of AKT and ERK. Accelerated cell cycle progression was achieved by increasing the levels of cyclins A, B, and E, as well as those of CDKs 1 and 2. Although triple negative cells lack estrogen receptor, they express high levels of EGFR. Therefore, further studies on HCC 1937 and another triple-negative cell line, HCC 38, were conducted using specific siRNA and an inhibitor of EGFR to determine whether EGFR was responsible for mediating the effect of Cd. The results revealed that in both cell types EGFR was not only activated upon Cd treatment, but was also essential for the downstream activation of AKT and ERK. Based on these observations, it is concluded that, in breast cancer cells lacking estrogen receptor, sub-micromolar concentration of Cd can promote cell proliferation. Furthermore, that EGFR plays a critical role in this process. - Highlights: • Sub-micromolar concentrations of Cd promote cell growth in breast cancer cells that lack ER, PR, and HER2. • The increase in cell number is not due to reduction in apoptosis. • Growth promotion involves AKT and ERK signaling and downstream stimulation of cell cycle progression. • Initiation of cell growth by Cd occurs at the cell membrane and requires the activation of EGFR.

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes.

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Arca, M J; Krauss, J C; Strome, S E; Cameron, M J; Chang, A E

1996-05-01

We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon gamma (IFN gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFN gamma and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFN gamma secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to upregulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor.

SOX2 regulates self-renewal and tumorigenicity of human melanoma-initiating cells.

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Santini, R; Pietrobono, S; Pandolfi, S; Montagnani, V; D'Amico, M; Penachioni, J Y; Vinci, M C; Borgognoni, L; Stecca, B

2014-09-18

Melanoma is one of the most aggressive types of human cancer, characterized by enhanced heterogeneity and resistance to conventional therapy at advanced stages. We and others have previously shown that HEDGEHOG-GLI (HH-GLI) signaling is required for melanoma growth and for survival and expansion of melanoma-initiating cells (MICs). Recent reports indicate that HH-GLI signaling regulates a set of genes typically expressed in embryonic stem cells, including SOX2 (sex-determining region Y (SRY)-Box2). Here we address the function of SOX2 in human melanomas and MICs and its interaction with HH-GLI signaling. We find that SOX2 is highly expressed in melanoma stem cells. Knockdown of SOX2 sharply decreases self-renewal in melanoma spheres and in putative melanoma stem cells with high aldehyde dehydrogenase activity (ALDH(high)). Conversely, ectopic expression of SOX2 in melanoma cells enhances their self-renewal in vitro. SOX2 silencing also inhibits cell growth and induces apoptosis in melanoma cells. In addition, depletion of SOX2 progressively abrogates tumor growth and leads to a significant decrease in tumor-initiating capability of ALDH(high) MICs upon xenotransplantation, suggesting that SOX2 is required for tumor initiation and for continuous tumor growth. We show that SOX2 is regulated by HH signaling and that the transcription factors GLI1 and GLI2, the downstream effectors of HH-GLI signaling, bind to the proximal promoter region of SOX2 in primary melanoma cells. In functional studies, we find that SOX2 function is required for HH-induced melanoma cell growth and MIC self-renewal in vitro. Thus SOX2 is a critical factor for self-renewal and tumorigenicity of MICs and an important mediator of HH-GLI signaling in melanoma. These findings could provide the basis for novel therapeutic strategies based on the inhibition of SOX2 for the treatment of a subset of human melanomas.

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44.

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Babina, Irina S; McSherry, Elaine A; Donatello, Simona; Hill, Arnold D K; Hopkins, Ann M

2014-02-10

Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally

B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

International Nuclear Information System (INIS)

Wu, Qiang; Kong, Xiang-jun; Xu, Xiao-chun; Lobie, Peter E; Zhu, Tao; Wu, Zheng-sheng; Liu, Xue; Yan, Hong; He, Yin-huan; Ye, Shan; Cheng, Xing-wang; Zhu, Gui-lu; Wu, Wen-yong; Wang, Xiao-nan

2014-01-01

B-cell lymphoma 6 (BCL6) protein, an evolutionarily conserved zinc finger transcription factor, showed to be highly expressed in various human cancers in addition to malignancies in the lymphoid system. This study investigated the role of BCL6 expression in breast cancer and its clinical significance in breast cancer patients. Expression of BCL6 protein was assessed using in situ hybridization and immunohistochemistry in 127 breast cancer patients and 50 patients with breast benign disease as well as in breast cell lines. Expression of BCL6 was restored or knocked down in two breast cancer cell lines (MCF-7 and T47D) using BCL6 cDNA and siRNA, respectively. The phenotypic change of these breast cancer cell lines was assessed using cell viability MTT, Transwell invasion, colony formation, and flow cytometry assays and in a xenograft mice model. Luciferase reporter gene, immunoblot, and qRT-PCR were used to investigate the molecular events after manipulated BCL6 expression in breast cancer cells. BCL6 protein was highly expressed in breast cancer cell lines and tissue specimens and expression of BCL6 protein was associated with disease progression and poor survival of breast cancer patients. In vitro, the forced expression of BCL6 results in increased proliferation, anchorage-independent growth, migration, invasion and survival of breast cancer cell lines, whereas knockdown of BCL6 expression reduced these oncogenic properties of breast cancer cells. Moreover, forced expression of BCL6 increased tumor growth and invasiveness in a nude mouse xenograft model. At the gene level, BCL6 was a target gene of miR-339-5p. Expression of BCL6 induced expression of CXCR4 and cyclinD1 proteins. The current study demonstrated the oncogenic property of BCL6 in breast cancer and further study could target BCL6 as a novel potential therapeutic strategy for breast cancer

Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells

International Nuclear Information System (INIS)

Yaswen, P.; Smoll, A.; Stampfer, M.R.; Peehl, D.M.; Trask, D.K.; Sager, R.

1990-01-01

A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4 kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo[α]pyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to calmodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type β increased its relative abundance. The protein encoded by NB-1 may have Ca 2 plus binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined

Exosomal MicroRNA MiR-1246 Promotes Cell Proliferation, Invasion and Drug Resistance by Targeting CCNG2 in Breast Cancer.

Science.gov (United States)

Li, Xiu Juan; Ren, Zhao Jun; Tang, Jin Hai; Yu, Qiao

2017-01-01

Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246) as a serum biomarker for breast cancer and understand the underlying mechanism in breast cancer progression. The expression levels of endogenous and exosomal miRNAs were examined by real time PCR, and the expression level of the target protein was detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study their uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-1246 was estimated by invasion assay and cell viability assay. In this study, we demonstrate that exosomes carrying microRNA can be transferred among different cell lines through direct uptake. miR-1246 is highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-malignant breast cells. Moreover, miR-1246 can suppress the expression level of its target gene, Cyclin-G2 (CCNG2), indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could enhance the viability, migration and chemotherapy resistance of non-malignant HMLE cells. Together, our results support an important role of exosomes and exosomal miRNAs in regulating breast tumor progression, which highlights their potential for applications in miRNA-based therapeutics. © 2017 The Author(s). Published by S. Karger AG, Basel.

Exosomal MicroRNA MiR-1246 Promotes Cell Proliferation, Invasion and Drug Resistance by Targeting CCNG2 in Breast Cancer

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Xiu Juan Li

2017-12-01

Full Text Available Background/Aims: Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246 as a serum biomarker for breast cancer and understand the underlying mechanism in breast cancer progression. Methods: The expression levels of endogenous and exosomal miRNAs were examined by real time PCR, and the expression level of the target protein was detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study their uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-1246 was estimated by invasion assay and cell viability assay. Results: In this study, we demonstrate that exosomes carrying microRNA can be transferred among different cell lines through direct uptake. miR-1246 is highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-malignant breast cells. Moreover, miR-1246 can suppress the expression level of its target gene, Cyclin-G2 (CCNG2, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could enhance the viability, migration and chemotherapy resistance of non-malignant HMLE cells. Conclusions: Together, our results support an important role of exosomes and exosomal miRNAs in regulating breast tumor progression, which highlights their potential for applications in miRNA-based therapeutics.

The Human Cell Surfaceome of Breast Tumors

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da Cunha, Júlia Pinheiro Chagas; Galante, Pedro Alexandre Favoretto; de Souza, Jorge Estefano Santana; Pieprzyk, Martin; Carraro, Dirce Maria; Old, Lloyd J.; Camargo, Anamaria Aranha; de Souza, Sandro José

2013-01-01

Introduction. Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. Methods. We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. Results. We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. Conclusions. The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors. PMID:24195083

Stem cells in the human breast

DEFF Research Database (Denmark)

Petersen, Ole William; Polyak, Kornelia

2010-01-01

The origins of the epithelial cells participating in the development, tissue homeostasis, and cancer of the human breast are poorly understood. However, emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner, these generate the two main...... mammary cell lineages, producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from...... nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area, whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides...

Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail.

Science.gov (United States)

Wei, Zhengxi; Shan, Zhongguo; Shaikh, Zahir A

2018-04-01

Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail. Copyright © 2018 Elsevier Inc. All rights reserved.

Squamous cell carcinoma of the breast: a case report

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Hofstee Mans

2008-12-01

Full Text Available Abstract Background Squamous cells are normally not found inside the breast, so a primary squamous cell carcinoma of the breast is an exceptional phenomenon. There is a possible explanation for these findings. Case presentation A 72-year-old woman presented with a breast abnormality suspected for breast carcinoma. After the operation the pathological examination revealed a primary squamous cell carcinoma of the breast. Conclusion The presentation of squamous cell carcinoma could be similar to that of an adenocarcinoma. However, a squamous cell carcinoma of the breast could also develop from a complicated breast cyst or abscess. Therefore, pathological examination of these apparent benign abnormalities is mandatory.

Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

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Santini, Roberta; Vinci, Maria C; Pandolfi, Silvia; Penachioni, Junia Y; Montagnani, Valentina; Olivito, Biagio; Gattai, Riccardo; Pimpinelli, Nicola; Gerlini, Gianni; Borgognoni, Lorenzo; Stecca, Barbara

2012-09-01

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. Copyright © 2012 AlphaMed Press.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Valgardur Sigurdsson

Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

The non-specific symptoms of breast implant-associated anaplastic large cell lymphoma resulting in delayed diagnosis: A case-based review

Directory of Open Access Journals (Sweden)

Reem Dina Jarjis

2015-12-01

Full Text Available Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL is a rare entity that has become known as a distinct clinical condition recently. In general, BIA-ALCL patients with a history of breast implants present with non-specific implant-related complications, resulting in delayed diagnosis and appropriate treatment because of the lack of awareness of BIA-ALCL. The cause and pathogenesis have still not been identified, and there are no evidence-based guidelines on how this condition should be detected, treated or followed up because of the rarity of available data. We present the first published Danish case of anaplastic lymphoma kinase negative BIA-ALCL, and review the current literature to raise awareness of and discuss management options for this rare clinical entity.

Myoepithelial Cells: Any role in aspiration cytology smears of breast tumors?

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Pattari Sanjib

2008-04-01

Full Text Available Abstract Aims and Objective To study the role of myoepithelial (ME cells in distinguishing benign, proliferative breast diseases (PBD and frank malignant breast lesions. Materials and methods In this study, histology proven 71 cases of fine needle aspiration cytology (FNAC of palpable breast lesions were selected. There were 30 invasive carcinomas (24 infiltrating duct carcinoma and 6 infiltrating lobular carcinoma, 25 cases of benign lesion (21 fibroadenomas and 4 fibrocystic lesions and 11 proliferative breast diseases (other than carcinoma in situ and five cases of carcinoma in situ. The number of ME cells were estimated in respect to 1000 ductal cells. In every case at least 20 high power fields (× 40 were studied. Quantitative estimation of ME cell was correlated with the final diagnosis. Corresponding histopathology cases were also evaluated for diagnostic confirmation along with the pattern of distribution of ME cells. The ME cells were also quantitated on histopathology sections on smooth muscle actin (SMA immunostained sections. Results The mean number of ME cells per 1000 ductal cells on cytology smears was 5.1 ± 5.5, 30.8 ± 25, 28.3 ± 20.2, and 38.4 ± 38.8 in malignant, carcinoma in situ, PBD and benign breast lesions respectively. The non parametric Mann Whitney test showed significant difference in number of the ME cells between benign and malignant groups (p .01. In SMA stained histopathology sections, ME cell in benign, PBD, carcinoma in situ and malignant cases were 741.12 ± 248, 238 ± 172, 121.6 ± 115 and 15.6 ± 25.1 respectively. Statistical analysis showed significantly different number of ME cell between benign versus PBD group, carcinoma in situ and malignant group. It was also significant between PBD versus malignant, and carcinoma in situ versus malignant (p Conclusion The number of ME cell in breast lesions may be helpful in distinguishing PBD versus invasive malignant tumors on FNAC smears. However it is not

T cell recognition of breast cancer antigens

DEFF Research Database (Denmark)

Petersen, Nadia Viborg; Andersen, Sofie Ramskov; Andersen, Rikke Sick

Recent studies are encouraging research of breast cancer immunogenicity to evaluate the applicability ofimmunotherapy as a treatment strategy. The epitope landscape in breast cancer is minimally described, thus it is necessary to identify T cell targets to develop immune mediated therapies.......This project investigates four proteins commonly upregulated in breast cancer and thus probable tumor associated antigens (TAAs). Aromatase, prolactin, NEK3, and PIAS3 contribute to increase growth, survival, and motility of malignant cells. Aspiring to uncover novel epitopes for cytotoxic T cells, a reverse...... recognition utilizing DNA barcode labeled MHC multimers to screen peripheral blood lymphocytes from breast cancer patients and healthy donor samples. Signif-icantly more TAA specific T cell responses were detected in breast cancer patients than healthy donors for both HLA-A*0201 (P

Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

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Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2017-08-02

The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

The CD44(high tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34a expression.

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Saroj K Basak

Full Text Available Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states, and aggressive behavioral properties (including high tumorigenic potentials. We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE. Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44(hi (CD44-high cancer cell subsets display higher clonal, colony forming potential than CD44(lo cells (n=3 and are also tumorigenic (n=2/2 when transplanted in mouse xenograft model. The CD44(hi subsets express different levels of embryonal (de-differentiation markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n=5/7 than Adeno Carcinoma (n=1/12. MPE cancer cells and a lung cancer cell line (NCI-H-2122 exhibit chromosomal abnormalities and 1p36 deletion (n=3/3. Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44(hi cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44(hi cells are enriched for cells in the G2 phase of cell cycle.

Early retirement and non-employment after breast cancer

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Lindbohm, M-L; Kuosma, E; Taskila, T

2014-01-01

This study examined whether workplace support, sociodemographic factors and co-morbidity are associated with early retirement or non-employment due to other reasons among breast cancer survivors. We also compared quality of life and chronic symptoms (pain, fatigue, anxiety and depression) among...... employed, retired and other non-employed breast cancer survivors....

Analysis of HP1α regulation in human breast cancer cells

DEFF Research Database (Denmark)

Thomsen, Rune; Christensen, Dennis B; Rosborg, Sanne

2011-01-01

The three mammalian HP1 proteins, HP1α/CBX5, HP1β/CBX1, and HPγ/CBX3, are involved in chromatin packing and gene regulation. The HP1α protein is down-regulated in invasive compared to non-invasive breast cancer cells and HP1α is a suppressor of cell migration and invasion. In this report, we...... examined the background for HP1α protein down-regulation in invasive breast cancer cells. We identified a strict correlation between HP1α down-regulation at the protein level and the mRNA level. The HP1α mRNA down-regulation in invasive cancer cells was not caused by mRNA destabilization. Chromatin...... immunoprecipitation analysis of the HP1α gene showed a decrease in the histone mark for transcriptional activity H3-K36 tri-methylation and RNA polymerase II in invasive breast cancer cells which correlated with a decreased abundance of basal transcription factors at the HP1α promoter. E2F transcription factors...

Cytotoxicity and anti-tumor effects of new ruthenium complexes on triple negative breast cancer cells.

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Cecília P Popolin

Full Text Available Triple-negative breast cancer (TNBC is a highly aggressive breast cancer subtype. The high rate of metastasis associated to the fact that these cells frequently display multidrug resistance, make the treatment of metastatic disease difficult. Development of antitumor metal-based drugs was started with the discovery of cisplatin, however, the severe side effects represent a limitation for its clinical use. Ruthenium (Ru complexes with different ligands have been successfully studied as prospective antitumor drugs. In this work, we demonstrated the activity of a series of biphosphine bipyridine Ru complexes (1 [Ru(SO4(dppb(bipy], (2 [Ru(CO3(dppb(bipy], (3 [Ru(C2O4(dppb(bipy] and (4 [Ru(CH3CO2(dppb(bipy]PF6 [where dppb = 1,4-bis(diphenylphosphinobutane and bipy = 2,2'-bipyridine], on proliferation of TNBC (MDA-MB-231, estrogen-dependent breast tumor cells (MCF-7 and a non-tumor breast cell line (MCF-10A. Complex (4 was most effective among the complexes and was selected to be further investigated on effects on tumor cell adhesion, migration, invasion and in apoptosis. Moreover, DNA and HSA binding properties of this complex were also investigated. Results show that complex (4 was more efficient inhibiting proliferation of MDA-MB-231 cells over non-tumor cells. In addition, complex (4 was able to inhibit MDA-MB231 cells adhesion, migration and invasion and to induce apoptosis and inhibit MMP-9 secretion in TNBC cells. Complex (4 should be further investigated in vivo in order to stablish its potential to improve breast cancer treatment.

A rare case of breast carcinoma co-existing with axillary mantle cell lymphoma

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Scally John

2003-12-01

Full Text Available Abstract Background Mantle cell lymphoma (MCL is a rare variety of non-Hodgkin's lymphoma which originates from CD5+ B-cell population in the mantle zones of lymphoid follicles. Coexistence of such tumours in the axillary lymph nodes with invasive breast cancers without prior history of adjuvant chemotherapy or radiotherapy has not been previously reported in literature. Case report We report a rare case of breast cancer co-existing with stage I mantle cell lymphoma of the ipsilateral axillary lymph node detected fortuitously by population screening. Conclusion Though some studies have tried to prove breast carcinomas and lymphomas to share a common molecular or viral link, more research needs to be done to establish whether such a link truly exists.

Effects of biosurfactants on the viability and proliferation of human breast cancer cells.

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Duarte, Cristina; Gudiña, Eduardo J; Lima, Cristovao F; Rodrigues, Ligia R

2014-01-01

Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l(-1) surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l(-1) BioEG for 48 h decreased cancer cells' viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.

Drinking water disinfection byproduct iodoacetic acid induces tumorigenic transformation of NIH3T3 cells.

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Wei, Xiao; Wang, Shu; Zheng, Weiwei; Wang, Xia; Liu, Xiaolin; Jiang, Songhui; Pi, Jingbo; Zheng, Yuxin; He, Gengsheng; Qu, Weidong

2013-06-04

Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 μg/L IAA and 0.86 μg/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 μM exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 μM). IAA, but not IF, induced a concentration-dependent DNA damage measured by γ-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern.

Effect of radiation dosage changes on the cell viability and the apoptosis induction on normal and tumorigenic cells

International Nuclear Information System (INIS)

Park, In Woo; Choi, Soon Chul; Lee, Sam Sun; Heo, Min Suk

1999-01-01

The study was aimed to detect the differences in the cell viability and the apoptosis induction after irradiation on normal and tumorigenic cells. The study, that was generated for two human normal cells (RHEK, HGF-1) and two human tumor cells (KB, HT-1080), was tested using MTT assay at 1 day and 3 day after irradiation and TUNEL assay under confocal laser scanning microscope at 1 day after irradiation. Single irradiation of 0.5, 1, 2, 4, and 8 Gy were applied to the cells. The two fractions of 1, 2, 4, and 8 Gy were separated with a 4 hour time interval. The irradiation was done with 5.38 Gy/min dose rate using Cs-137 irradiator at room temperature. 1. In 3-day group, the cell viability of HGF-1 cell was significantly decreased at 2, 4 and 8 Gy irradiation, the cell viability of KB cell was significantly decreased at 8 Gy irradiation and the cell viability of HT-1080 cell was significantly decreased at 4 and 8 Gy irradiation. 2. There was significant difference between RHEK and KB cell line in the cell viability of 3-day group at 8 Gy irradiation. There was significant difference between RHEK and HGF-1 cell line in the cell viability of 3-day group at 4 and 8 Gy irradiation. 3. There was a significantly decreased cell viability in 3-day group than those in 1-day group at 2, 4 and 8 Gy on HGF-1 cell, at 4 and 8 Gy on HT-1080 cell, at 8 Gy on KB cell.4. We could detect DNA fragmented cells only on KB cell. Number of apoptotic cells of KB cell was significantly increased at 4 and 8 Gy irradiation. However, there was no correlation between cell viability and apoptosis.5. On all 4 cell lines, there were no differences between single and split irradiation method in cell viability and apoptosis.

Planar cell polarity gene expression correlates with tumor cell viability and prognostic outcome in neuroblastoma

International Nuclear Information System (INIS)

Dyberg, Cecilia; Papachristou, Panagiotis; Haug, Bjørn Helge; Lagercrantz, Hugo; Kogner, Per; Ringstedt, Thomas; Wickström, Malin; Johnsen, John Inge

2016-01-01

The non-canonical Wnt/Planar cell polarity (PCP) signaling pathway is a major player in cell migration during embryonal development and has recently been implicated in tumorigenesis. Transfections with cDNA plasmids or siRNA were used to increase and suppress Prickle1 and Vangl2 expression in neuroblastoma cells and in non-tumorigenic cells. Cell viability was measured by trypan blue exclusion and protein expression was determined with western blotting. Transcriptional activity was studied with luciferase reporter assay and mRNA expression with real-time RT-PCR. Immunofluorescence stainings were used to study the effects of Vangl2 overexpression in non-tumorigenic embryonic cells. Statistical significance was tested with t-test or one-way ANOVA. Here we show that high expression of the PCP core genes Prickle1 and Vangl2 is associated with low-risk neuroblastoma, suppression of neuroblastoma cell growth and decreased Wnt/β-catenin signaling. Inhibition of Rho-associated kinases (ROCKs) that are important in mediating non-canonical Wnt signaling resulted in increased expression of Prickle1 and inhibition of β-catenin activity in neuroblastoma cells. In contrast, overexpression of Vangl2 in MYC immortalized neural stem cells induced accumulation of active β-catenin and decreased the neural differentiation marker Tuj1. Similarly, genetically modified mice with forced overexpression of Vangl2 in nestin-positive cells showed decreased Tuj1 differentiation marker during embryonal development. Our experimental data demonstrate that high expression of Prickle1 and Vangl2 reduce the growth of neuroblastoma cells and indicate different roles of PCP proteins in tumorigenic cells compared to normal cells. These results suggest that the activity of the non-canonical Wnt/PCP signaling pathway is important for neuroblastoma development and that manipulation of the Wnt/PCP pathway provides a possible therapy for neuroblastoma. The online version of this article (doi:10.1186/s

Clinical Trial Design for Testing the Stem Cell Model for the Prevention and Treatment of Cancer

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Reddy, Rishindra M.; Kakarala, Madhuri; Wicha, Max S.

2011-01-01

The cancer stem cell model introduces new strategies for the prevention and treatment of cancers. In cancers that appear to follow the stem cell model, pathways such as Wnt, Notch and Hedgehog may be targeted with natural compounds such as curcumin or drugs to reduce the risk of initiation of new tumors. Disease progression of established tumors could also potentially be inhibited by targeting the tumorigenic stem cells alone, rather than aiming to reduce overall tumor size. These new approaches mandate a change in the design of clinical trials and biomarkers chosen for efficacy assessment for preventative, neoadjuvant, adjuvant, and palliative treatments. Cancer treatments could be evaluated by assessing stem cell markers before and after treatment. Targeted stem cell specific treatment of cancers may not result in “complete” or “partial” responses radiologically, as stem cell targeting may not reduce the tumor bulk, but eliminate further tumorigenic potential. These changes are discussed using breast, pancreatic, and lung cancer as examples

Clinical Trial Design for Testing the Stem Cell Model for the Prevention and Treatment of Cancer

Energy Technology Data Exchange (ETDEWEB)

Reddy, Rishindra M., E-mail: reddyrm@med.umich.edu [Medical Center, University of Michigan, 1500 E. Medical Center Drive, 2120 Taubman Center, Ann Arbor, MI 48109 (United States); Kakarala, Madhuri; Wicha, Max S. [Comprehensive Cancer Center, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109 (United States)

2011-06-20

The cancer stem cell model introduces new strategies for the prevention and treatment of cancers. In cancers that appear to follow the stem cell model, pathways such as Wnt, Notch and Hedgehog may be targeted with natural compounds such as curcumin or drugs to reduce the risk of initiation of new tumors. Disease progression of established tumors could also potentially be inhibited by targeting the tumorigenic stem cells alone, rather than aiming to reduce overall tumor size. These new approaches mandate a change in the design of clinical trials and biomarkers chosen for efficacy assessment for preventative, neoadjuvant, adjuvant, and palliative treatments. Cancer treatments could be evaluated by assessing stem cell markers before and after treatment. Targeted stem cell specific treatment of cancers may not result in “complete” or “partial” responses radiologically, as stem cell targeting may not reduce the tumor bulk, but eliminate further tumorigenic potential. These changes are discussed using breast, pancreatic, and lung cancer as examples.

Gain of glucose-independent growth upon metastasis of breast cancer cells to the brain

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Chen, Jinyu; Lee, Ho-Jeong; Wu, Xuefeng; Huo, Lei; Kim, Sun-Jin; Xu, Lei; Wang, Yan; He, Junqing; Bollu, Lakshmi Reddy; Gao, Guang; Su, Fei; Briggs, James; Liu, Xiaojing; Melman, Tamar; Asara, John M.; Fidler, Isaiah J.; Cantley, Lewis C.; Locasale, Jason W.; Weihua, Zhang

2014-01-01

Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain-metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the non-oxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBPs) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis, and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients. PMID:25511375

Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells

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2017-10-01

AWARD NUMBER: W81XWH-15-1-0644 TITLE: Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: Chun-Ju...Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0644 5c. PROGRAM ELEMENT...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Cancer stem cells (CSCs), a cell population with acquired perpetuating self-renewal properties which

Transformed human mesenchymal stem cells are more radiosensitive compared to their cells of origin in normoxia and in physiological hypoxia

International Nuclear Information System (INIS)

Worku, M.; Fersht, N.; Martindale, C.; Funes, J.M.; Shah, S.; Short, S.C.

2013-01-01

The full text of the publication follows. Purpose: The presence of hypoxic regions in tumours is associated with the recurrence of solid tumours after treatment with radiotherapy and thought to be an important element in defining the stem cell niche. We studied the effect of hypoxia on the response to radiation in sequentially transformed human mesenchymal stem cells (MSC) to investigate how the genetic events that lead to tumorigenicity influence the cellular response to radiation under hypoxic and normoxic conditions. Experimental Design: Human bone marrow derived SH2+, SH4+, Stro-1+ MSC were transformed step-wise by retroviral transfection of hTERT, HPV-16 E6 and E7, SV40 small T antigen and oncogenic H-ras. Cells were grown and irradiated with 0, 1 to 5 Gy, X-Ray at 20%, 5% and 1% oxygen tensions. Cytotoxicity, DNA double-strand break (DSB) repair and checkpoint signalling were compared between cells at three different stages of transformation and in different oxygen concentrations. Results: MSCs became more radiosensitive at each point during step-wise transformation, and this effect persisted when cells were irradiated in physiological hypoxia. Increased cytotoxicity of radiation was associated with increased residual DNA DSB at 24 post X-irradiation assessed by gamma-H2AX foci. Growth and irradiation in 1% but not 5% oxygen promoted increased radioresistance compared to growth in 20% oxygen but did not change the relative sensitivity of tumorigenic cells compared to parental cells. Activation of checkpoint signalling before and after single radiation doses is more marked in tumorigenic cells compared to parental lines, and is not altered when cells are irradiated and grown in hypoxic conditions. Conclusions: These data show that tumorigenic cells are more radiosensitive compared to non-tumorigenic parental cells in both normoxic and hypoxic conditions. 1% hypoxia promotes radioresistance in all cells. Checkpoint signalling is up-regulated in tumorigenic

Primary anaplastic large cell lymphoma of the breast arising in reconstruction mammoplasty capsule of saline filled breast implant after radical mastectomy for breast cancer: an unusual case presentation

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Sur Monalisa

2009-04-01

Full Text Available Abstract Background Primary non-Hodgkin lymphoma (NHL of the breast represents 0.04–0.5% of malignant lesions of the breast and accounts for 1.7–2.2% of extra-nodal NHL. Most primary cases are of B-cell phenotype and only rare cases are of T-cell phenotype. Anaplastic large cell lymphoma (ALCL is a rare T-cell lymphoma typically seen in children and young adults with the breast being one of the least common locations. There are a total of eleven cases of primary ALCL of the breast described in the literature. Eight of these cases occurred in proximity to breast implants, four in relation to silicone breast implant and three in relation to saline filled breast implant with three out of the eight implant related cases having previous history of breast cancer treated surgically. Adjuvant postoperative chemotherapy is given in only one case. Secondary hematological malignancies after breast cancer chemotherapy have been reported in literature. However in contrast to acute myeloid leukemia (AML, the association between lymphoma and administration of chemotherapy has never been clearly demonstrated. Case Presentation In this report we present a case of primary ALCL of the breast arising in reconstruction mamoplasty capsule of saline filled breast implant after radical mastectomy for infiltrating ductal carcinoma followed by postoperative chemotherapy twelve years ago. Conclusion Primary ALK negative ALCL arising at the site of saline filled breast implant is rare. It is still unclear whether chemotherapy and breast implantation increases risk of secondary hematological malignancies significantly. However, it is important to be aware of these complications and need for careful pathologic examination of tissue removed for implant related complications to make the correct diagnosis for further patient management and treatment. It is important to be aware of this entity at this site as it can be easily misdiagnosed on histologic grounds and to exclude

CYP1A1 expression in breast milk cells of Japanese population

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Yonemoto, Junzo; Shiizaki, Kazuhiro; Sone, Hideko; Morita, Masatosi [National Institute for Environmental Studies, Tsukuba (Japan); Uechi, Hiroto [Uechi Obstetrics and Gynecology Clinic, Utsunomiya (Japan); Masuzaki, Yuko; Koizumi, Atsuko; Matzumura, Toru [Metocean Environment Inc., Ohigawa (Japan)

2004-09-15

Dioxins are persistent, lipophilic compounds that are ubiquitous in the environment. Concern over the reproductive and developmental toxicity of dioxins has been growing since they have endocrine-disrupting properties and have adversely affected the health of offspring in experimental and epidemiological studies. Monitoring of maternal body burdens of dioxins and their biological responses to dioxin exposure is needed to estimate the potential health risk to their offspring. Breast milk has been used for monitoring dioxins in humans for decades. Breast milk has some advantages in exposure monitoring. Sampling is non-invasive, and dioxin levels are relatively high because of the high lipid content. It is assumed that mammary glands are exposed to a higher level of dioxins than other tissues since mammary glands synthesize and store milk fat. Breast milk contains leukocytes and exfoliated ductal epithelial cells. If these cells responded to dioxins and expressed CYP enzymes, a sensitive biomarker for dioxin exposure, they would be useful as biomarkers for dioxin exposure. In the present study, the expression of CYP enzymes in intact milk cells or cells cultured with TCDD was investigated. In addition, breast milk samples were collected from mothers within one week of childbearing, and the expression of CYP1A1 mRNA in milk cells was determined. The relationship between CYP1A1 mRNA expression in milk cells and dioxin levels in the cream layer of breast milk was analyzed.

Cytokeratin 19 (KRT19) has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells

Science.gov (United States)

Kim, Kyeongseok; Yang, Gwang-Mo; Choi, Hye Yeon

2018-01-01

Cytokeratin 19 (KRT19) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, KRT19 is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of KRT19 as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of KRT19 in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of KRT19 was attenuated in several patients-derived breast cancer tissues and patients with a low expression of KRT19 were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including ALDH1, CXCR4, and CD133, but a much lower expression of KRT19 than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of KRT19 in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of KRT19 in KU-CSLCs resulted in the significant attenuation of cancer properties. KRT19 regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers (ALDH1, CXCR4, and CD133), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of KRT19 expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment. PMID:29747452

Cytokeratin 19 (KRT19) has a Role in the Reprogramming of Cancer Stem Cell-Like Cells to Less Aggressive and More Drug-Sensitive Cells.

Science.gov (United States)

Saha, Subbroto Kumar; Kim, Kyeongseok; Yang, Gwang-Mo; Choi, Hye Yeon; Cho, Ssang-Goo

2018-05-09

Cytokeratin 19 ( KRT19 ) is a cytoplasmic intermediate filament protein, which is responsible for structural rigidity and multipurpose scaffolds. In several cancers, KRT19 is overexpressed and may play a crucial role in tumorigenic transformation. In our previous study, we revealed the role of KRT19 as signaling component which mediated Wnt/NOTCH crosstalk through NUMB transcription in breast cancer. Here, we investigated the function of KRT19 in cancer reprogramming and drug resistance in breast cancer cells. We found that expression of KRT19 was attenuated in several patients-derived breast cancer tissues and patients with a low expression of KRT19 were significantly correlated with poor prognosis in breast cancer patients. Consistently, highly aggressive and drug-resistant breast cancer patient-derived cancer stem cell-like cells (konkuk university-cancer stem cell-like cell (KU-CSLCs)) displayed higher expression of cancer stem cell (CSC) markers, including ALDH1 , CXCR4 , and CD133 , but a much lower expression of KRT19 than that is seen in highly aggressive triple negative breast cancer MDA-MB231 cells. Moreover, we revealed that the knockdown of KRT19 in MDA-MB231 cells led to an enhancement of cancer properties, such as cell proliferation, sphere formation, migration, and drug resistance, while the overexpression of KRT19 in KU-CSLCs resulted in the significant attenuation of cancer properties. KRT19 regulated cancer stem cell reprogramming by modulating the expression of cancer stem cell markers ( ALDH1 , CXCR4 , and CD133 ), as well as the phosphorylation of Src and GSK3β (Tyr216). Therefore, our data may imply that the modulation of KRT19 expression could be involved in cancer stem cell reprogramming and drug sensitivity, which might have clinical implications for cancer or cancer stem cell treatment.

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

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Magnusson Magnus K

2010-07-01

Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

Isolation, Characterization and Anticancer Potential of Cytotoxic Triterpenes from Betula utilis Bark.

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Tripti Mishra

Full Text Available Betula utilis, also known as Himalayan silver birch has been used as a traditional medicine for many health ailments like inflammatation, HIV, renal and bladder disorders as well as many cancers from ages. Here, we performed bio-guided fractionation of Betula utilis Bark (BUB, in which it was extracted in methanol and fractionated with hexane, ethyl acetate, chloroform, n-butanol and water. All six fractions were evaluated for their in-vitro anticancer activity in nine different cancer cell lines and ethyl acetate fraction was found to be one of the most potent fractions in terms of inducing cytotoxic activity against various cancer cell lines. By utilizing column chromatography, six triterpenes namely betulin, betulinic acid, lupeol, ursolic acid (UA, oleanolic acid and β-amyrin have been isolated from the ethyl acetate extract of BUB and structures of these compounds were unraveled by spectroscopic methods. β-amyrin and UA were isolated for the first time from Betula utilis. Isolated triterpenes were tested for in-vitro cytotoxic activity against six different cancer cell lines where UA was found to be selective for breast cancer cells over non-tumorigenic breast epithelial cells (MCF 10A. Tumor cell selective apoptotic action of UA was mainly attributed due to the activation of extrinsic apoptosis pathway via up regulation of DR4, DR5 and PARP cleavage in MCF-7 cells over non-tumorigenic MCF-10A cells. Moreover, UA mediated intracellular ROS generation and mitochondrial membrane potential disruption also play a key role for its anti cancer effect. UA also inhibits breast cancer migration. Altogether, we discovered novel source of UA having potent tumor cell specific cytotoxic property, indicating its therapeutic potential against breast cancer.

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

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Eun Hyun Ahn

Full Text Available Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS methods have high error rates. We have established a new method termed Duplex Sequencing (DS, which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

The tumor macroenvironment and systemic regulation of breast cancer progression.

Science.gov (United States)

Castaño, Zafira; Tracy, Kristin; McAllister, Sandra S

2011-01-01

Breast cancer is the most common malignancy among women worldwide and is the most common cause of death for women between 35 and 50 years of age. Women with breast cancer are at risk of developing metastases for their entire lifetime and, despite local and systemic therapies, approximately 30% of breast cancer patients will relapse (Jemal et al., 2010). Nearly all breast cancer related deaths are due to metastatic disease, even though metastasis is considered to be an inefficient process. In some cases, tumor cells disseminate from primary sites at an early stage, but remain indolent for protracted periods of time before becoming overt, life-threatening tumors. Little is known about the mechanisms that cause these indolent tumors to grow into malignant disease. Because of this gap in our understanding, we are unable to predict which breast cancer patients are likely to experience disease relapse or develop metastases years after treatment of their primary tumor. A better understanding of the mechanisms and signals involved in the exit of tumor cells from dormancy would not only allow for more accurate selection of patients that would benefit from systemic therapy, but could also lead to the development of more targeted therapies to inhibit the signals that promote disease progression. In this review, we address the systemic, or "macroenvironmental", contribution to tumor initiation and progression and what is known about how a pro-tumorigenic systemic environment is established.

Krüppel-like factor 4 promotes c-Met amplification-mediated gefitinib resistance in non-small-cell lung cancer.

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Feng, Wei; Xie, Qianyi; Liu, Suo; Ji, Ying; Li, Chunyun; Wang, Chunle; Jin, Longyu

2018-06-01

Gefitinib has been widely used in the first-line treatment of advanced EGFR-mutated non-small-cell lung cancer (NSCLC). However, many NSCLC patients will acquire resistance to gefitinib after 9-14 months of treatment. This study revealed that Krüppel-like factor 4 (KLF4) contributes to the formation of gefitinib resistance in c-Met-overexpressing NSCLC cells. We observed that KLF4 was overexpressed in c-Met-overexpressing NSCLC cells and tissues. Knockdown of KLF4 increased tumorigenic properties in gefitinib-resistant NSCLC cell lines without c-Met overexpression, but it reduced tumorigenic properties and increased gefitinib sensitivity in gefitinib-resistant NSCLC cells with c-Met overexpression, whereas overexpression of KLF4 reduced gefitinib sensitivity in gefitinib-sensitive NSCLC cells. Furthermore, Western blot analysis revealed that KLF4 contributed to the formation of gefitinib resistance in c-Met-overexpressing NSCLC cells by inhibiting the expression of apoptosis-related proteins under gefitinib treatment and activating the c-Met/Akt signaling pathway by decreasing the inhibition of β-catenin on phosphorylation of c-Met to prevent blockade by gefitinib. In summary, this study's results suggest that KLF4 is a promising candidate molecular target for both prevention and therapy of NSCLC with c-Met overexpression. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Production of prostate-specific antigen by a breast cancer cell line, Sk-Br-3

International Nuclear Information System (INIS)

Kamali Sarvestani, E.; Ghaderi, A.

2002-01-01

Prostate-specific antigen is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce prostate-specific antigen and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study prostate-specific antigen production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB-2. For this purpose we investigated the ability of prostate-specific antigen production in five different cell lines, including two breast cancer cell lines, Sk-Br-3 and MDA-MB-453. The prostate-specific antigen in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-prostate-specific antigen producer breast cancer cell line, MDA-MB-453. Progesterone receptor was expressed by both prostate-specific antigen-positive and -negative cell lines and only the intensity of staining and the number of positive cells in Sk-Br-3 population was higher than MDA-MB-453. According to our findings prostate-specific antigen can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations

Exosome-mediated transfer of miR-10b promotes cell invasion in breast cancer.

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Singh, Ramesh; Pochampally, Radhika; Watabe, Kounosuke; Lu, Zhaohui; Mo, Yin-Yuan

2014-11-26

Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion. The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay. In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells. Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through

[Exploring a non-inflammatory clinical breast mass: Clinical practice guidelines].

Science.gov (United States)

Legendre, G; Guilhen, N; Nadeau, C; Brossard, A; Fauvet, R

2015-12-01

The aim of the study was to assess the diagnostic value of physical examination, radiologic explorations and percutaneous procedures of the breast in the exploration of a non-inflammatory palpable mass, in order to propose guidelines. A systematic literature review was conducted in the Medline and Cochrane library databases. International guidelines in French and English language were also consulted until April 30th 2015. Physical examination of a non-inflammatory palpable breast mass is not sufficient to eliminate a breast cancer (LE2). Mammography alone has a sensitivity between 70 and 95% for the diagnosis of breast cancer (LE3). Echography alone has a sensitivity of 98 to 100% for the diagnosis of breast cancer (LE2). The core needle biopsy has a better sensitivity and specificity than the fine-needle aspiration for breast cancer diagnosis (LE2). The association of mammography and 2D echography presents excellent sensitivity and negative predictive value (close to 100 %) to exclude a breast cancer (LE3). A double evaluation using mammography and echography is recommended in the exploration of a non-inflammatory palpable breast mass (grade B). Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

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Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T. [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Aftab, Blake T. [Department of Medical Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M.; Wong, John [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Rudin, Charles M. [Department of Medical Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Tran, Phuoc T. [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Department of Medical Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Hales, Russell K., E-mail: rhales1@jhmi.edu [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States)

2013-05-01

Purpose: Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials: We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of Kras{sup G12D}-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results: In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions: Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer.

ONC201 demonstrates anti-tumor effects in both triple negative and non-triple negative breast cancers through TRAIL-dependent and TRAIL-independent mechanisms

OpenAIRE

Ralff, Marie D.; Kline, Christina L.B.; Küçükkase, Ozan C; Wagner, Jessica; Lim, Bora; Dicker, David T.; Prabhu, Varun V.; Oster, Wolfgang; El-Deiry, Wafik S.

2017-01-01

Breast cancer is a major cause of cancer-related death. TRAIL has been of interest as a cancer therapeutic, but only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL. The small molecule ONC201 induces expression of TRAIL and its receptor DR5. ONC201 has entered clinical trials in advanced cancers. Here we show that ONC201 is efficacious against both TNBC and non-TNBC cells (n=13). A subset of TNBC and non-TNBC cells succumb to ONC201-induced cell death. In 2/8 TNBC cell...

Non-Invasive Radiofrequency Field Treatment of 4T1 Breast Tumors Induces T-cell Dependent Inflammatory Response.

Science.gov (United States)

Newton, Jared M; Flores-Arredondo, Jose H; Suki, Sarah; Ware, Matthew J; Krzykawska-Serda, Martyna; Agha, Mahdi; Law, Justin J; Sikora, Andrew G; Curley, Steven A; Corr, Stuart J

2018-02-22

Previous work using non-invasive radiofrequency field treatment (RFT) in cancer has demonstrated its therapeutic potential as it can increase intratumoral blood perfusion, localization of intravenously delivered drugs, and promote a hyperthermic intratumoral state. Despite the well-known immunologic benefits that febrile hyperthermia can induce, an investigation of how RFT could modulate the intra-tumoral immune microenvironment had not been studied. Thus, using an established 4T1 breast cancer model in immune competent mice, we demonstrate that RFT induces a transient, localized, and T-cell dependent intratumoral inflammatory response. More specifically we show that multi- and singlet-dose RFT promote an increase in tumor volume in immune competent Balb/c mice, which does not occur in athymic nude models. Further leukocyte subset analysis at 24, 48, and 120 hours after a single RFT show a rapid increase in tumoral trafficking of CD4+ and CD8+ T-cells 24 hours post-treatment. Additional serum cytokine analysis reveals an increase in numerous pro-inflammatory cytokines and chemokines associated with enhanced T-cell trafficking. Overall, these data demonstrate that non-invasive RFT could be an effective immunomodulatory strategy in solid tumors, especially for enhancing the tumoral trafficking of lymphocytes, which is currently a major hindrance of numerous cancer immunotherapeutic strategies.

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

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Pietilä, Mika [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Lehenkari, Petri [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Institute of Clinical Medicine, Division of Surgery, University of Oulu and Clinical Research Centre, Department of Surgery and Intensive Care, Oulu University Hospital, Aapistie 5a, P.O. Box 5000, FIN-90014 (Finland); Kuvaja, Paula [Institute of Biomedicine, Department of Anatomy and Cell Biology, University of Oulu, Aapistie 7, P.O. Box 5000, FIN-90014 (Finland); Department of Pathology, Oulu University Hospital, P.O. Box 50, FIN-90029 OYS, Oulu (Finland); Kaakinen, Mika [Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014 (Finland); Kaul, Sunil C.; Wadhwa, Renu [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan); Uemura, Toshimasa, E-mail: t.uemura@aist.go.jp [National Institute of Advanced industrial Sciences and Technology, Tsukuba, Ibaraki 305 8562 (Japan)

2013-11-01

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

International Nuclear Information System (INIS)

Rashid-Kolvear, Fariborz; Taboski, Michael AS; Nguyen, Johnny; Wang, Dong-Yu; Harrington, Lea A; Done, Susan J

2010-01-01

Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated. In this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference. We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's t test. To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined

Carboplatin treatment of antiestrogen-resistant breast cancer cells

DEFF Research Database (Denmark)

Larsen, Mathilde S; Yde, Christina Westmose; Christensen, Ib J

2012-01-01

Antiestrogen resistance is a major clinical problem in current breast cancer treatment. Therefore, biomarkers and new treatment options for antiestrogen-resistant breast cancer are needed. In this study, we investigated whether antiestrogen‑resistant breast cancer cell lines have increased...... sensitivity to carboplatin, as it was previously shown with cisplatin, and whether low Bcl-2 expression levels have a potential value as marker for increased carboplatin sensitivity. Breast cancer cells resistant to the pure antiestrogen fulvestrant, and two out of four cell lines resistant...... to the antiestrogen tamoxifen, were more sensitive to carboplatin treatment compared to the parental MCF-7 cell line. This indicates that carboplatin may be an advantageous treatment in antiestrogen‑resistant breast cancer; however, a marker for increased sensitivity would be needed. Low Bcl-2 expression...

APC selectively mediates response to chemotherapeutic agents in breast cancer

International Nuclear Information System (INIS)

VanKlompenberg, Monica K.; Bedalov, Claire O.; Soto, Katia Fernandez; Prosperi, Jenifer R.

2015-01-01

The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70 % of sporadic breast cancers depending on subtype; however, the effects of APC mutation on tumorigenic properties remain unexplored. Using the Apc Min/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we identified enhanced breast tumorigenesis and alterations in genes critical in therapeutic resistance independent of Wnt/β-catenin signaling. Apc mutation changed the tumor histopathology from solid to squamous adenocarcinomas, resembling the highly aggressive human metaplastic breast cancer. Mechanistic studies in tumor-derived cell lines demonstrated that focal adhesion kinase (FAK)/Src/JNK signaling regulated the enhanced proliferation downstream of Apc mutation. Despite this mechanistic information, the role of APC in mediating breast cancer chemotherapeutic resistance is currently unknown. We have examined the effect of Apc loss in MMTV-PyMT mouse breast cancer cells on gene expression changes of ATP-binding cassette transporters and immunofluorescence to determine proliferative and apoptotic response of cells to cisplatin, doxorubicin and paclitaxel. Furthermore we determined the added effect of Src or JNK inhibition by PP2 and SP600125, respectively, on chemotherapeutic response. We also used the Aldefluor assay to measure the population of tumor initiating cells. Lastly, we measured the apoptotic and proliferative response to APC knockdown in MDA-MB-157 human breast cancer cells after chemotherapeutic treatment. Cells obtained from MMTV-PyMT;Apc Min/+ tumors express increased MDR1 (multidrug resistance protein 1), which is augmented by treatment with paclitaxel or doxorubicin. Furthermore MMTV-PyMT;Apc Min/+ cells are more resistant to cisplatin and doxorubicin-induced apoptosis, and show a larger population of ALDH positive cells. In the human metaplastic breast cancer cell line MDA-MB-157, APC knockdown led to paclitaxel and cisplatin

Are Mast Cells MASTers in Cancer?

Science.gov (United States)

Varricchi, Gilda; Galdiero, Maria Rosaria; Loffredo, Stefania; Marone, Giancarlo; Iannone, Raffaella; Marone, Gianni; Granata, Francescopaolo

2017-01-01

Prolonged low-grade inflammation or smoldering inflammation is a hallmark of cancer. Mast cells form a heterogeneous population of immune cells with differences in their ultra-structure, morphology, mediator content, and surface receptors. Mast cells are widely distributed throughout all tissues and are stromal components of the inflammatory microenvironment that modulates tumor initiation and development. Although canonically associated with allergic disorders, mast cells are a major source of pro-tumorigenic (e.g., angiogenic and lymphangiogenic factors) and antitumorigenic molecules (e.g., TNF-α and IL-9), depending on the milieu. In certain neoplasias (e.g., gastric, thyroid and Hodgkin's lymphoma) mast cells play a pro-tumorigenic role, in others (e.g., breast cancer) a protective role, whereas in yet others they are apparently innocent bystanders. These seemingly conflicting results suggest that the role of mast cells and their mediators could be cancer specific. The microlocalization (e.g., peritumoral vs intratumoral) of mast cells is another important aspect in the initiation/progression of solid and hematologic tumors. Increasing evidence in certain experimental models indicates that targeting mast cells and/or their mediators represent a potential therapeutic target in cancer. Thus, mast cells deserve focused consideration also as therapeutic targets in different types of tumors. There are many unanswered questions that should be addressed before we understand whether mast cells are an ally, adversary, or innocent bystanders in human cancers.

Breast cancer cell lines: friend or foe?

International Nuclear Information System (INIS)

Burdall, Sarah E; Hanby, Andrew M; Lansdown, Mark RJ; Speirs, Valerie

2003-01-01

The majority of breast cancer research is conducted using established breast cancer cell lines as in vitro models. An alternative is to use cultures established from primary breast tumours. Here, we discuss the pros and cons of using both of these models in translational breast cancer research

Squamous cell carcinoma of the breast : a case report

NARCIS (Netherlands)

Flikweert, Elvira R.; Hofstee, Mans; Liem, Mike S. L.

2008-01-01

Background: Squamous cells are normally not found inside the breast, so a primary squamous cell carcinoma of the breast is an exceptional phenomenon. There is a possible explanation for these findings. Case presentation: A 72-year-old woman presented with a breast abnormality suspected for breast

The role of lipolysis stimulated lipoprotein receptor in breast cancer and directing breast cancer cell behavior.

Directory of Open Access Journals (Sweden)

Denise K Reaves

Full Text Available The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

International Nuclear Information System (INIS)

Lau, Wen Min; Doucet, Michele; Huang, David; Weber, Kristy L.; Kominsky, Scott L.

2013-01-01

Highlights: •The effects of elevated CITED2 on ER function in breast cancer cells are examined. •CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. •CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co

Polychlorinated biphenyls (PCBs enhance metastatic properties of breast cancer cells by activating Rho-associated kinase (ROCK.

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Sijin Liu

Full Text Available BACKGROUND: Polychlorinated biphenyls (PCBs are a family of structurally related chlorinated aromatic hydrocarbons. Numerous studies have documented a wide spectrum of biological effects of PCBs on human health, such as immunotoxicity, neurotoxicity, estrogenic or antiestrogenic activity, and carcinogenesis. The role of PCBs as etiologic agents for breast cancer has been intensively explored in a variety of in vivo, animal and epidemiologic studies. A number of investigations indicated that higher levels of PCBs in mammary tissues or sera correlated to breast cancer risk, and PCBs might be implicated in advancing breast cancer progression. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, we for the first time report that PCBs greatly promote the ROCK activity and therefore increase cell motility for both non-metastatic and metastatic human breast cancer cells in vitro. In the in vivo study, PCBs significantly advance disease progression, leading to enhanced capability of metastatic breast cancer cells to metastasize to bone, lung and liver. Additionally, PCBs robustly induce the production of intracellular reactive oxygen species (ROS in breast cancer cells; ROS mechanistically elevate ROCK activity. CONCLUSIONS/SIGNIFICANCE: PCBs enhance the metastatic propensity of breast cancer cells by activating the ROCK signaling, which is dependent on ROS induced by PCBs. Inhibition of ROCK may stand for a unique way to restrain metastases in breast cancer upon PCB exposure.

Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells.

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Pistone Creydt, Virginia; Fletcher, Sabrina Johanna; Giudice, Jimena; Bruzzone, Ariana; Chasseing, Norma Alejandra; Gonzalez, Eduardo Gustavo; Sacca, Paula Alejandra; Calvo, Juan Carlos

2013-02-01

Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

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Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

2014-01-01

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

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Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J.

2014-01-01

To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells

Non-coding RNAs as epigenetic regulator of glioma stem-like cell differentiation

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Keisuke eKatsushima

2014-02-01

Full Text Available Glioblastomas show heterogeneous histological features. These distinct phenotypic states are thought to be associated with the presence of glioma stem cells (GSCs, which are highly tumorigenic and self-renewing sub-population of tumor cells that have different functional characteristics. Differentiation of GSCs may be regulated by multi-tiered epigenetic mechanisms that orchestrate the expression of thousands of genes. One such regulatory mechanism involves functional non-coding RNAs (ncRNAs, such as microRNAs (miRNAs; a large number of ncRNAs have been identified and shown to regulate the expression of genes associated with cell differentiation programs. Given the roles of miRNAs in cell differentiation, it is possible they are involved in the regulation of gene expression networks in GSCs that are important for the maintenance of the pluripotent state and for directing differentiation. Here, we review recent findings on ncRNAs associated with GSC differentiation and discuss how these ncRNAs contribute to the establishment of tissue heterogeneity during glioblastoma tumor formation.

Modulation of the BRCA1 Protein and Induction of Apoptosis in Triple Negative Breast Cancer Cell Lines by the Polyphenolic Compound Curcumin

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Danica L. Rowe

2009-09-01

Full Text Available In the current study, we sought to examine the effects of curcumin in a specific type of breast cancer called triple negative breast cancer. These cancers lack expression of the estrogen and progesterone receptors and do not over-express HER2. Current treatment for triple negative breast cancers is limited to cytotoxic chemotherapy, and upon relapse, there are not any therapies currently available. We demonstrate here that the bioactive food compound curcumin induces DNA damage in triple negative breast cancer cells in association with phosphorylation, increased expression, and cytoplasmic retention of the BRCA1 protein. In addition, curcumin promotes apoptosis and prevents anchorage-independent growth and migration of triple negative breast cancer cells. Apoptosis and BRCA1 modulation were not observed in non-transformed mammary epithelial cells, suggesting curcumin may have limited non-specific toxicity. This study suggests that curcumin and potentially curcumin analogues should be tested further in the context of triple negative breast cancer. These results are novel, having never been previously reported, and suggest that curcumin could provide a novel, non-toxic therapy, which could lead to improved survival for patients with triple negative breast cancer. Curcumin should be studied further in this subset of breast cancer patients, for whom treatment options are severely limited.

Quantitative changes in endogenous DNA damage correlate with conazole mutagenicity and tumorigenicity.

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The mouse liver tumorigenic conazolefungicides triadimefon and propiconazole have previously been shown to be in vivo mouse liver mutagens in the Big Blue" transgenic mutation assay when administered in feed at tumorigenic doses, whereas the nontumorigenic conazole myclobutanil w...

Alterations in the Immune Cell Composition in Premalignant Breast Tissue that Precede Breast Cancer Development.

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Degnim, Amy C; Hoskin, Tanya L; Arshad, Muhammad; Frost, Marlene H; Winham, Stacey J; Brahmbhatt, Rushin A; Pena, Alvaro; Carter, Jodi M; Stallings-Mann, Melody L; Murphy, Linda M; Miller, Erin E; Denison, Lori A; Vachon, Celine M; Knutson, Keith L; Radisky, Derek C; Visscher, Daniel W

2017-07-15

Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). Experimental Design: A breast tissue matched case-control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm 2 for CD4 + T cells, CD8 + T cells, CD20 + B cells, and CD68 + macrophages and quantification of positive pixel measure for CD11c (dendritic cells). Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8 + T cells, CD11c + dendritic cells, CD20 + B cells, and CD68 + macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20 + cell density ( P = 0.04). Nearly 42% of BBD cases had no CD20 + B cells in evaluated lobules compared with 28% of BBD controls ( P = 0.02). The absence of CD20 + cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4-23.1) for subsequent breast cancer risk. Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945-52. ©2017 AACR . ©2017 American Association for Cancer Research.

Anti-tumor effects of an engineered “killer” transfer RNA

International Nuclear Information System (INIS)

Zhou, Dong-hui; Lee, Jiyoung; Frankenberger, Casey; Geslain, Renaud; Rosner, Marsha; Pan, Tao

2012-01-01

Highlights: ► tRNA with anti-cancer effects. ► tRNA induced protein misfolding. ► tRNA as anti-tumor agent. -- Abstract: A hallmark of cancer cells is their ability to continuously divide; and rapid proliferation requires increased protein translation. Elevating levels of misfolded proteins can elicit growth arrest due to ER stress and decreased global translation. Failure to correct prolonged ER stress eventually results in cell death via apoptosis. tRNA Ser (AAU) is an engineered human tRNA Ser with an anticodon coding for isoleucine. Here we test the possibility that tRNA Ser (AAU) can be an effective killing agent of breast cancer cells and can effectively inhibit tumor-formation in mice. We found that tRNA Ser (AAU) exert strong effects on breast cancer translation activity, cell viability, and tumor formation. Translation is strongly inhibited by tRNA Ser (AAU) in both tumorigenic and non-tumorigenic cells. tRNA Ser (AAU) significantly decreased the number of viable cells over time. A short time treatment with tRNA Ser (AAU) was sufficient to eliminate breast tumor formation in a xenograft mouse model. Our results indicate that tRNA Ser (AAU) can inhibit breast cancer metabolism, growth and tumor formation. This RNA has strong anti-cancer effects and presents an opportunity for its development into an anti-tumor agent. Because tRNA Ser (AAU) corrupts the protein synthesis mechanism that is an integral component of the cell, it would be extremely difficult for tumor cells to evolve and develop resistance against this anti-tumor agent.

Performance comparison of digital microRNA profiling technologies applied on human breast cancer cell lines.

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Erik Knutsen

Full Text Available MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.