Role of Epithelial Mesenchymal Plasticity associated cancer subpopulations in mammary tumourigenisis and chemoresistance

© 2014 Dr. Cletus A. PintoTumour heterogeneity is a key characteristic of cancer and has significant implications relating to tumour response to chemotherapy as well as patient prognosis and potential relapse. It is increasingly accepted that tumours are clonal in origin, suggestive of a tumour arising from a deregulated or mutated cell. Cancer stem cells (CSC) possess/propagate these capabilities, and with appropriate intracellular triggers and/or signalling from extracellular environments, can ‘differentiate’ to initiate tumour formation. Additionally through epithelial mesenchymal plasticity (EMP), where cells gain and maintain characteristics of both epithelial and mesenchymal cell types, epithelial-derived tumour cells have been shown to ‘de-differentiate’ to acquire cancer stem attributes, which also imparts chemotherapy resistance. This new paradigm places EMP centrally in the process of tumour formation, propagation, progression and metastasis, as well as modulating drug response to current forms of chemotherapy. Furthermore, EMP and CSCs have been identified in cancers arising from different tissue types making them a possible generic therapeutic target in cancer biology. In this study, we expand on the relationship between tumour heterogeneity, EMP and CSC in BrCa through the identification and characterisation of epithelial and mesenchymal subpopulations within two BrCa cell lines. In addition, we demonstrate the plasticity that allows these cell populations to effectively regenerate the other cell populations with a particular emphasis on the CSC phenotype. Through a functional genomics screen, the importance of the mesenchymal phenotype in tumour initiation is demonstrated. Taken together, this study demonstrates that heterogeneity exists at a cell line level and this heterogeneity differs in different cellular systems. We also find evidence to suggest that BrCa cell lines can use multiple mechanisms to achieve an outcome such as tumour initiation or mammosphere formation, and subsequently emphasize the importance of phenotype specific drugs. This ideology of drug repurposing to identify phenotype specific drugs is explored through the use of the connectivity map database to identify new uses for previously established drugs to target these subpopulations find preliminary evidence for the role of HDACi to affect these EMP associated subpopulations in BrCa cell lines

Breast cancer stem cells and epithelial mesenchymal plasticity - Implications for chemoresistance

Tumour heterogeneity is a key characteristic of cancer and has significant implications relating to tumour response to chemotherapy as well as patient prognosis and potential relapse. It is being increasingly accepted that tumours are clonal in origin, suggestive of a tumour arising from a deregulated or mutated cell. Cancer stem cells (CSC) possess these capabilities, and with appropriate intracellular triggers and/or signalling from extracellular environments, can purportedly differentiate to initiate tumour formation. Additionally through epithelial mesenchymal plasticity (EMP), where cells gain and maintain characteristics of both epithelial and mesenchymal cell types, epithelial-derived tumour cells have been shown to de-differentiate to acquire cancer stem attributes, which also impart chemotherapy resistance. This new paradigm places EMP centrally in the process of tumour progression and metastasis, as well as modulating drug response to current forms of chemotherapy. Furthermore, EMP and CSCs have been identified in cancers arising from different tissue types making it a possible generic therapeutic target in cancer biology. Using breast cancer (BrCa) as an example, we summarise here the current understanding of CSCs, the role of EMP in cancer biology - especially in CSCs and different molecular subtypes, and the implications this has for current and future cancer treatment strategies

Interrogation of phenotypic plasticity between epithelial and mesenchymal states in breast cancer

Dynamic interconversions between transitional epithelial and mesenchymal states underpin the epithelial mesenchymal plasticity (EMP) seen in some carcinoma cell systems. We have delineated epithelial and mesenchymal subpopulations existing within the PMC42-LA breast cancer cell line by their EpCAM expression. These purified but phenotypically plastic states, EpCAMHigh (epithelial) and EpCAMLow (mesenchymal), have the ability to regain the phenotypic equilibrium of the parental population (i.e., 80% epithelial and 20% mesenchymal) over time, although the rate of reversion in the mesenchymal direction (epithelial-mesenchymal transition; EMT) is higher than that in the epithelial direction (mesenchymal-epithelial transition; MET). Single-cell clonal propagation was implemented to delineate the molecular and cellular features of this intrinsic heterogeneity with respect to EMP flux. The dynamics of the phenotypic proportions of epithelial and mesenchymal states in single-cell generated clones revealed clonal diversity and intrinsic plasticity. Single cell-derived clonal progenies displayed differences in their functional attributes of proliferation, stemness marker (CD44/CD24), migration, invasion and chemo-sensitivity. Interrogation of genomic copy number variations (CNV) with whole exome sequencing (WES) in the context of chromosome count from metaphase spread indicated that chromosomal instability was not influential in driving intrinsic phenotypic plasticity. Overall, these findings reveal the stochastic nature of both the epithelial and mesenchymal subpopulations, and the single cell-derived clones for differential functional attributes

Interrogation of Phenotypic Plasticity between Epithelial and Mesenchymal States in Breast Cancer

Dynamic interconversions between transitional epithelial and mesenchymal states underpin the epithelial mesenchymal plasticity (EMP) seen in some carcinoma cell systems. We have delineated epithelial and mesenchymal subpopulations existing within the PMC42-LA breast cancer cell line by their EpCAM expression. These purified but phenotypically plastic states, EpCAMHigh (epithelial) and EpCAMLow (mesenchymal), have the ability to regain the phenotypic equilibrium of the parental population (i.e., 80% epithelial and 20% mesenchymal) over time, although the rate of reversion in the mesenchymal direction (epithelial-mesenchymal transition; EMT) is higher than that in the epithelial direction (mesenchymal-epithelial transition; MET). Single-cell clonal propagation was implemented to delineate the molecular and cellular features of this intrinsic heterogeneity with respect to EMP flux. The dynamics of the phenotypic proportions of epithelial and mesenchymal states in single-cell generated clones revealed clonal diversity and intrinsic plasticity. Single cell-derived clonal progenies displayed differences in their functional attributes of proliferation, stemness marker (CD44/CD24), migration, invasion and chemo-sensitivity. Interrogation of genomic copy number variations (CNV) with whole exome sequencing (WES) in the context of chromosome count from metaphase spread indicated that chromosomal instability was not influential in driving intrinsic phenotypic plasticity. Overall, these findings reveal the stochastic nature of both the epithelial and mesenchymal subpopulations, and the single cell-derived clones for differential functional attributes

Identifying therapies to combat epithelial mesenchymal plasticity-associated chemoresistance to conventional breast cancer therapies using an shRNA library screen

Background: Breast cancer (BC) is a heterogeneous disease for which the commonly used chemotherapeutic agents primarily include the anthracyclines (doxorubicin, epirubicin), microtubule inhibitors (paclitaxel, docetaxel, eribulin), and alkylating agents (cyclophosphamide). While these drugs can be highly effective, metastatic tumours are frequently refractory to treatment or become resistant upon tumour relapse. Methods: We undertook a cell polarity/epithelial mesenchymal plasticity (EMP)-enriched short hairpin RNA (shRNA) screen in MDA-MB-468 breast cancer cells to identify factors underpinning heterogeneous responses to three chemotherapeutic agents used clinically in breast cancer: Doxorubicin, docetaxel, and eribulin. shRNA-transduced cells were treated for 6 weeks with the EC10 of each drug, and shRNA representation assessed by deep sequencing. We first identified candidate genes with depleted shRNA, implying that their silencing could promote a response. Using the Broad Institute’s Connectivity Map (CMap), we identified partner inhibitors targeting the identified gene families that may induce cell death in combination with doxorubicin, and tested them with all three drug treatments. Results: In total, 259 shRNAs were depleted with doxorubicin treatment (at p Conclusions: Taken together, a cell polarity/EMP-enriched shRNA library screen identified relevant gene products that could be targeted alongside current chemotherapeutic agents for the treatment of invasive BC.</p

Abstract 3428: Coordinated regulation of mesenchymal epithelial transition in the PMC42-LA breast cancer cell line variant

Abstract PMC42 cells were derived from a breast cancer pleural effusion and exhibit stem cell-like features when first characterized (Whitehead, Bertoncello et al. 1983; Whitehead, Monaghan et al. 1983). The PMC42-LA subline (Ackland, Michalczyk et al. 2001; Ackland, Newgreen et al. 2003) is distinctly less mesenchymal than parental PMC42-ET cells, but both exhibit EGF- and TGFβ-inducible mesenchymal-like change. Together they provide an ideal model in which to study the regulation of epithelial-mesenchymal plasticity in a breast cancer context. We have assessed in vitro parameters including migration, 3D colony formation and EMT marker expression, and undertook both miRNA profiling (mirVana probe set V1, Ambion) and alternative splice usage (Affymetrix human 1.0 ST whole genome exon arrays) analysis. Several miRNAs were expressed differently in the two sublines and/or up- or down-regulated in response to EMT-inducing treatments. Relative to the mesenchymal ET subline, the, miR-200 family members were elevated in the epithelial LA subline, where a corresponding loss of Zeb1 expression was seen. The LA subline also showed reduced E-Cadherin promoter methylation, and increased methylation of the Zeb1 promoter. Manipulation of the miR 200 family affected the EMT marker expression in these cells, as did Zeb1 suppression with shRNA. Matrigel invasion was also inhibited with Zeb1 knockdown. Additional gene expression changes and alternative splice usage, promoter methylation changes, and miR expression changes are under study. These studies may identify molecules and pathways that are important in cell specification across the epithelial mesenchymal axis, and represent diagnostic and therapeutic targets in breast cancer. Ackland, M. L., A. Michalczyk, et al. (2001). “PMC42, A novel model for the differentiated human breast.” Exp Cell Res 263(1): 14-22. Ackland, M. L., D. F. Newgreen, et al. (2003). “Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells.” Lab Invest 83(3): 435-48. Whitehead, R. H., I. Bertoncello, et al. (1983). “A new human breast carcinoma cell line (PMC42) with stem cell characteristics. I. Morphologic characterization.” J Natl Cancer Inst 70(4): 649-61. Whitehead, R. H., P. Monaghan, et al. (1983). “A new human breast carcinoma cell line (PMC42) with stem cell characteristics. II. Characterization of cells growing as organoids.” J Natl Cancer Inst 71(6): 1193-203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2011-3428</jats:p

Direct repression of MYB byDirect repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

Introduction: Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells.Methods: MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05).Results: Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity.Conclusions: This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells

Genome-wide gain-of-function screen for genes that induce epithelial-to-mesenchymal transition in breast cancer

Epithelial to mesenchymal transition (EMT) is a developmental program that has been implicated in progression, metastasis and therapeutic resistance of some carcinomas. To identify genes whose overexpression drives EMT, we screened a lentiviral expression library of 17000 human open reading frames (ORFs) using high-content imaging to quantitate cytoplasmic vimentin. Hits capable of increasing vimentin in the mammary carcinoma-derived cell line MDA-MB-468 were confirmed in the non-tumorigenic breast-epithelial cell line MCF1OA. When overexpressed in this model, they increased the rate of cell invasion through Matrigel'TM, induced mesenchymal marker expression and reduced expression of the epithelial marker E-cadherin. In gene-expression datasets derived from breast cancer patients, the expression of several novel genes correlated with expression of known EMT marker genes, indicating their in vivo relevance. As EMT-associated properties are thought to contribute in several ways to cancer progression, genes identified in this study may represent novel targets for anti-cancer therapy