Abstract CN12-03: Epithelial-mesenchymal transition in human breast cancer progression: cancer stem cell attributes, dissemination, and dormancy

CN12-03

Epithelial mesenchymal transition (EMT) occurs developmentally to generate the primary mesenchyme, and then subsequently in the ectoderm to form muscle, bone, nerve and connective tissues (1,2). In many cases, the EMT occurs transiently, and is followed by the reverse transition (MET) at the destination, to result in epithelial structures (1). During EMT otherwise sessile collectives of epithelial cells down-regulate cell junctional machinery and gain motility (3).

The role of EMT in the metastatic process of malignancy has long been proposed (4-6). EMT may allow maintenance of residual cancer cells, since the CDH1 repressor SNAI was shown to be central to the emergence of residual disease into local recurrence after oncogene silencing (7). Mesenchymal derivatives of carcinoma cells show a number of attributes which would favor metastasis, such as separation from the collective as individual cells, increased migratory and invasive potential, increased survival in suspension and resistance to apoptosis in response to chemotherapy. It has long been recognised that circulating tumor cells (CTCs) show reduced expression of specific cytokeratins (8), and recently, cell lines derived from breast cancer micrometastases stably express the mesenchymal marker VIM (9). EMT biology has grown to encompass resistance to anoikis (10), enhanced survival (11), genomic instability (12) and resistance to chemotherapies (13).

One of the strongest evidences for EMT in the clinical scenario is the loss of epithelial features in colon cancer cells at the invasive front, with nuclear β-catenin, lack of E-cadherin and vimentin expression (14). Microarray analysis of these cells isolated by laser capture microdissection revealed ‘migrating stem cell’ phenotypes (15).

Human breast cancer cell lines show clear evidence of EMT, with a growing number of cell lines exhibiting mesenchymal features or undergoing EMT in response to an ever-growing collection of stimuli (reviewed in (16)). Building on gene expression profiling of breast cancer cell lines (17), we devised a literature-based classifier (EMT-SIG) and independently confirmed an EMT association within the invasive subgroup (Basal B/ Mesenchymal). EMT-SIG was notably under-expressed in luminal breast cancer cell lines. This coordinate expression of EMT-SIG genes in basaloid cells supports both a role for EMT in defining the phenotype and regulating the biology of aggressive breast tumour subtypes.

Breast cancer stem cells (BCSC) have been defined largely by a relative lack of the luminal marker CD24 and overexpression of CD44 (CD44hiCD24lo: (18,19)). CD44hiCD24lo/- cells have been reported to have higher expression levels of proinvasive genes (20,21), enhanced tumorigenicity compared to sister cells from the same tumour (22), and the capacity to generate a heterogenous tumour cell population (23). Gene expression (SAGE) analysis of CD44hiCD24lo compared to CD24hi cells from the same clinical samples generated a signature rich in TGF-ß pathway genes which was also predictive of patient survival (24). Along similar lines, Liu et al. (2007) generated a 186 gene “invasiveness gene signature (IGS)” based on CD44hiCD24lo sorted cancer cells compared to normal breast epithelium, and found this was predictive of patient outcome (21). Perhaps along similar lines, breast tumors resistant to Lapatinib showed higher ratios of BCSC phenotype and EMT-like signatures (25).

We observed that the Basal B cell lines are characterised by the CD44hiCD24lo/- phenotype which has been used to isolate and characterise breast cancer stem cells (BCSC; (18,19)). We found strong concordance between gene products associated with the EMT phenotype in HBC cell lines and cells sorted from clinical specimens (24), consistent with EMT attributes being advantageous for stem cells. Loss of CD24 correlated tightly with degree of mesenchymal gene expression, and 27 of 60 gene products correlating tightly with CD24 status in Basal B cell lines were also differentially expressed in the Shipitsin data set from isolated BCSC (P < 0.0001). The Weinberg laboratory also recently showed that the CD44hi/CD24lo cells which have high malignant potential (so called breast cancer stem cells; BCSC (18,19)) purified from normal and malignant mammary tissues, exhibited EMT features, and that human mammary cells induced to undergo EMT exhibited increased mammospheric and malignant potential (26). These new data confirm and extend the importance of EMT and the value of analysing established HBC cell lines for new leads in this area.

We are particularly interested in the PMC42 human breast cancer cell line. When first isolated, it showed expression of predominantly luminal cytokeratins 8 and 18, and stem-like capacity producing 8 morphological subtypes in culture after cloning (27). Parental PMC42 cells (PMC42-ET) are 100% VIM-positive and respond to EGF with increased VIM expression, and a further reduction in their already low CDH1 levels (27). The PMC42-LA epithelial subline develops acini-like structures in 3-dimensional Matrigel culture which produce milk proteins in response to lactogenic hormone, and elaborate myoepithelial markers in peripheral cells when grown as 3-dimensional clusters (28,29). Stimulation of PMC42-LA cells with EGF leads to EMT marker expression both in 2D monolayer culture (30) and 3D collagen cultures (unpublished data), and 3D Matrigel cultures of PMC42-LA show increased expression of these markers when treated with factors selectively secreted by carcinoma-associated fibroblasts over normal mammary fibroblasts (28). Thus, the PMC42 system provides a spectrum of EMT progression stages, and could provide important leads into the identification of markers that indicate EMT and/or a propensity for BrCa cells to undergo an EMT. EGF-regulated EMT has also been reported in the MCF-10A cells (31), and more recently in MDA-MB-468 cells (32).

EMT may represent an important target in the prevention of recurrence and metastasis. Understanding the mechanisms that maintain mesenchymal phenotype in breast cancer cells may provide new opportunities for clearance of disseminated tumour cells and prevention of recurrence. The emergence of EMT through tumour evolution will be discussed, in the context of the relationship with BCSC.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN12-03.