Cancer Prevention Research

Cancer Prevention Research 1 (7 Supplement), CN12-03, November 1, 2008. doi: 10.1158/1940-6207.PREV-08-CN12-03
© 2008 American Association for Cancer Research

http://cancerpreventionresearch.aacrjournals.org/cgi/content/short/1/7_MeetingAbstracts/CN12-03?rss=1

http://preventionportal.aacrjournals.org/misc/08Prevention_InvitedAbs.pdf

Evolution in Neoplastic Progression and Epithelial-Mesenchynal Transition

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

Tony Blick, Edwin Widodo, Honor Hugo, Angels Fabra-Fres, Razan Wafai, Devika Gunasinghe, Mark Waltham, Marc Lenburg, Rich Neve, Don Newgreen, Leigh Ackland and Erik Thompson

St. Vincent’s Institute, Fitzroy, Australia, Univ. of Melbourne Dept. Surgery, St. Vincent’s Hospital, Fitzroy, Australia, IDIBEL, Molecular Oncology, Hospital Duran Reynals, Barcelona, Spain, Univ. of Melbourne Dept. Surgery, St. Vincent’s Hospital and St. Vincent’s Institute, Fitzroy, Australia, Department of Genetics and Genomics, Boston University School of Medicine, Boston, MA, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, Embryology Laboratory, Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Australia, Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood, Australia

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.

http://cancerpreventionresearch.aacrjournals.org/cgi/content/short/1/7_MeetingAbstracts/CN12-03?rss=1

http://preventionportal.aacrjournals.org/misc/08Prevention_InvitedAbs.pdf

Publication at the Conference : Era of Hope – Department of Defense – Breast Cancer Research Program

https://cdmrpcures.org/ocs/index.php/eoh/eoh2008/paper/view/697

GENE EXPRESSION PATTERNS WHICH DISTINGUISH BASAL B (MESENCHYMAL) HUMAN BREAST CANCER CELL LINES FROM BASAL A OR LUMINAL ARE EXHIBITED BY PURIFIED HUMAN BREAST CANCER STEM CELLS. EVIDENCE OF EPITHELIAL MESENCHYMAL TRANSITION IN CLINICAL SPECIMENS
BC021320

Erik Thompson,1 Tony Blick,2 Edwin Widodo,1 Honor Hugo,3 Don F. Newgreen,3 M. Leigh Ackland,4 Marc E. Lenburg,5 Richard M. Neve,6 and Mark Waltham2

1University of Melbourne, 2St. Vincent’s Institute of Medical Research, 3Murdoch Children’s Research Institute, 4Deakin University, Burwood, Australia, 5Boston University School of Medicine, and 6Lawrence Berkeley National Laboratory

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed “Basal B”/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed “Luminal”) or mixed basal/luminal (termed “Basal A”) features (Neve et al., Cancer Cell, 2006). Expression of genes in a literature-derived EMT signature (EMT SIG) showed an EMT (positive) signature in the Basal B lines, and an EMT (negative) signature in the Luminal lines. The coordinate expression of EMT SIG genes in these cell lines supports a role for EMT in the differences between these HBC subtypes and an important biological role for EMT in different breast cancer subtypes. Further analysis of these data has implicated specific transcriptional programs in the HBC EMT, with Zeb1 and TCF-4 showing highest selective expression in the Basal B group. Snail 2 and TWIST were overexpressed in Basal B lines, but also in some Basal A and Luminal lines, respectively. We also identified potential new markers/regulators of EMT in the Neve dataset. We have examined these associations further in the HBC cell line PMC42ET and its epithelial counterpart (PMC42LA), which undergo EGF-induced EMT (Ackland et al., Lab Invest, 2003). Each line displays typical morphology, gene expression and immunohistochemistry with regard to epithelial (CDH1, occludin, claudin-1, mucin-1, with membranous beta catenin) and mesenchymal genes (vimentin, fibronectin, MMP-2, Snail1, Snail2, Zeb1/delta EF1). Transfection and siRNA analysis of several EMT regulators confirmed a pre-eminent role for Zeb1 in the PMC42 HBC system. Other relationships have also been examined further in the context of the PMC42 cell system and complement our recent work demonstrating that breast cancer-associated fibroblasts selectively stimulate EMT-related changes in the PMC42 cell line in 3-dimensional culture (Lebret et al., In Vitro Cellular & Developmental Biology, 2007 and Breast Cancer Research, 2007). Strong concordance was also found between gene products associated with the EMT phenotype in HBC cell lines and cells sorted from clinical specimens (Shipitsin et al., Cancer Cell, 2006), consistent with EMT attributes being advantageous for stem cells. Loss of CD24 is a defining attribute of the Basal B subgroup, where the level of CD24 correlates tightly with degree of mesenchymal gene expression. Of 60 gene products correlating tightly with CD24 status in Basal B cell lines, 27 were identified as differentially expressed in the Shipitsin data set (P < 0.0001). These new data confirm and extend the importance of EMT and the value of analyzing established HBC cell lines for new leads in this area. At a time when EMT remains somewhat controversial as a clinical entity, evidence of EMT-like gene expression in cells purified from breast cancers adds valuable support to a growing number of reports associating EMT parameters with poor clinical outcome in breast cancer.

This work was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-03-1-0416 and University of Melbourne.

https://cdmrpcures.org/ocs/index.php/eoh/eoh2008/paper/view/697

Curriculum vitae

Edwin Widodo

Currently studying Masters in the University of Melbourne since March 2007 and hopefully will finish in March 2009.

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