Estrogen Receptor Silencing Induces Epithelial to Mesenchymal Transition in Human Breast Cancer Cells

Abstract

We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in trans-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from a keratin/actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated geometric fold changes ≥3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. These data support our hypothesis that induced loss of estrogen receptor in previously estrogen/antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may offer a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity.

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells

“…5A) and triggered a mesenchymal-like morphology (Fig. 5B), as previously reported (1,5). Decreased apo-ERα binding and mRNA expression of target genes (Fig.…”

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer

“…Both processes share similar pathways of activation. Our recent data (Luqmani et al, 2009;Al Saleh, 2010;Al Saleh et al, 2011a) suggests that there may also be causal links between the development of endocrine resistance and the onset of EMT. In this report we summarise the molecular pathways of ER activity, the mechanisms proposed to account for resistance and finally review the evidence for the above hypothesis.…”

“…At a molecular level there is a certain uniformity of changes. Cells that have lost ER function and consequently acquired endocrine independence, in this case by shRNA-induced down-regulation (Al Saleh, 2010), show both the morphological appearance as well as the phenotypic changes that are characteristic of cells undergoing EMT. Several differences are indicated between MCF7 and pII cells that parallel those seen during EMT.…”

“…It has also been reported that elevated AXL levels are needed for maintaining breast cancer invasiveness, growth in foreign microenvironments and metastatic potential. Endocrine-resistant breast cancer cells show highly elevated expression of AXL (Al Saleh et al, 2010).…”

“…Identification of ligands, receptors and downstream signaling molecules with increased activity in the resistant phenotype, both in cell culture and in tumour biopsies, has highlighted a bewildering collection of molecules that may play a direct causative role, be a consequence or simply innocent bystanders in the progressive cellular change towards endocrine independence. For the purposes of therapeutic discrimination, attempts have been made to reduce this plethora, generated principally by microarray analyses (eg Charafe-Jauffret et al, 2006 ;Luqmani et al, 2009;Al Saleh, 2010) to a manageable number, and given the designation of 'gene signature' by virtue of selectively circumscribing a particular sub-group of patients. In a separate scenario, new insights have been gained into our understanding of cell differentiation from studies that have demonstrated that epithelial cells have the potential to trans-differentiate into mesenchymal cells (epithelial to mesenchymal transition: EMT) and vice versa (mesenchymal to epithelial transition: MET).…”