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.

We have established several breast cancer cell lines that exhibit a permanent ER-depleted phenotype, induced by shRNA transfection of MCF-7 cells, which afford a useful model for studying acquired endocrine resistance. Previously we showed that MDA-231 as well as ER-silenced cells could invade through simulated extracellular matrix components. However, the contribution of individual serum components responsible for cell invasion was not determined. In the present study, an under-agarose gel assay was used to quantitatively assess the invasive movement of two ER-silenced cell lines (pII and YS2.5) in comparison to the parental MCF-7, the ER negative MDA-231, and normal HBL100 cells, as well as a line that was ER-shRNA transfected but failed to exhibit ER down-regulation (YS1.2). We also examined the effect of the growth factors EGF, IGF-1, TGFβ, PDGFC and RANTES on pII cell invasion and proliferation. All breast cancer cell lines which had reduced ER expression exhibited a serum-dependent invasive ability related to the degree of induced ER loss. TGFβ treatment inhibited pII cell proliferation and enhanced their invasive ability but at a relatively high dose. IGF-1 and EGF enhanced pII cell proliferation, with the latter playing the major role in promoting cell invasion. PDGFC did not affect either process although it is highly expressed in pII cells. Differential effects were observed on activation of Akt and ERK1/2 suggesting their involvement as intracellular mediators of EGF induced invasion, in part through the regulation of matrix metalloproteinase activity. Targeting EGF receptor tyrosine kinase activity by erlotinib resulted in significant inhibition of both pII cell proliferation and directional invasion towards EGF suggesting that this drug has potential therapeutic usefulness for preventing spread of particularly endocrine resistant breast cancer.

Abstract. Both de novo and acquired endocrine resistance constitute a major therapeutic problem for treatment of hormone-positive breast cancer. Multiple explanatory mechanisms have been proposed through the study of cellular models which focus principally on receptor tyrosine kinase mediated signalling pathways utilizing sRC, PI3K, MAPK and sMAds. Many of the transducing molecules, particularly nuclear transcription factors such as sNAIL, TwIsT, sNAIL2, ZEB, FOXC2, TCF/LEF and GOOsECOId are participants in proliferation as well as invasion and metastasis, involving a process of orchestrated cellular remodeling which is being likened to the process of epithelial to mesenchymal transition that takes place during embryonic development. we review the accumulating evidence that points towards the occurrence of this phenomenon as a consequence of the loss of endocrine control, with both processes being similarly characterized by depletion of cell adhesion proteins, E-cadherin, catenins and cytokeratins, increased association with the extracellular matrix through induction of metalloproteinases, fibronectin and collagen, and a switch to a mobile vimentin-based cytoskeletal structure with loss of apical basal polarity. IntroductionEndocrine therapy represents the most effective form of treatment for the majority of breast cancer patients whose tumours over-express the estrogen receptor (ER). In addition to ablative procedures (ovariectomy) and administration of antiendocrine agents to inhibit ovarian function, treatment is reliant predominantly upon anti-hormonal agents termed selective estrogen modulators (sERMs). Until recent introduction of agents such as toremifene and raloxifene, tamoxifen has been the mainstay of treatment (1) inducing objective response or disease stabilization in over half of previously untreated metastatic breast cancer patients with ER + tumours (2). Further options include the use of pure anti-estrogens such as fulvestrant (Faslodex) which achieves its effects through receptor degradation, and application of aromatase inhibitors that reduce extra-gonadal peripheral estrogen synthesis from the adrenals and adipose tissue, including the breast. Both types of agents improve relapse-free survival and reduce incidence of contralateral breast cancers in women with early-stage cancer and increase overall survival in patients with advanced disease (3,4). Unfortunately, following initial response to sERMs and second line therapy with aromatase inhibitors, most patients subsequently develop resistance to both classes of drugs and become refractive to further attempts at endocrine manipulation. Added to the de novo resistance in patients whose tumours express levels of ER <10 fmol/mg protein, this presents a serious therapeutic problem, particularly in view of the increased aggressiveness of hormone insensitive breast cancers. Estrogen receptor actionThe classical mode of action of ER is related to the regulation of expression of genes with estrogen response elements (ERE) in their promoters through t...

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