**5. Endocrine resistance and EMT**

It is becoming increasingly apparent that acquired endocrine resistance is a multi-factorial stepwise progression that can be triggered through a number of distinct pathways, that *in vitro*, can be manipulated. Whether it is the actual loss of ER due to transcriptional or translational down-regulation, or functional redundancy of ER (which seems to be the more frequent occurrence *in vivo*), either scenario would have the same end result in terms of

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 471

renewal of epithelial characteristics as typified by increased levels of E-cadherin and decrease of N-cadherin, vimentin and MMP9 with parallel reduction of tumour forming capacity of MDAMB231 cells injected into xenografted mice. These studies elegantly support the notion that ER regulated events is intimately involved in the same processes that lead to

Another significant group of genes variously implicated in EMT that is elevated in pII cells is included in the '24 gene signature' of genes proposed as predictive of invasiveness (Zajchowski et al., 2001): integrin, TIMP-2 and TIMP-3, MT1-MMP, PAI-1, Osteonectin/SPARC, thrombospondin-1, collagen (VI) α1 and collagen (I) α2. pII also display the '9 gene signature' of down-regulated or low expressing genes (E-cadherin, CLDN7, CRB3, KRT8, TACSTD1, IRF6, SPINT2, MAL2 and MARVELD3) that was found by Katz et al., (2011) to be common between their C35 transfected cells and claudin-low tumours. Evidence that the latter represent EMT is now substantial and supported by *in* 

Substantial reduction in ER expression has been observed in modified MCF7 sub-lines resistant to the mitotic inhibitors paclitaxel and docetaxel and the anthracycline doxorubicin (Iseri et al., 2011). Microarray analysis showed up-regulation of SNAIL2, CDH2, VIM, CLDN1, CLDN11, EGFR, FGFR1, SMAD3 and TGFBR2 and down-regulation of E-cadherin, OCLN, CLDN3, CLDN4, and CLDN7. This data bears remarkable resemblance to the profile

This brings us finally to the group of transcriptional repressors that have been coined as the 'mediators of EMT' and discussed above, so far a relatively smaller group that unify a much larger and diverse array of signalling molecules involved in their regulation. Of the key factors identified in cadherin switching, ZEB1, ZEB2/SIP1 and SNAIL2 (Onder et al, 2008) are all significantly elevated in our endocrine resistant pII cells. These observations lead us to conclude that there is a high degree of synonimity between endocrine resistance and EMT, both effected by functional loss of ER and both resulting in increased propensity for tumour dissemination through the actions of a common set of mediators. The repression of SNAIL by the ER dependent MTA3 (Fujita et al., 2003), a subunit of the Mi-2/NuRD histone deacetylase complex, which could well be regarded, among others, as a guardian of the epithelial phenotype (?) may be worthy of further attention. Interestingly, another family member, MTA1, is described as a potent inhibitor of nuclear ER function through cytoplasmic sequestration of the receptor and this may provide an explanation for resistance in ER+ cells as MTA1 would indirectly reduce the levels of MTA3 thereby relieving SNAIL

There have also been intriguing suggestions regarding the origin of the mesenchymal-like cells, with the attractive view of these as a possibly slow growing pre-existing CSC subpopulation within the tumour (Lim et al., 2010; May et al., 2011). In such a scenario there is no induced EMT as such, but a gradual emergence of a group of cells already bearing these properties, to become the dominant group. Similar ideas have often been suggested to explain the re-emergence of 'drug–regressed' tumours as an expansion of a pre-existing intrinsically resistant cell population once the sensitive cells have been eliminated. However, attractive as this may be, in the alternative scheme elaborated by May et al., (2011) there would be a reversion of such 'MaSCs' back to an epithelial phenotype at the site of metastatic growth in a reverse MET transition, which raises the question that If cells can undergo MET then why not EMT, and there is no necessity to postulate the existence of *a priori* mesenchymal cells. Moreover, the *in vitro* data demonstrates quite clearly that an

EMT and very crucially, that these events are reversible.

*vitro* observations (Prat et al., 2010; Taube et al., 2010).

for pII cells with the common denominator being loss of ER.

repression.

independence from estrogen. It is therefore pertinent to ask what happens to a cell that experiences loss of ER. As described in preceding sections this issue has been addressed by various cell models that have been made endocrine resistant by exposure to antiestrogens or by deprivation of estradiol, but rarely by the direct prevention of ER synthesis.

We have explored this avenue by modifying MCF7 cells by transfection with shRNA generating plasmids targeting the ER mRNA (Al Azmi, 2006; Luqmani et al., 2009; Al Saleh et al., 2011a). As expected, stably transfected cell lines with constitutive reduction of ER (termed pII) exhibit a loss of response to either estradiol or tamoxifen/fulvestrant and hypersensitivity to EGF and IGF1 (Salloum, 2010). There is reduction in the classical ERregulated markers such as pS2, cathepsin D, PR and PRLR. Like the tumour-derived naturally ER-ve MDAMB231 cell line, these (acquired) endocrine resistant cells show increased motility and ability to invade simulated components of the ECM mimicking the behaviour of aggressive ER-ve/EGFR+ve tumours. Both of these activities as well as cellular proliferation are reduced by various tyrosine kinase inhibitors that are known to block, in particular, EGFR and VEGFR phosphorylation (Al Saleh, 2010) supporting the data mentioned in preceding sections. However, the most striking features of pII cells was initially noted in their morphological appearance (see Fig 3), assuming a more elongated spindly shape and failure to form the compact colonies characteristic of MCF7 cells, with rearrangement of the actin cytoskeleton giving rise to increased incidence of lamellipodia and microspikes, features closely associated with cellular motility (Parker et al., 2002).

Microarray analysis confirmed that pII cells had assumed a phenotype that is generally seen for mesenchymal cells, with transcriptional loss of genes normally associated with epithelial cells. Lack of colony formation can be explained by loss of E-cadherin and many other factors responsible for normal cell-cell adhesion including catenins, laminin, type IV collagen, desmogleins, desmocollins, occludins, connexion 2b claudins and MUC1. Likewise, archetypical epithelial components such as keratins 8, 18 and 19 and tissue inhibitors of metallo-proteinases are all reduced. On the other hand, we observed an increased expression of mesenchymal markers such as N cadherin, vimentin, fibronectin, integrins β4 and α5, tenascin, SPARC, PLAU, VEGF, CD68, FSP1/S100A4, LCN2 and various metalloproteinases In short, we are seeing all the hallmarks of cells undergoing EMT with acquisition of the phenotype characterising the group of basal-like 'claudin low' tumours such as the triple negative (ER-ve, PR-ve, ERBB2-ve) metaplastic tumours described by Hennessy et al., (2009). A similar conclusion was reached by Gadalla et al., (2005) who observed an EMT-like transition with loss of E-cadherin and reduction in CD24 induced by ER silencing. However, they did not observe the increase in CD44 that we and others have widely reported.

An interesting molecule whose expression was found to be substantially repressed in our pII cells **(**Al Saleh et al., 2011a) is GATA3, a zinc finger transcription factor that plays an important role as a regulator of mammary gland formation and development (Kouros-Mehr et al., 2008) and has been implicated in both EMT and breast cancer metastasis. GATA3 is a positive transcriptional regulator of ER expression whilst simultaneously itself being a target gene for the ER complex. Its expression has been linked to favourable outcome of endocrine therapy (Parikh et al., 2005). Several studies have shown association of GATA3 with ER+ tumours (eg, Mehra et al., 2005). Yan et al., (2010) recently demonstrated that not only was GATA3 expression abolished in ER-ve cell lines but also correlated with Ecadherin. siRNA-induced silencing of GATA3 resulted in fibroblastic-like transformation of MCF7 cells. On the other hand, restoration of GATA3 expression in ER-ve cells led to

independence from estrogen. It is therefore pertinent to ask what happens to a cell that experiences loss of ER. As described in preceding sections this issue has been addressed by various cell models that have been made endocrine resistant by exposure to antiestrogens or

We have explored this avenue by modifying MCF7 cells by transfection with shRNA generating plasmids targeting the ER mRNA (Al Azmi, 2006; Luqmani et al., 2009; Al Saleh et al., 2011a). As expected, stably transfected cell lines with constitutive reduction of ER (termed pII) exhibit a loss of response to either estradiol or tamoxifen/fulvestrant and hypersensitivity to EGF and IGF1 (Salloum, 2010). There is reduction in the classical ERregulated markers such as pS2, cathepsin D, PR and PRLR. Like the tumour-derived naturally ER-ve MDAMB231 cell line, these (acquired) endocrine resistant cells show increased motility and ability to invade simulated components of the ECM mimicking the behaviour of aggressive ER-ve/EGFR+ve tumours. Both of these activities as well as cellular proliferation are reduced by various tyrosine kinase inhibitors that are known to block, in particular, EGFR and VEGFR phosphorylation (Al Saleh, 2010) supporting the data mentioned in preceding sections. However, the most striking features of pII cells was initially noted in their morphological appearance (see Fig 3), assuming a more elongated spindly shape and failure to form the compact colonies characteristic of MCF7 cells, with rearrangement of the actin cytoskeleton giving rise to increased incidence of lamellipodia and

by deprivation of estradiol, but rarely by the direct prevention of ER synthesis.

microspikes, features closely associated with cellular motility (Parker et al., 2002).

others have widely reported.

Microarray analysis confirmed that pII cells had assumed a phenotype that is generally seen for mesenchymal cells, with transcriptional loss of genes normally associated with epithelial cells. Lack of colony formation can be explained by loss of E-cadherin and many other factors responsible for normal cell-cell adhesion including catenins, laminin, type IV collagen, desmogleins, desmocollins, occludins, connexion 2b claudins and MUC1. Likewise, archetypical epithelial components such as keratins 8, 18 and 19 and tissue inhibitors of metallo-proteinases are all reduced. On the other hand, we observed an increased expression of mesenchymal markers such as N cadherin, vimentin, fibronectin, integrins β4 and α5, tenascin, SPARC, PLAU, VEGF, CD68, FSP1/S100A4, LCN2 and various metalloproteinases In short, we are seeing all the hallmarks of cells undergoing EMT with acquisition of the phenotype characterising the group of basal-like 'claudin low' tumours such as the triple negative (ER-ve, PR-ve, ERBB2-ve) metaplastic tumours described by Hennessy et al., (2009). A similar conclusion was reached by Gadalla et al., (2005) who observed an EMT-like transition with loss of E-cadherin and reduction in CD24 induced by ER silencing. However, they did not observe the increase in CD44 that we and

An interesting molecule whose expression was found to be substantially repressed in our pII cells **(**Al Saleh et al., 2011a) is GATA3, a zinc finger transcription factor that plays an important role as a regulator of mammary gland formation and development (Kouros-Mehr et al., 2008) and has been implicated in both EMT and breast cancer metastasis. GATA3 is a positive transcriptional regulator of ER expression whilst simultaneously itself being a target gene for the ER complex. Its expression has been linked to favourable outcome of endocrine therapy (Parikh et al., 2005). Several studies have shown association of GATA3 with ER+ tumours (eg, Mehra et al., 2005). Yan et al., (2010) recently demonstrated that not only was GATA3 expression abolished in ER-ve cell lines but also correlated with Ecadherin. siRNA-induced silencing of GATA3 resulted in fibroblastic-like transformation of MCF7 cells. On the other hand, restoration of GATA3 expression in ER-ve cells led to renewal of epithelial characteristics as typified by increased levels of E-cadherin and decrease of N-cadherin, vimentin and MMP9 with parallel reduction of tumour forming capacity of MDAMB231 cells injected into xenografted mice. These studies elegantly support the notion that ER regulated events is intimately involved in the same processes that lead to EMT and very crucially, that these events are reversible.

Another significant group of genes variously implicated in EMT that is elevated in pII cells is included in the '24 gene signature' of genes proposed as predictive of invasiveness (Zajchowski et al., 2001): integrin, TIMP-2 and TIMP-3, MT1-MMP, PAI-1, Osteonectin/SPARC, thrombospondin-1, collagen (VI) α1 and collagen (I) α2. pII also display the '9 gene signature' of down-regulated or low expressing genes (E-cadherin, CLDN7, CRB3, KRT8, TACSTD1, IRF6, SPINT2, MAL2 and MARVELD3) that was found by Katz et al., (2011) to be common between their C35 transfected cells and claudin-low tumours. Evidence that the latter represent EMT is now substantial and supported by *in vitro* observations (Prat et al., 2010; Taube et al., 2010).

Substantial reduction in ER expression has been observed in modified MCF7 sub-lines resistant to the mitotic inhibitors paclitaxel and docetaxel and the anthracycline doxorubicin (Iseri et al., 2011). Microarray analysis showed up-regulation of SNAIL2, CDH2, VIM, CLDN1, CLDN11, EGFR, FGFR1, SMAD3 and TGFBR2 and down-regulation of E-cadherin, OCLN, CLDN3, CLDN4, and CLDN7. This data bears remarkable resemblance to the profile for pII cells with the common denominator being loss of ER.

This brings us finally to the group of transcriptional repressors that have been coined as the 'mediators of EMT' and discussed above, so far a relatively smaller group that unify a much larger and diverse array of signalling molecules involved in their regulation. Of the key factors identified in cadherin switching, ZEB1, ZEB2/SIP1 and SNAIL2 (Onder et al, 2008) are all significantly elevated in our endocrine resistant pII cells. These observations lead us to conclude that there is a high degree of synonimity between endocrine resistance and EMT, both effected by functional loss of ER and both resulting in increased propensity for tumour dissemination through the actions of a common set of mediators. The repression of SNAIL by the ER dependent MTA3 (Fujita et al., 2003), a subunit of the Mi-2/NuRD histone deacetylase complex, which could well be regarded, among others, as a guardian of the epithelial phenotype (?) may be worthy of further attention. Interestingly, another family member, MTA1, is described as a potent inhibitor of nuclear ER function through cytoplasmic sequestration of the receptor and this may provide an explanation for resistance in ER+ cells as MTA1 would indirectly reduce the levels of MTA3 thereby relieving SNAIL repression.

There have also been intriguing suggestions regarding the origin of the mesenchymal-like cells, with the attractive view of these as a possibly slow growing pre-existing CSC subpopulation within the tumour (Lim et al., 2010; May et al., 2011). In such a scenario there is no induced EMT as such, but a gradual emergence of a group of cells already bearing these properties, to become the dominant group. Similar ideas have often been suggested to explain the re-emergence of 'drug–regressed' tumours as an expansion of a pre-existing intrinsically resistant cell population once the sensitive cells have been eliminated. However, attractive as this may be, in the alternative scheme elaborated by May et al., (2011) there would be a reversion of such 'MaSCs' back to an epithelial phenotype at the site of metastatic growth in a reverse MET transition, which raises the question that If cells can undergo MET then why not EMT, and there is no necessity to postulate the existence of *a priori* mesenchymal cells. Moreover, the *in vitro* data demonstrates quite clearly that an

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