**4.3 E-cadherin and its transcriptional repressors**

E-cadherin is a critical switch in EMT during early embryonic development. Its downregulation in epithelial cells triggers acquisition of a fibroblastic phenotype, dissociation from the epithelium sheets and migration, vital steps in gastrulation, neural crest formation and organ development (Thiery, 2003). E-cadherin expression is often lost in aggressive breast cancers acquiring EMT which would result in the disassembly of intercellular adhesion complexes, loosening contacts between neighbouring epithelial cells and thus disrupting the overall tissue architecture. E-cadherin loss also causes the liberation of β-catenin to the nucleus and its subsequent activation of WNT signalling of other EMT inducers as described above. Furthermore, E-cadherin loss mediates EMT through the induction of its own transcriptional repressors, SNAIL, TWIST and ZEB1 (EF1), in a feedforward loop that sustains E-cadherin repression and potentiates EMT (Onder et al., 2008).

An interesting connection between endocrine resistance and EMT is established through the connection between SNAIL, E-cadherin and metastasis-associated protein 3 (MTA3). MTA3, which is directly activated by ER, is a repressor of SNAIL, thereby also repressing EMT (Al Saleh et al., 2011). We have recently shown that down-regulation of ER in MCF7 cells leads to a reduction in both MTA1 and MTA3 and a concurrent rise in SNAIL2 **(**Al Saleh et al., 2011a).

Reduction of E-cadherin expression correlates with poor differentiation, invasiveness, aggressive metastatic behaviour, and an unfavourable prognosis (Berx et al., 2001; Wheelock et al., 2003); experimental knockdown of E-cadherin is sufficient to establish metastasis but not fully reverse EMT by itself. Interestingly, the down regulated expression of E-cadherin during EMT is a reversible process that arises through hypermethylation of the E-cadherin promoter or transcriptional repression although many lobular breast cancers appear to have lost the expression of E-cadherin through inactivating mutations and loss of heterozygosity (Berx et al, 2001).

might participate in initiation of EMT such as the WNT and NOTCH pathways (Polyak and Weinberg, 2009). Figure 4 illustrates the major events that are thought to be critical in the

As mentioned earlier, receptor tyrosine kinase activity is altered in breast cancer and is considered to be an important factor in endocrine resistance. These molecules are also implicated in EMT since they already play a pivotal role in embryogenesis. One interesting member of the TAM (Tyro-AXL-MER) receptor tyrosine kinases is AXL which exerts diverse effects in regulating cellular responses that include cell proliferation, cell survival, migration, autophagy, angiogenesis, natural killer cell differentiation and platelet aggregation (Linger et al., 2008). AXL was reported to be associated with EMT since it is activated in many signal transduction pathways including AKT, MAPK, NFKB, and STAT. (Hafizi et al., 2006). Furthermore, AXL expression alone is considered as a predictive marker for poor overall patient survival. 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

E-cadherin is a critical switch in EMT during early embryonic development. Its downregulation in epithelial cells triggers acquisition of a fibroblastic phenotype, dissociation from the epithelium sheets and migration, vital steps in gastrulation, neural crest formation and organ development (Thiery, 2003). E-cadherin expression is often lost in aggressive breast cancers acquiring EMT which would result in the disassembly of intercellular adhesion complexes, loosening contacts between neighbouring epithelial cells and thus disrupting the overall tissue architecture. E-cadherin loss also causes the liberation of β-catenin to the nucleus and its subsequent activation of WNT signalling of other EMT inducers as described above. Furthermore, E-cadherin loss mediates EMT through the induction of its own transcriptional repressors, SNAIL, TWIST and ZEB1 (EF1), in a feedforward loop that sustains E-cadherin repression and potentiates EMT (Onder et al., 2008). An interesting connection between endocrine resistance and EMT is established through the connection between SNAIL, E-cadherin and metastasis-associated protein 3 (MTA3). MTA3, which is directly activated by ER, is a repressor of SNAIL, thereby also repressing EMT (Al Saleh et al., 2011). We have recently shown that down-regulation of ER in MCF7 cells leads to a reduction in both MTA1 and MTA3 and a concurrent rise in SNAIL2 **(**Al Saleh et al.,

Reduction of E-cadherin expression correlates with poor differentiation, invasiveness, aggressive metastatic behaviour, and an unfavourable prognosis (Berx et al., 2001; Wheelock et al., 2003); experimental knockdown of E-cadherin is sufficient to establish metastasis but not fully reverse EMT by itself. Interestingly, the down regulated expression of E-cadherin during EMT is a reversible process that arises through hypermethylation of the E-cadherin promoter or transcriptional repression although many lobular breast cancers appear to have lost the expression of E-cadherin through inactivating mutations and loss of heterozygosity

trans-differentiation of epithelial cells.

expression of AXL (Al Saleh et al., 2010).

**4.3 E-cadherin and its transcriptional repressors** 

**4.2 AXL** 

2011a).

(Berx et al, 2001).

Fig. 4. Transduction pathways and effectors contributing to processes leading to EMT. A variety of growth factors (EGF, TGFα, IGFI, II, PDGF, HGF, FGF) binding to receptor tyrosine kinases (RTK) activate the central RAS pathway to promote transcription of SNAIL through the RAF/MAPK, the PI3K/AKT/NFKB or the SRC/LIV pathways. AKT, as well as WNTs acting through the FRIZZLED receptor, promote inhibition of GSK3 through DSH to promote re-localisation of β-catenin and generate TCF/LEF that also increases SNAIL. DELTA/JAGGED signalling through NOTCH also increases SNAIL via CSL as well as TWIST through HIF1. TGFβ signals through its receptor to increase SMAD family members that co-operatively promote both SNAIL as well as SIP1/ZEB2. It also acts through PAR6 to up-regulate the ubiquitin ligase SMURF that degrades RHO which is a key promoter of tight junctions, The transcriptional repressors SNAIL, TWIST, GSC, ZEB1,2 and TFC/LEF effectively down-regulate E-cadherin and associated molecules, which leads to loss of cell adhesion, permitting cell scattering, cellular motility and invasion through the action of upregulated proteases. Not shown here, for clarity, is HEDGEHOG signalling which through GLI integrates with the RTK and WNT pathways to up-regulate SNAIL family members Evidence for the interactions illustrated is summarised in excellent reviews by Huber et al., 2005; Moustakes & Heldin, 2007 and Sabbah et al., 2008 and references therein.

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 465

also found to play a major role in cell regulation, proliferation, differentiation and regulation of EMT. It's over-expression in human breast cancer cells can cause up-regulation of vimentin and fibronectin while E-cadherin is down regulated (Yang et al., 2009). Furthermore, LCN2 over-expression significantly increases cell motility and invasiveness in previously non-invasive MCF-7 cells. Interestingly, siRNA-mediated LCN2 silencing inhibited cell migration and development of the mesenchymal phenotype in aggressive breast cancer cells. It was also reported that reduced expression of ER and increased expression of SNAIL2 was correlated with LCN2 expression while over-expression of ER in LCN-2 expressing cells was able to reverse EMT and reduce SNAIL2 expression, suggesting

An interesting physiological mechanism that can cause EMT is hypoxia. It has been reported that tumour progression and metastasis is promoted by the stabilisation of the hypoxiainducible factor-1α (HIF-1α). This transcription factor was shown to be associated with TWIST in inducing both EMT and tumour metastasis by hypoxia or over-expression of the former. Furthermore, the expression of TWIST was found to be regulated by HIF-1 binding to the hypoxia-response element (HRE) in the TWIST proximal promoter and is associated with it in inducing EMT or metastasis (Yang et al, 2008). Interestingly, the HIF-1α null mice phenotype resembles TWIST deficient mice. In addition, patients with head and neck cancer whose tumours co-express TWIST and HIF-1 had very poor prognosis suggesting a major

Another important set of genes in regulating EMT is the homeobox (HOX) gene family, master players in regulating embryonic development and maintaining homeostasis through strictly regulated expression in various tissues and organs during adult life. Several studies have demonstrated the association of HOX genes in the pathogenesis of multiple cancers. For example, HOXA7 and HOXD13 have been associated with lung cancer (Lechner et al., 2001), HOXC4 and HOXC8 in prostate cancer (Miller et al., 2003), HOXB7 in ovarian cancer (Naora et al, 2001) and HOXA10 in endometrial cancer (Yoshida et al., 2006). In one study 60% of their breast cancers had no HOXA5 expression (Raman et al., 2000) which causes p53-dependent apoptosis. HOXA5 was reported to cause cell death through the activation of the caspase pathways in HS578T cells expressing mutant p53 (Chen et al., 2004). HOXD10 was extensively reduced as malignancy increased in epithelial cells, and restoring its expression in MDA-MB-231 could significantly reduce the migration capacity of these highly aggressive cells (Carrio et al., 2005). HOXB13 over-expression was associated with increased MCF10A cell motility and invasion *in vitro*, while its ratio to interleukin-17β receptor was predictive of tumour recurrence during adjuvant tamoxifen monotherapy. HOXB7 is involved in tissue remodeling of the normal mammary gland (Ma et al., 2004) and is expressed at higher levels in metastatic breast tumours (Care et al., 1998, 2001). Furthermore, regulation of the expression of several growth and angiogenic factors, including basic FGF, VEGF, IL8, ANG1, ANG2, and MMP9 in SKBR3 breast cancer cells, depends on the over-expressed levels of HOXB7 which can result in the formation of vascularised tumours when grown as xenografts in nude mice. HOXB9 like HOXB7 can lead

that ER negatively regulates LCN2-induced EMT (Yang et al, 2009).

role for these two genes in regulating EMT.

to increased cell motility and EMT (Hayashida et al., 2010).

**4.6 Hypoxia** 

**4.7 HOX genes** 

The appearance of another mesenchymal marker, N-cadherin (CDH12) and/or cadherin-11 (CDH11), in a process termed 'cadherin switching', is also a well documented event in EMT (Gjerdrum et al., 2010; Sarrio et al., 2008; Sphyris and Mani, 2009; Wheelock et al., 2008). The expression of these mesenchymal markers during EMT is induced by SNAIL, ZEB2/SIP1 and SNAIL2 (Cano et al., 2000; Sarrio et al., 2008; Vandewalle et al., 2005). N-cadherin is reported to be highly expressed in invasive and metastatic human breast cancer cell lines and tumours and to correlate with aggressive clinical behaviour. Nevertheless, N-cadherin expression can be triggered in E-cadherin expressing cells and it could in fact cause EMT, impacting on their epithelial phenotype, suggesting a dominating role for this cadherin over the other, possibly in synergy with FGF2 (Hazan et al., 2000, 2004). MCF7 cells that have acquired endocrine independence through induced loss of ER expression also display cadherin switching which is accompanied by increased motility, F-actin cytoskeletal rearrangement and the loss of cellular adhesion molecules. It is suggested that endocrine resistance is a major event influencing the cells to move and invade into the surrounding tissues (Al Saleh, 2010; Al Saleh et al., 2011a).

#### **4.4 Vimentin**

A marker that is commonly used to characterise EMT is vimentin, a component of type III intermediate filaments and the archetypal mesenchymal marker (Trimboli et al., 2008). Elevated vimentin expression correlates well with increased cell migration, invasion and EMT induction in several breast cancer cell lines (Al Saleh, 2010; Al Saleh et al., 2011a; Gilles et al., 2003) in co-ordination with other mesenchymal markers such as tenascin C (Dandachi et al., 2001; Polette et al., 2007), which has been associated with over-expressed ERBB2 and down-regulated ER. The molecular events triggering vimentin expression during EMT are less well delineated in comparision to the mechanisms inducing E-cadherin downregulation. The expression of vimentin is considered to be a late occurrence in EMT in a temporal sequence of genetic events starting from loss of epithelial markers followed by appearance of mesenchymal markers (Polette et al., 2007). Direct activation of vimentin expression in human breast tumour cells (Gilles et al., 2003) by β-catenin/T-cell factor/lymphocyte enhancer factor-1 is consistent with the activation of β-catenin as a downstream event from consequential loss of E-cadherin. The indirect promotion of vimentin expression by ZEB2/SIP1 during EMT in a β-catenin-independent manner (Bindels et al., 2006) suggests the existence of some trans-activators driving EMT which are associated with vimentin expression.

#### **4.5 Matrix metalloproteinases and lipocalin**

In order for cancer cells to metastasise, they need to penetrate into and through the extracellular matrix (ECM). This process is facilitated by the activity of matrix metalloproteinases (MMPs). A family of more than 28 MMPs have been reported to be upregulated in nearly every tumour type and are closely involved in cancer progression through cleavage and release of bioactive molecules that inhibit apoptosis and stimulate cancer invasion and metastasis. For example, treatment of cells with MMP-3 results in an increased expression of the activated splice variant RAC1b, elevating the levels of cellular reactive oxygen species which, in turn, lead to increased expression of SNAIL and EMT initiation (Orlichenko et al., 2008). An MMP-9 associated protein, Lipocalin2 (LCN2), was also found to play a major role in cell regulation, proliferation, differentiation and regulation of EMT. It's over-expression in human breast cancer cells can cause up-regulation of vimentin and fibronectin while E-cadherin is down regulated (Yang et al., 2009). Furthermore, LCN2 over-expression significantly increases cell motility and invasiveness in previously non-invasive MCF-7 cells. Interestingly, siRNA-mediated LCN2 silencing inhibited cell migration and development of the mesenchymal phenotype in aggressive breast cancer cells. It was also reported that reduced expression of ER and increased expression of SNAIL2 was correlated with LCN2 expression while over-expression of ER in LCN-2 expressing cells was able to reverse EMT and reduce SNAIL2 expression, suggesting that ER negatively regulates LCN2-induced EMT (Yang et al, 2009).
