**4. Epithelial mesenchymal transition**

The phenomenon of epithelial cells undergoing a transition towards a mesenchymal phenotype was first identified as programmed events occurring during embryonic developmental processes (Greenberg & Hay, 1982). Since then EMT has since been described in various pathological conditions. During the process of cancer metastasis, a minority of

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 461

The transformation of epithelial cells into a mesenchymal-like form requires the participation of a complex network of both extra- and intra-cellular signals., Amongst the many identified are TGFβ, HGF, FGF, EGFR family members, IGF1 and 2, and PDGF (Thiery et al., 2002). An array of embryonic transcription factors such as the homeobox protein GOOSECOID (GSC), TCF3 (E47), the zinc-nger proteins SNAIL1 and SNAIL2 (previously SLUG), the basic helix-loop-helix protein TWIST1, the forkhead box proteins FOXC1 and FOXC2 , and the zinc-nger E-box-binding proteins ZEB1 and ZEB2 (SIP1) , are generated by the activity of these growth factor pathways, each of which is capable, on its

There is increasing evidence of extensive crosstalk between these molecules, permitting the formation of an extensive signalling network responsible for establishing and maintaining a mesenchymal phenotype. (Moreno-Bueno et al., 2008; Peinado et al., 2007). In addition, some of these transcriptional activators such as TWIST are pivotal factors in overcoming cellular senescence (Ansieau et al., 2008) and in generating tumourigenic cancer stem cells (Mani et al., 2008). Interestingly, EMT-inducing transcription factors also confer stem cell characteristics on epithelial cells. For example, the receptor KIT which is an important factor for maintaining the stem cell state in the haematopoietic system has been shown to induce SNAIL2 expression in both mice (Perez-Losada et al., 2002) and humans (Sanchez-Martin et al., 2002). Many of these transcription factors exert repressive functions by binding to conserved E-box sequences in the promoter regions of such critical genes as CDHI (Gilles et

TGFβ can independently promote an EMT phenotype in mouse mammary epithelial cells (Thuault et al., 2006; Waerner et al., 2006). This cytokine induces EMT by both SMADdependent and independent signalling events (Berx et al., 2007; Das et al, 2009; Santisteban et al., 2009). In advanced disease, TGF-β can stimulate invasion and metastasis of tumours that have become TGF-β insensitive which can be inhibited by ectopic expression of dominant negative TGF-β receptors (Ozdamar et al., 2005). TGF-β1 ligand activates a heteromeric receptor of two transmembrane serine/threonine kinases, type I and II receptors (TβRI and TβRII). TβRII transphosphorylates TβRI, activating its kinase function to exert its signalling effects. Activated TβRI phosphorylates the intracellular proteins SMAD 2 and 3 which then associate with SMAD 4, translocating to the nucleus where the complex interacts with other transcriptional co-activators and co-repressors to regulate expression of several genes (Onder et al., 2008). This type of signalling that depends on SMAD, up-regulates the expression of many transcription factors such as SNAIL1, SNAIL2, TWIST, and members of the ZFH family, ZEB1 and ZEB2 (Sarrio et al., 2008; Vandewalle et al., 2005; Yang et al., 2004) that are considered to be primary transcriptional inducers of EMT. TGFβ can also phosphorylate certain cytoplasmic proteins regulating cell polarity and tight junction formation. These include RAS/MAPK (Xue et al., 2003), integrin β-1 (Blanco et al., 2002), integrin-linked kinase (Hartwell et al., 2006), p38 MAPK (Mani et al., 2007), RHOA kinase (ROCK) (Moody et al., 2005), PI3K (Martin et al., 2003), JAGGED1/NOTCH (Come et al., 2006), SARA (Laffin et al., 2008), NFKB (Lester et al., 2007), PAR6 (Berx et al., 2001; Storci et al., 2008), pAR66A and ERK (Wu et al., 2009). Furthermore, EMT induced by the oncogenic stimulation by RAS and/or RAF activation in mammary, kidney and skin epithelial tissue was found to depend almost completely on TGF-β signaling (Moustakas and Heldin, 2009). TGFβ can also induce the activation of other signalling pathways that

own, of inducing an EMT.

al., 2003; Pieper et al., 1992).

**4.1 Transforming Growth Factor β**

epithelial cells lose their apico-basal polarity, detach from adjacent cells, scatter and acquire increased motility and are able to invade into the extracellular matrix with subsequent penetration into the vasculature. This process is facilitated by a morphological transformation into a broblastoid structure that has all the hallmark features of EMT, Both processes share remarkable similarities, with characteristic phenotypic changes. These include the loss of cell-cell adhesion as a result of reduced E-cadherin in adherens junctions, occludins (OCLN) and claudins (CLDN) in tight junctions and desmoplakin (DSP) in desmosomes and down regulation of epithelial cytokeratins (KRT8, KRT18, and KRT19) and up-regulation of mesenchymal proteins most notably vimentin (VIM) and fibronectin and sometimes alpha smooth muscle actin (ACTA2) along with many other changes.

Fig 3 depicts the changes occurring during EMT. Multiple molecular mechanisms underlie EMT initiation and its reversal process, MET, which cancer cells are thought to undergo at sites where they form metastases, in order to re-establish cohesive colonies and initiate neovascularisation.

Fig. 3. Epithelial to mesenchymal transition. Loss of epithelial characteristics and breakdown of tissue architecture through dissolution of cell-cell junctions and loss of apico-basal polarity by detachment from the basement membrane can be initiated through a variety of diverse cellular insults which lead to transformation into a cell type that displays mesenchymal–like features. 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.

The transformation of epithelial cells into a mesenchymal-like form requires the participation of a complex network of both extra- and intra-cellular signals., Amongst the many identified are TGFβ, HGF, FGF, EGFR family members, IGF1 and 2, and PDGF (Thiery et al., 2002). An array of embryonic transcription factors such as the homeobox protein GOOSECOID (GSC), TCF3 (E47), the zinc-nger proteins SNAIL1 and SNAIL2 (previously SLUG), the basic helix-loop-helix protein TWIST1, the forkhead box proteins FOXC1 and FOXC2 , and the zinc-nger E-box-binding proteins ZEB1 and ZEB2 (SIP1) , are generated by the activity of these growth factor pathways, each of which is capable, on its own, of inducing an EMT.

There is increasing evidence of extensive crosstalk between these molecules, permitting the formation of an extensive signalling network responsible for establishing and maintaining a mesenchymal phenotype. (Moreno-Bueno et al., 2008; Peinado et al., 2007). In addition, some of these transcriptional activators such as TWIST are pivotal factors in overcoming cellular senescence (Ansieau et al., 2008) and in generating tumourigenic cancer stem cells (Mani et al., 2008). Interestingly, EMT-inducing transcription factors also confer stem cell characteristics on epithelial cells. For example, the receptor KIT which is an important factor for maintaining the stem cell state in the haematopoietic system has been shown to induce SNAIL2 expression in both mice (Perez-Losada et al., 2002) and humans (Sanchez-Martin et al., 2002). Many of these transcription factors exert repressive functions by binding to conserved E-box sequences in the promoter regions of such critical genes as CDHI (Gilles et al., 2003; Pieper et al., 1992).
