**4.1 Transforming Growth Factor β**

460 Breast Cancer – Focusing Tumor Microenvironment, Stem Cells and Metastasis

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

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 neo-

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

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

diverse cellular insults which lead to transformation into a cell type that displays

sometimes alpha smooth muscle actin (ACTA2) along with many other changes.

vascularisation.

during EMT.

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

Endocrine Resistance and Epithelial Mesenchymal Transition in Breast Cancer 463

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.

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 trans-differentiation of epithelial cells.

### **4.2 AXL**

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