**4.6 Hypoxia**

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

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

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

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

tissues (Al Saleh, 2010; Al Saleh et al., 2011a).

associated with vimentin expression.

**4.5 Matrix metalloproteinases and lipocalin** 

**4.4 Vimentin** 

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 role for these two genes in regulating EMT.
