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1.6.1.6. EMT, Cancer Invasiveness, Drug resistance and TG2

In normal physiology, Epithelial-Mesenchymal Transition (EMT) is a dynamic and essential process required in reprogramming epithelial cells during embryonic development. However persistent reactivation of EMT in adulthood is associated with various pathologic conditions including cancer and fibrosis (Kumar and Mehta 2013, Nyabam 2016). Recent studies have implicated EMT as the first necessary step in metastatic dissemination and tumour progression. Cells undergoing EMT acquire the ability to degrade the basement membrane as a result of increased activity of matrix metalloproteases (such as MMP2, MMP3, and MMP9); and these cells migrate through the ECM to populate different areas during cancer progression (Kumar and Mehta, 2013). Ilyas et al., (1997) suggests that EMT is essential for the development of drug-resistant phenotypes in cancer cells, and enables them to become invasive at the early stage of the disease.

EMT can be induced by a variety of different stimuli (growth factors / cytokines) including Hepatocyte Growth Factor (HGF), Transforming Growth Factor (TGFβ), Platelet-Derived Growth Factor Receptor (PDGFR), Wnt and Hedgehog (Christiansen and Rajasekaran, 2006). TGFβ is a major inducer of EMT, resulting in suppressed expression of various epithelial proteins such as E-cadherin and enhanced mesenchymal proteins such as fibronectin and vimentin. TGFβ operates as both a tumour suppressor and promoter by acting via Smad (e.g. Smad 2 and 4) or non-Smad (e.g. c-Myc, PI3-kinase and MAPK) pathways (Morris et al., 2010). Key targets of TGFβ signalling are the transcriptional repressors of E- cadherin; upregulation of Snail, Slug and Twist, Zeb-1 and Zeb-2. Wnt signalling is also a key regulator of EMT during cancer progression. The canonical catenin-dependent Wnt signalling pathway results in the nuclear accumulation of -catenin. This causes transcriptional activation of target genes, involved in EMT, through the lymphoid enhancer factor 1 (LEF-1)/TCF transcription factors. These include vimentin and fibronectin, which are both markers of a mesenchymal morphology (Nawshad et al., 2007).

1.6.1.6.2 Key Alterations and Factors That Govern EMT

A number of key alterations occur during EMT, and several markers have been identified, that distinguish epithelial and mesenchymal cells (Figure 1.7). During EMT, the polarized epithelial cells, which normally interact with the basement membrane via its basal surface, undergo multiple biochemical changes that enable them to acquire a mesenchymal phenotype. Initially the perturbation of cellular junctions is required. This requires the loss of adherens junctions, loss of tight junctions, gap junctions and desmosomes (Voulgari and Pintzas, 2009). The ‘cadherin switch’ where epithelial cells downregulate E-cadherin and up-regulate N-cadherin is often associated with EMT. This results in the breakdown, or loosening, of cell-cell contacts and the cells adopt a more migratory phenotype due to the pro-migratory role of N-cadherin (Wheelock et al., 2008). Changes in the cytoskeleton is also common to promote the necessary structural machinery for cellular migration. Another key marker of EMT in this regard, is vimentin, an intermediate filament family member which is commonly used as a mesenchymal cell marker. In order to detach from the ECM and initiate migration or invasion during EMT, cells require matrix metalloproteinases- a set of enzymes that cleave structural proteins of the ECM enable degradation of the basement membrane. MMPs, specifically MMP2 and MMP9 are associated with mesenchymal cells and are upregulated in EMT. For example, MMP activities enable the degradation of the basement membrane and ECM to allow the more motile and invasive N-cadherin expressing mesenchymal-like colon cancer cells to infiltrate into the surrounding tissues and body circulatory systems (Wheelock et al., 2008, Curran and Murray, 2000). The transcriptional control of EMT is maintained by Zeb-1, Zeb-2, Twist1 Snail, and Slug which effectively turn on the EMT process (Agnihotri et al., 2013).

Figure 1.7 Cellular changes during EMT. Epithelial cells lose cell polarity, epithelial cell tight junctions with significant cytoskeletal reorganisation. The TGF, Wnt and Notch signalling process play a role in activating Snail, Slug and Zeb 1/2-transcription factors that regulate EMT. (Modified from Aroeira et al., 2007).

1.6.6.3.3 The Role of TG2 in EMT

TG2 as a proinflammatory protein has been shown in certain cancer and fibrotic models to induce promote EMT (Agnihotri et al., 2013, Eckert et al., 2014, Ku et al., 2014, Condello et al., 2013, Nyabam et al., 2016, Verderio et al., 2004), although, other studies also report that TG2 role in the ECM may perturb EMT (Kotsakis and Griffin 2007). In general TG2-mediated EMT in cancer cells have been reported to also confer metastasis , invasiveness, drug resistance, apoptotic resistance and a tumorigenic phenotype (Budillon et al., 2013). In breast cancer, Mehta and colleagues found that that Stable expression of TG2 in mammary epithelial cells resulted in the loss of epithelial markers (E- cadherin) and gain of mesenchymal markers (vimentin, fibronectin, N-cadherin, e.t.c). More so TG2-expression exhibited a significant increase in Snail1, Twist1, and Zeb1 transcription repressors which was

accompanied by increased invasiveness and the ability to form colonies in agarose (Agnihotri et al., 2013). Similar findings have been reported in the breast (Kumar et al., 2010a, Morin et al., 1997), Squamous (Fisher et al., 2015a), prostrate, and ovarian (Cao et al., 2008) cancer. The inhibitory role of TG2 in EMT in certain cell context has been shown with cell surface TG2 which modulates cell migration in an ECM dependent way and proteolytic degradation of cell surface TG2 by membrane- type matrix metalloproteinases (MT-MMPs) in gliomas and fibrosarcomas, resulting in increased cellular motility of tumour cells on FN (Kotsakis and Griffin 2007). Taken together TG2 may play an important role in EMT in cancer cells either by enhancing cell signalling processes that promote EMT in on the one hand or inhibiting cell migration by crosslinking the matrix.

1.6.1.6.4 TG2 enhances autophagy resistance in Tumour cells

Studies by Akar et al., (2007) have identified a potential new role of TG2 in tumour cells autophagy resistance. It was demonstrated that protein kinase C-delta (PKCδ) constitutively protected cells from autophagy by up-regulating TG2 expression in pancreatic cancer cells and significant autophagic cell death was observed following downregulation of TG2 by siRNA or by PKCδ inhibition.

Figure 1.8. TG2 is involved in multiple cellular mechanisms that drive inflammation and EMT in fibrosis and cancer. TG2 interacts with fibronectin and induces integrin clustering, which leads to activation of Src, PIP3 and downstream activation of NFκB via phosphorylation of AKT. Extracellular TG2, via interaction with the ECM, can induce mechanical sheer stress that releases TGFβ from its latent TGFβ complex. Canonical TGFβ operates via Smad2/3 and activates TGFβ activated kinase 1 (TAK1), which in turn activates NFκB. Intracellular TG2 can bind NFκB, thus preventing its interaction with NFκB inhibitory unit (IKBα). Activated NFκB can translocate into the nucleus where it upregulates inflammation, EMT, or induces TG2 by binding to the TGM2 promoter. Alternatively, intracellular TG2 can complex NFκB and TG2 facilitates the translocation of the complex into the nucleus to induce Hypoxia inducible factor 1 alpha (HIF 1α). Non-canonical TGFβ signalling activates extracellular kinase (ERK) which induces c Fos and proliferation. Canonical TGFβ signalling activates Smad4 which also induces TG2 expression by binding to the TGM2 promoter site. Extracellular TG2 interaction with fibronectin and integrin activates Src, which inhibits proteasomal degradation of β-catenin. In addition, extracellular TG2 interaction with LRP5/6, a co-receptor with frizzled in canonical Wnt signalling facilitates β-catenin stability and nuclear translocation and β-catenin acts as a transcription factor for cell cycle regulators and EMT.

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