Condición de análisis en relación a la Ley 20

GBM is one such malignancy in which hypoxic regions, hypoxia-induced necrosis and neovascularization are very common and are thought to play an important role in pathological diagnosis. Indeed hypoxic necrotic foci associated with pseudopalasading cells is a WHO recommended classification feature for GBM. The physiological O2 concentration in the brain ranges from 0.5% to 7% (Bar et al., 2010). Using the method of EF5 binding and Eppendorf needle electrodes, Evans and colleagues measured O2 levels in gliomas. They noted that the hypoxia levels varied drastically between patients and they grouped them as mild hypoxia (0.5% to 2.5% O2), moderate hypoxia (0.1% to 0.5% O2) and severe hypoxia/anoxia (0.1% O2 or less). They noted that the patients with moderate hypoxia had reduced survival while the patients with severe hypoxic gliomas had the worst outcome among all (Evans et al., 2008). Many other studies also showed that increased levels of hypoxic regions are associated with invasive, resistant phenotype of GBM that has very poor patient survival (Sathornsumetee et al., 2008; Vaupel and Mayer, 2007).

Although hypoxia or microenvironment induced EMT is considered as a major modulator of metastasis formation in many epithelial solid tumours, there are only very few reports on EMT process occurring in neuroepithelial environment or in CNS tumours. In spite of being extensively hypoxygenated tumours, there is no robust evidence for the existence of EMT process or its role in invasion or progression in the case of GBMs. Recent analysis of The Cancer Genome Atlas (TCGA) data by Cheng and colleagues, demonstrated that GBM has a strong

143 association between an EMT expression signature and the expression of the CSC markers and early recurrence following initial treatment (Cheng et al., 2012). The activity of EMT transcription factors like SNAI1, TWIST1, ZEB2 are reported to be significantly elevated in GBM tissue compared to normal brain and positively correlate with tumour grading. These TFs are thought to promote invasion and proliferation in GBM by modulating genes such as MMPs, HGF, FGF, Wnt and -catenin (Yang et al., 2010; Mikheeva et al., 2010). These TFs are known to suppress the epithelial marker E-cadherin and in parallel increase the mesenchymal markers like N-cadherin, vimentin and cadherin-11. But E- cadherin expression is very rare in both normal and glioma tissues of the brain (Motta et al., 2008). On the other hand, N-cadherin and cadherin-11 are frequently expressed at a low level in normal brain tissue and their expression is elevated in malignant gliomas (Asano et al., 2004). The loss of E-cadherin and its correlation with glioma‟s grade was reported in only two studies and therefore there is no definitive evidence for classical cadherin switch in malignant gliomas, except in some specific cases (Motta et al., 2008; Lewis- Tuffin et al., 2010). However, Lu and colleagues recently reported an alternative cadherin switch in GBMS, different from the classic E-cadherin-to-N-cadherin switch (Lu et al., 2012). They observed that another atypical member of the cadherin family called T-cadherin was downregulated when Snail and N- cadherin increased in GBM cells. T-cadherin expression is absent in most high grade gliomas including invasive GBMs and is also associated with a poorer prognosis (Andreeva and Kutuzov, 2010). Zeb-1 has been shown to suppress T-cadherin to aid the acquisition of EMT phenotype and it has been

144 demonstrated that ectopic overexpression of T-cadherin in GBM cells inhibits proliferation, migration and invasion (Huang et al., 2003).

It is becoming increasingly evident that EMT plays an important role in regulating GBM CSCs, GBM invasion and its intrinsic resistance but a large part of it remains unexplored due to complex feedback loop systems and missing links between them. It is likely that the elevation of mesenchymal signature genes in GBM could be due to an ongoing EMT process due to hypoxia or could be the reminiscent of an earlier mesenchymal shift during gliomagenesis. Due to the remarkable comprehensive analysis of TCGA data by Philips and colleagues, the WHO classification system has added the mesenchymal like GBM as another subtype of GBM owing to the solid evidences of EMT in GBM (Phillips et al., 2006; Louis et al., 2007). Given the established role in progression of other hypoxygenated solid tumours, signalling pathways like NF- κB, HIFs, notch, hedgehog-GLI, WNT/β-catenin, EGFR, RTKs and PI3K-AKT may play important roles in hypoxia induced EMT program of GBM. All these facts together indicate that GBM share several features with EMT but more research and perfect evidence are required to propose this as a therapeutic target in GBM.

4.1.4 Hypoxia induced EMT and CSCs

It is interesting to know that hypoxia induced EMT mesenchymal cells carry molecular characteristics similar to progenitor or stem cells and are thought to initiate or propagate tumour acting as seeds for relapse of tumour after therapy.

145 Multiple evidence from researchers working on various solid tumours argue that the CSC phenotypes inside a tumour are probably these EMT induced mesenchymal cells. EMT phenotypes are often confused with cancer initiating mutated progenitor cells and hence are referred to as tumour initiating cells or cancer stem like cells. Joo et al., (2008) showed that a mesenchymal subtype genetic signature was seen specifically in CD133-negative GBM CSCs when compared with CD133-positive GBM CSCs and they initiated tumours with an invasive growth. Similarly, Lottaz et al., (2010) demonstrated that GBM CSCs which are positive for the EMT marker CD44 carry a mesenchymal gene signature and produced enhanced invasive growth in vitro and in vivo compared with CD44 negative cells regardless of CD133 expression.

Many researchers still believe that a mutated progenitor cell could become a CSC and establish a tumour, while other believe that the EMT phenotypes are resistant stem like population or metastasis initiating cells induced by microenvironment and may not be the actual CSCs that initiated the tumours. Regardless of whether CSCs arise from progenitors or are induced by an EMT program, it is well established that hypoxia supports the survival and maintenance of these CSCs. A hypoxic niche is well known for its role in maintaining normal NSCs in the CNS. This raises the possibility that if CSCs are present in GBM, the intra-tumoural hypoxia provides the hypoxic niche for these cells along with cytokines and growth factors from the tumour stroma to maintain their undifferentiated state (Panchision, 2009).

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