In vitro models of glioblastoma targeting cancer metabolism

2017-05-19T02:30:17Z (GMT) by Wilding, Alexander
Glioblastoma (GBM) is a highly lethal and incurable primary tumour of the brain and central nervous system (CNS). While the genetic alterations in GBM are highly heterogeneous, most tumours exhibit changes promoting activation of RTK signalling and inactivation of Rb and p53 signalling pathways. As with other cancer types, the metabolism of rapidly proliferating tumour cells in GBM is dramatically different to that of their normal, non-proliferating counterparts. The most notable alteration is that of ‘aerobic glycolysis’, the propensity to metabolise glucose to lactate, even in the presence of oxygen. These changes are believed to support the increased proliferation rate and resistance to therapy that typify this and other types of cancer. Our understanding of how metabolic changes support tumour growth is still poor. Nevertheless, the conservation of these changes across diverse tumour types highlights their importance and suggests that cancer metabolism is a potential therapeutic target. In particular PKM2, an isozyme of the glycolytic enzyme pyruvate kinase is highly expressed in most cancers including GBM and appears to facilitate many metabolic features of cancer including aerobic glycolysis. Universal expression of PKM2 in cancer makes it an appealing target for therapy and both activators and inhibitors of PKM2 have been investigated as potential therapeutic options. Using in vitro models, this thesis explores the effects of a number of compounds that target or otherwise modulate cell metabolism. It is hoped that an understanding of how these compounds affect cancer cell metabolism will contribute to the development of novel therapeutic strategies for the treatment of glioma, as well as other cancers. In Chapter 3, a methodology is developed for assaying the anti-proliferative effects of combinations of drugs on glioma cells in vitro, comparing different approaches to assess antagonistic and synergistic drug interactions. In Chapter 4 of this dissertation, the factors that regulate tetramer formation and catalytic activity of PKM2 were examined in glioblastoma cell lines. Novel methods for the detection of PKM2 complex formation, pyruvate kinase activity and glycolytic rate were developed. The ability of a novel PKM2 activator to induce PKM2 tetramer formation and catalytic activity was assesses. We then attempted to exploit the effects of the PKM2 activator to affect cell metabolism in an attempt to identify a therapeutic application of pharmacologic PKM2 activation. In Chapter 5, investigating the effects of TORC1 inhibition using Temsirolimus on PKM2 tetramer formation led to the observation that TORC1 inhibition results in rapid inhibition of glycolysis and a reversal of the Crabtree effect. While the precise target could not be identified, glucose metabolism appeared to be at the level of hexokinase or glucose transport, as both glycolysis and the PPP were affected. We then go on to demonstrate that this block in glucose metabolism induces a striking sensitivity to oxidative stress as would be encountered by glioma cells exposed to IR therapy. In Chapter 6, the multi-kinase inhibitor Sorafenib was identified as exhibiting rapid and potent inhibitory effects on mitochondrial function in glioma cells leading to a reliance on glycolysis for ATP production. Detailed analysis of mitochondrial respiratory function the utility of simultaneous inhibition of respiration and glycolysis using Sorafenib and the glycolysis inhibitor 2-deoxyglucose as a therapeutic strategy is then evaluated in vitro and in vivo.