Targeting cancer metabolism

Abstract

The Warburg effect occurs in 90% of tumors, where glycolysis is favored despite the presence of oxygen. The activity of pyruvate dehydrogenase kinases (PDKs) is increased in cancer cells, suppressing glucose oxidation. Inhibition of PDKs can redirect glucose flux into the mitochondria, reversing the Warburg effect. Dichloroacetate (DCA) is a relatively non-toxic PDK inhibitor that inhibits all four isoforms of PDK with differing potency. We examined the determinants of DCA sensitivity, the ability of DCA to enhance apoptosis, and how DCA regulates cell metabolism. We found in a range of cancer types, that all four PDK isoforms can be expressed and contribute to tumor metabolism. DCA inhibited cancer cell growth in vivo and in vitro, activated PDH and reduced lactate production. The magnitude of DCA growth inhibition correlated with the PDK expression profiles of the cells, with PDK3 (highest Ki for DCA) conferring low sensitivity towards DCA. PDK2 siRNA-knockdown inhibited growth to a similar extent to DCA, whilst PDK3 knockdown significantly increased sensitivity to DCA, confirming sensitivity to DCA is determined by PDK expression. Further examination of PDKs in patient samples will increase the likelihood of DCA being successfully translated into clinical use, with the PDK expression profile being a biomarker for sensitivity to DCA. The mechanisms by which DCA can sensitize cancer cells towards apoptosis were investigated, as we observed that DCA increased hypoxia-induced apoptosis. DCA enhanced the effects of 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid (PENAO; a novel anti-mitochondrial agent) in vitro. DCA increased ROS levels, which were fully responsible for enhancing PENAO apoptosis in MDA-MB-231 cells, but only partially in T-47D cells. The two cell lines were metabolically distinct, potentially explaining the different mechanisms by which DCA enhanced apoptosis. PDK knockdown experiments revealed that DCA could enhance apoptosis via off-target effects. The pro-apoptotic proteins Noxa and Puma were investigated, however DCA did not alter their expression. DCA however depolarized the mitochondrial membrane potential in T-47D cells, suggesting that this is an additional mechanism of DCA apoptosis enhancement in these cells. Nevertheless, DCA sensitized all cancer cell lines tested towards apoptosis. Thus DCA has potential to be used in combination with other cytotoxic agents in order to reduce their adverse effects. To understand the mechanism of action, the metabolic effects of DCA on cancer cells were investigated. Gas chromatography-mass spectrometry metabolomics revealed a general trend of increase in the proportion of anabolic metabolites upon DCA treatment. Furthermore, long-term DCA treatment resulted in DCA-resistance, which was accompanied by correlating changes in the PDK expression profile. These findings thus open avenues of further exploration on the mechanism of DCA action and resistance factors. Our findings provide potential biomarkers for sensitivity to DCA, and evidence that DCA can be combined with other therapies for enhanced anti-cancer effects. We have opened future directions of identifying DCA-resistance factors. This will allow using drugs in combination with DCA that specifically target these resistance factors. Collectively our work will allow DCA to have greater success in clinical trials by being able to target patients most likely to respond.