De novo lipogenese als doelwit voor anti-kanker therapy: een in vitrostudie naar de haalbaarheid en onderliggende mechanismen

Even though significant improvement has been made in the field of cancer treatment over the past decades, many cancers still respond poorly to available antineoplastic therapies or acquire resistance. For these reasons, cancer remains a leading cause of death worldwide. In order to improve the perspectives of cancer patients, unraveling the underlying mechanisms of cancer resistance to current therapies and the identification of new antineoplastic targets is indispensible. Previous studies conducted by our and other research groups have elucidated the de novo fatty acid synthesis pathway as a potential new chemotherapeutic target. Inhibition of this pathway both in vitro and in vivo induces cell death specifically in cancer cells. However, essential questions as to the underlying mechanism of this cell death induction and the relative importance of denovo synthesized versus exogenous fatty acids for cancer cell survival and response to lipogenesis inhibitors remain. In this thesis we tried to get more insight into these questions in order to further explore the real potential of the lipogenesis pathway as target for therapeutic cancer intervention.In a first part of this thesis, we examined the interrelationship between de novo synthesized lipids and exogenous lipids and their relative role in cancer cell proliferation and therapeutic response to lipogenic inhibitors. Contrary to earlier reports that lipogenic activation in cancer cells occurs independently of exogenous lipids, we demonstrated that the lipogenic activity of cancer cells is potently affected by exogenous lipids. In fact, mere lipid restriction, as thought to occur in growing intact tumors because of their reduced access to circulating lipids, is sufficient to activate this pathway. Interestingly, the extent to which cancer cells could activate their lipogenic pathway in these lipid-reduced conditions determined their growth rate and survival under these conditions. Moreover, cancer cells in lipid-restricted conditions, became more dependent on de novo lipid synthesis and consequently more sensitive to lipogenesis inhibitors, like Soraphen A and simvastatin. These data show that limitation of access to exogenous lipids activates the lipogenic pathways in cancer cells; this helps them to survive under these conditions and sensitizes them to lipogenesis inhibitors. Conversely, these in vitro findings imply that exogenous lipids may compromise the therapeutic potential of lipogenic inhibitors and have important implications for the optimization of antineoplastic treatments targeting the lipogenic pathways. In a second part of this thesis, we assessed the underlying mechanism of cell death induction after inhibition of the fatty acid synthesis pathway in cancer cells. We could demonstrate that inhibition of this pathway in lipogenic cancer cells, by the use of the chemical inhibitor Soraphen A, increased the content and poly-unsaturation of the mitochondrial phospholipids, including the cardiolipins. These changes in phospholipid composition made the cancer cell’s mitochondria more sensitive to cardiolipin oxidation by exogenous and endogenous reactive oxygen species (ROS) and enhanced mitochondrial outer membrane permeabilization by truncated Bid (tBid). Importantly, this SoraphenA-induced increase in mitochondrial sensitivity could be exploited in combination with other cell death inducers that induced ROS or tBid, likerotenone or TRAIL. Moreover, reversal of Soraphen A-induced phospholipid poly-unsaturation by supplementation of the culture media with saturated and mono-unsaturated fatty acids, reversed the sensitizing effect of Soraphen A on the mitochondria and the cancer cell’s susceptibility to cell death inducers. Together these data implicate mitochondrial phospholipid poly-unsaturation in the mechanism by which lipogenesis inhibition sensitizes cancer cells to cell death induction and show the utility of lipogenic inhibitors as anti-cancer agents.Taken together, the findings presented in this thesis provide significant novel insights into howthe lipogenic pathway may serve as a potential target for antineoplastic intervention, as mono-therapy or in combination with other chemotherapeutic agents that trigger the mitochondrial pathway of apoptosis. Our data also imply that to reach maximal therapeutic effects with lipogenesisinhibitors, additional measures might be needed to diminish the exchange of lipids between the cancer cells and their environment.List of abbreviations III CHAPTER 1: General introduction 1.1 Cancer 1 1.2 Altered metabolism in cancer cells 1 1.2.1 Lipid metabolism 3 1.2.1.1 Fatty acids and cholesterol 3 1.2.1.2 Uptake of fatty acids and cholesterol 5 1.2.1.3 De novo lipid synthesis 6 1.2.1.3.1 De novo fatty acid synthesis 6 1.2.1.3.2 De novo cholesterol synthesis 8 1.2.1.4 Regulation of de novo lipid synthesis 10 1.2.4.1 Regulation of de novo fatty acid synthesis 10 1.2.4.2 Regulation of de novo cholesterol synthesis 13 1.2.2 Dysregulation of lipid metabolism in cancer cells 14 1.2.2.1 Dysregulation of fatty acid metabolism 14 1.2.2.2 Dysregulation of cholesterol metabolism 18 1.2.3 Lipid metabolism as targets for antineoplastic therapy 18 1.3 Membranes 21 1.3.1 Structure and composition of membranes 21 1.3.2 Phospholipids metabolism and transport 24 1.3.2.1 Phospholipid synthesis 24 1.3.2.2 Phospholipid inter-organelle transport 28 1.3.2.3 Phospholipid fatty acyl chain remodeling 28 1.4 Mitochondria 31 1.4.1 Apoptotic cell death 31 1.4.2 Mitochondrial membranes in apoptotic cell death 33 1.5 Aims and objectives 39 CHAPTER 2: Cancer cells differentially activate and thrive on de novo lipid synthesis pathways in a low-lipid environment Abstract 43 2.1. Introduction 44 2.2. Materials and methods 45 2.3. Results 49 2.4. Discussion 60 Acknowledgements 64 References 64 Supplementary information 67 CHAPTER 3: Targeting de novo lipogenesis in cancer cells increases their susceptibility to cell death stimuli by modulation of mitochondrial cardiolipin saturation Abstract 77 3.1. Introduction 78 3.2. Materials and methods 79 3.3. Results 83 3.4. Discussion 95 Acknowledgements 98 References 98 Supplementary information 102 CHAPTER 4: General discussion and perspectives 111 4.1 Cancer cells differentially activate and thrive on de novo lipid synthesis pathways in a low-lipid environment 113 4.2 Targeting de novo lipogenesis in cancer cells increases their susceptibility to cell death stimuli by modulation of mitochondrial cardiolipin saturation 117 Summary 125 Samenvatting 127 References general introduction and general discussion 131 Curriculum Vitaenrpages: 140status: publishe