Investigating Sphingolipid Metabolism in Glioblastoma

Abstract

Glioblastoma (GBM), a genetically heterogeneous disease, has a significant burden on our society. Currently, the standard treatment for newly diagnosed GBM patients consists of surgery followed by concomitant radiotherapy and temozolomide chemotherapy, resulting in a median survival of 12-15 months. Targeted therapies are being developed to inhibit oncogenes based upon GBM molecular profiling, though have not been as successful as expected. Our understanding of DNA and RNA alterations in GBM has grown considerably over the past few years. However, our understanding of the lipid biology, specifically sphingolipids, in GBM is lagging and may prove useful in the arsenal of targeted therapies. The sphingolipid pathway contains lipid signalling molecules, which modulate cellular survival through the balance of ceramide, a pro-apoptotic metabolite, and Sphingosine-1-Phosphate (S1P), a pro-survival metabolite. Herein, I characterise for the first time the sphingolipid profile of normal grey matter (NGM), diffuse astrocytomas (AII), anaplastic astrocytomas (AIII), and GBM using liquid chromatography tandem mass spectrometry. The lipid profile is supported by an enzyme expression profile favouring ceramide catabolism and S1P formation, including upregulation of acid ceramidase (ASAH1) and sphingosine kinase 1 (SPHK1), and a down regulation of S1P phosphatase 2 (SGPP2). Significantly, C18 ceramide was reduced 5-fold in GBM compared to NGM, while S1P was increased in GBM by approximately 9-fold compared to NGM. Based on the sphingolipid profiles, ASAH1 and SPHK1 were assessed for functional relevance in vitro. Using gene silencing and pharmacological inhibition, I found SPHK1 to be critical for U87MG-induced angiogenesis through S1P paracrine signalling, which was independent of VEGF levels. EGFR mutations were associated with increased C16 and C22 ceramide levels. For the first time, I measured sphingolipid metabolites in plasma extracted from GBM patients and healthy controls. Elevated levels of S1P were found in GBM plasma and together with tumour S1P levels, were associated with a poor survival outcome. In contrast low S1P levels in tissue combined with MGMT methylation was associated with a good survival outcome. Overall, the data presented in this thesis reaffirm the importance of sphingolipid metabolism in GBM biology, reflected by a shift in the ceramide-S1P balance, favouring the pro-angiogenic S1P. Additionally, sphingolipid interactions with altered genetic pathways and potential biomarker capacity are novel findings that require further validation, with the hope of informing and monitoring therapeutic responses for GBM patients.