Targeting autophagy and glycolysis: In vitro and in vivo studies in Glioblastoma. (Doctoral thesis)

The aim of this work was to identify the survival pathways activated in glioblastomas as the result of increased hypoxia after bevacizumab treatment. This thesis is organised into 5 separate chapters. Chapter 1 and 2 represent the introduction to the topics presented in the thesis, chapter 3 describes the aim of the thesis, chapter 4 represents a description of the main experimental work and chapter 5 consists of a general discussion of the thesis. In particular, chapter 1 introduces the characteristics of glioblastoma at the histological and molecular level. We will specifically discuss angiogenesis in glioblastoma, the current anti-angiogenic therapies as well as the potential resistance mechanisms of glioma towards anti-angiogenic therapy. Based on this and earlier work from the laboratory, we postulated that glioma cells use glycolysis and autophagy as survival mechanisms in a hypoxic environment. In chapter 1, we will therefore provide a detailed introduction to the molecular process of autophagy and its role in cancer progression.
Tumor cell metabolism and glycolysis is introduced in chapter 2. This constitutes a review on the topic that we published in 2011 in the journal of Expert Opinion on Therapeutic Targets. Here we also review recent knowledge on the possible tumorigenic mechanism of mutant isocitrate dehydrogenase (IDH) and provide a detailed overview of cancer specific metabolic enzymes associated with glycolysis and intracellular pH regulation. We also discuss the available drugs that may serve as a basis for novel drug development to target metabolic transformation in gliomas. Some of these drugs have been tested in our in vitro and in vivo models of glioblastoma, which are described in chapter 4.
In chapter 3, we state the general aims of the thesis as well as our working hypothesis. Chapter 4 represents our experimental work where we demonstrate the higher glycolytic nature of glioma cells under hypoxia, which is further corroborated by the higher sensitivity of glioma cells towards the glycolysis inhibitors as compared to astrocytes. We will also show that hypoxia strongly induced autophagy in glioma cells, suggesting the potential role of inhibiting autophagy in hypoxic glioma cells both in vitro and in vivo. We further show that primary glioblastoma spheroids are a more appropriate in vitro model for drug testing since we were able to recapitulate the differences of sensitivities of different primary glioblastomas in vivo. Finally we present our in vivo results in xenograft models derived from patient glioblastoma using clotrimazole, chloroquine alone or in combination with bevacizumab. The data presented in this chapter is currently being prepared for publication.
In chapter 5, the general discussion, we conclude and make critical evaluations of our findings compared to current ideas and propose future perspectives of this work. Finally the annexes contain additional unpublished preliminary results related to the topic (Annexes I, II, III). Annex IV is a summary of the previous work of the lab that was published in 2011 (by Keunen et al. 2011) and where I was involved to carry out gene expression analysis of the glioblastoma xenografts in bevacizumab-treated and non-treated samples. This publication laid the basis of the current work. Annex V is a manuscript from the initial project that I was involved in and which is currently being prepared for publication. This work investigates the feasibility of integrating proteomics and transcriptomics data from our glioblastoma xenograft model using gene set enrichment based analysis in order to enhance the biological interpretability of the data relative to a more traditional analysis of the two datasets individually. This work is an extension of my initial project which was based on proteomics and gene expression studies in invasive and angiogenic glioblastoma xenografts carried out in the laboratory (Rajcevic et al. 2009; Sakariassen et al. 2006). I initially focused on the functional validation of novel biomarkers using quantitative PCR and gene knockdown strategies. Since the functional studies of the genes of interest did not show conclusive roles in influencing tumor invasion and/or angiogenesis, I, therefore, moved my focus towards targeting cancer metabolism and autophagy as a potential therapeutic option in the management of glioblastoma.