Improving treatment of glioblastoma: new insights in targeting cancer stem cells effectively

Glioblastoma is the most common primary malignant brain tumour in the adult population. Despite multimodality treatment with surgery, radiotherapy and chemotherapy, outcomes are very poor, with less than 15% of patients alive after two years. Increasing evidence suggests that glioblastoma stem cells (GSCs) are likely to play an important role in the biology of this disease and are involved in treatment resistance and tumour recurrence following standard therapy. My thesis aims to address two main aspects of this research area: 1) optimization of methods to evaluate treatment responses of GSCs and their differentiated counterparts (non-GSCs), with a particular focus on a tissue culture model that resembles more closely the tumoral niche; 2) characterization of cell division and centrosome cycle of GSCs, investigating possible differences between these cells and non-GSCs, that would allow the identification of targets for new therapeutic strategies against glioblastomas. In the first part of my project, I optimized a clonogenic survival assay, to compare sensitivity of GSCs and non-GSCs to various treatments, and I developed the use of a 3-dimentional tissue culture system, that allows analysis of features and radiation responses of these two subpopulations in the presence of specific microenvironmental factors from the tumoral niche. In the second part, I show that GSCs display mitotic spindle abnormalities more frequently than non-GSCs and that they have distinctive features with regards to the centrosome cycle. I also demonstrate that GSCs are more sensitive than non-GSCs to subtle changes in Aurora kinase A activity, which result in a rapid increase in polyploidy and subsequently in senescence, with a consistent reduction in clonogenic survival. Based on these findings, I propose that kinases involved in the centrosome cycle need to be explored as a novel strategy to target GSCs effectively and improve outcomes of glioblastoma patients

Contribution of 3q26-29 gene cluster to glioma invasion

Diffuse gliomas are the most common and lethal brain tumors. Cell invasion into the surrounding brain tissue is a hallmark feature of glioma. Understanding the mechanism of glioma invasion could lead to the discovery of novel therapeutic strategies to treat affected patients. Earlier gene expression analyses on human glioma biopsies showed that SOX2 is associated with glioma invasiveness. The gene for transcription factor SOX2 localizes to 3q26.3 in the human genome amid oncogene PIK3CA and genes regulating mitochondrial fusion, MFN1 and OPA1. Increasing evidence points to a role for 3q26-29 genes in tumor invasion. We hypothesized that SOX2 regulates the 3q26-29 candidate genes as effectors of glioma cell invasion. We used SOX2 expressing human glioma cell lines, LN319 and U373 to test our hypothesis in vitro. Lentiviruses expressing shRNAs against PIK3CA, MFN1, OPA1 or SOX2 were used for genetic knockdown. Engineered cells were assayed for invasion and migration using Boyden chamber and wound healing assays, respectively. Chromatin immuno-precipitation and luciferase assays were used to demonstrate protein-DNA interactions and trans-activation of 3q26-29 genes by SOX2. Our results show that cells downregulated for 3q26-29 genes exhibited enhanced invasion and migration, while shSOX2 and shPIK3CA cells exhibited reduced proliferation rates compared to sh scramble controls. Furthermore, we show that SOX2 knockdown reduced gene and protein expression of PIK3CA, MFN1 and OPA1 except for PIK3CA at the protein level. Chromatin immuno-precipitation assays suggested that SOX2 binds to the upstream region of 3q26-29 gene promoters in the glioma cells. Preliminary luciferase assays in HEK293 cells suggested that SOX2 trans-activates PIK3CA and OPA1. Preliminary immunofluorescence analysis showed that cells knocked-down for 3q26-29 genes demonstrated altered mitochondrial morphology compared to sh scramble controls. Overall, our results show that SOX2, PIK3CA, MFN1 and OPA1 contribute to glioma invasion and that SOX2 is a potential regulator of the 3q26-29 genes

Fishing for cures : the zebrafish as a powerful tool to identify novel therapies against glioblastoma by targeting MTH1 and beyond

Glioblastoma (GBM) is the most aggressive form of brain cancer. Despite today’s combinatory therapy consisting of surgery, radio- and chemotherapy, the prognosis remains dismal. Fostered by extensive tumor heterogeneity, cancer cell plasticity and the presence of cancer stem cells, GBM evades almost any therapeutic strategy, leading to high mortality. Thus, the development of novel therapies is of urgent need. With the identification of the Hallmarks of Cancer several cancer specific characteristics have been described that could serve as promising anti-cancer targets, including the combination of an elevated proliferation rate, crucial changes in cancer metabolism and consequently, an altered redox environment. Cancer cells and GBM in particular depend on effective anti- oxidant defense systems and non-oncogenic addiction enzymes such as MTH1, an enzyme that detoxifies oxidized bases to prevent DNA damage and subsequent cell death. While potential anti-cancer targets are constantly being identified, the development of novel therapies against GBM is, amongst other reasons, hampered by the lack of orthotopic animal models that support large drug discovery screens. During the last decade, the zebrafish has been introduced as a clinically relevant model for human malignancies including cancer. Owing its biological and technical advantages, the zebrafish is the only vertebrate animal suitable for automated drug discovery screens to facilitate the identification and validation of novel cancer therapies. In this thesis, we primarily focused on complementing established biochemical and cellular assays with a broad application of the zebrafish model to: 1. Describe factors that render cancer cells sensitive to MTH1 inhibitors 2. Validate MTH1 as a arget in GBM and GBM stem cells 3. Develop a new orthotopic in vivo model for GBM In Paper I we have demonstrated that the cellular redox environment and activation of the hypoxia signaling axis determine sensitivity to MTH1 inhibition in vitro and in vivo, thus suggesting that MTH1 inhibition may present a promising approach to treat cancers characterized by deregulated hypoxia signaling and redox imbalance. In Paper II we have tested this hypothesis and showed that depletion or inhibition of MTH1 efficiently reduces viability of patient-derived GBM cultures independent of aggressiveness i in vitro and in vivo, thus providing supporting data that MTH1 represents a promising target for GBM therapy in particular. In Paper III we addressed the lack of an orthotopic animal model for GBM which is suitable for large drug discovery screens. We found that GBM cultures transplanted into the blastoderm of zebrafish embryos form a congregated tumor in the central nervous system, fully recapitulating the human disease. As no intracranial transplantation is required, we have developed an orthotopic animal model for GBM that could readily be implemented in fully automatable drug discovery screens in order to accelerate the identification and development of novel therapies against GBM

Proteomics and metabolomics approach in adult and pediatric glioma diagnostics.

The diagnosis of glioma is mainly based on imaging methods that do not distinguish between stage and subtype prior to histopathological analysis. Patients with gliomas are generally diagnosed in the symptomatic stage of the disease. Additionally, healing scar tissue may be mistakenly identified based on magnetic resonance imaging (MRI) as a false positive tumor recurrence in postoperative patients. Current knowledge of molecular alterations underlying gliomagenesis and identification of tumoral biomarkers allow for their use as discriminators of the state of the organism. Moreover, a multiomics approach provides the greatest spectrum and the ability to track physiological changes and can serve as a minimally invasive method for diagnosing asymptomatic gliomas, preceding surgery and allowing for the initiation of prophylactic treatment. It is important to create a vast biomarker library for adults and pediatric patients due to their metabolic differences. This review focuses on the most promising proteomic, metabolomic and lipidomic glioma biomarkers, their pathways, the interactions, and correlations that can be considered characteristic of tumor grade or specific subtype.post-print2427 K

\u3ci\u3e Expression of HCMV IE1 in the U87MG Cell Line Augments Resistance to Temozolomide \u3c/i\u3e

INTRODUCTION: Human cytomegalovirus (HCMV) DNA and protein are found in gliomas but not in normal brain or other primary brain tumors. The role of HCMV infection in glioma biology is unclear. While it is unlikely that HCMV infection causes glioma, viral proteins might impart a proliferative and antiapoptotic phenotype that confers a survival advantage. Does this oncomodulation translate into a clinically relevant effect in glioma cells? To answer this question, we compared the response of the U87IE1 and U87MG malignant glioma cell lines to temozolomide. U87IE1 cells are U87MG cells that have been genetically engineered to produce HCMV IE1 protein. (The U87IE1 cell line is a generous gift from Charles Cobbs.) METHODS: Approximately 5,000 U87IE1 and U87MG cells in normal culture media were placed into wells of a 96-well plate. After 24 hours, the media was replaced with culture media containing temozolomide in increasing concentration. After 48 hours, cell viability was assessed using a luminescent assay. A dose-response curve for each cell line was generated using statistical software. The concentration of temozolomide resulting in 50% of cell death (the EC50 value) for each cell line was determined. Results: The EC50 for temozolomide in the U87MG cell line is 565.6 micromolar, while in the U87IE1 cell line it is 1319 micromolar. This difference is statistically significant (p \u3c 0.0001) and indicates that the U87IE1 cells are more resistant to temozlomide than are the U87MG cells. CONCLUSION: HCMV IE1 expression by U87MG cells enhances their proliferation and survival. In this study, we show that this oncomodulatory effect is clinically relevant: the U87IE1 cell line is more resistant than the U87MG cell line to temozolomide. This finding suggests that HCMV is a viable treatment target for patients with glioma