Surgery (RMH) - Theses
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ItemThe role of quiescence in treatment resistance and malignancy in glioblastoma multiforme( 2018)Glioblastoma multiforme (GBM) represents the most malignant incarnation of glial tumours – a World Health Organisation (WHO) grade IV brain malignancy. GBM is the most common primary brain tumour in adults, accounting for 78% of all malignant central nervous system (CNS) tumours, and affecting 2-3 people per 100,000 in Europe and North America, with an average survival of only 14.6 months. Despite continued research and incremental advances in imaging, surgery, and chemoradiotherapy, patient survival has stagnated in the past decade, with several promising lines of investigation failing to fully deliver on their anticipated translational outcomes. Recent advances in genetic sequencing and computational biology have allowed the simultaneous comparison of large numbers of patient cancer cell genomes and identified several GBM subtypes. It is hoped that such stratification will one day allow clinicians to tailor treatments specific to each GBM subtype as has already happened in cancers like medulloblastoma. However, despite best efforts, GBM remains highly aggressive, infiltrative, and treatment-resistant, rendering it incurable by current treatment modalities. Invasion of tumour cells into normal brain prohibits a surgical cure, while a high cancer stem cell (CSC) component resists treatment with radiation and temozolomide (TMZ) – both of which are more effective against rapidly dividing cells – and relapse remains the rule. Molecular mechanisms underlie GBM’s treatment resistance, and elucidating the key drivers that garner inherent resistance to the quiescent, stem-like fraction of cells that lead to treatment failure therefore presents as an exciting area of research that may uncover new potential drug targets that improve patient outcomes. This study has shown that the proliferation rate of GBM cells is spectral, approximating a positively skewed normal distribution, with highly proliferative cells at one end and quiescent cells at the other. The quiescent cell fraction was subsequently shown to be inherently more resistant to chemoradiotherapy than the proliferative fraction. The quiescent fraction also displayed increased size, complexity, rates of migration and invasion, secretion of extracellular matrix-degrading enzymes, and invadopodia activity than their proliferative counterparts. Similarly, quiescent cells proliferated slower as intracranial tumours but displayed significantly greater invasive properties than a subset of proliferative cells grown in vivo. mRNA expression analysis revealed the genetic signature that underpins the disparity in proliferation rate between quiescent and proliferative cells, and the putative genes that are responsible for the malignant properties identified in both populations. This body of work has uncovered the inherently dichotomous treatment response of quiescent and rapidly dividing GBM cells, as well as the difference in their abilities to migrate and invade. This study has also shed light on the fundamental molecular mechanisms that are at the root of treatment resistance and malignancy in this disease. It is hoped that this expression signature will help to inform future studies and treatments that target these differences and make GBM less of a death sentence and more of manageable, chronic disease.
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ItemDeveloping novel therapies using glioma stem cells( 2016)Glioblastoma multiforme (GBM) is a heterogeneous and malignant brain tumour with poor survival. Despite current best therapies most patients die within 15 months of diagnosis. These aggressive tumours are thought to recur after initial therapy due to their infiltrative nature. GBM recurrence and heterogeneity is also thought to be due to glioma stem cells (GSC). A panel of patient-derived cell lines (PDCL) isolated at The Royal Melbourne Hospital were previously subjected to in vitro analysis to confirm self-renewal and differentiation capacity. The stemness of the PDCLs was further evaluated with elevated mRNA expression of stem cell markers (nestin, SOX2, Oct4 and prominin-1) identified. The stem-like PDCLs were then tagged with luciferase recapitulated in vivo bioluminescent tumours within the brains of balb-c nude mice. The epidermal growth factor receptor (EGFR) pathway and its downstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signalling are often dysregulated in GBM and have been shown to be involved in cell proliferation, survival and migration. A selection of patient-derived glioma stem cells (GSC) were chosen for their mutant expression of EGFR, PI3K and PTEN proteins. The expression of the mutations was assessed as being stable over multiple passages in cell culture. The efficacy of PI3K inhibition was then assessed (in vitro and in vivo), but irrespective of the EGFR and/or PTEN status, standalone PI3K inhibition lacked cytotoxicity in GSC despite demonstrating target inhibition of phospho-Akt. Complex interactions within and between signalling pathways exist. Lysophosphatidic acid (LPA) signalling has been reported to interact with PI3K signalling and has been shown to stimulate migration and invasion. LPA signalling components have also been shown to be dysregulated in GBM and our GSC demonstrate a stable increase in mRNA expression of LPA receptor 1 (LPAR1). GSC expressing elevated levels of LPAR1 mRNA migrated in response to stimulation with LPA and this response was ameliorated using a selective LPA1/3 receptor antagonist (Ki16425). LPA appeared to stimulate migration in the GSC via a combination of PI3K and ROCK signalling. This interaction between LPA and PI3K signalling may help explain treatment resistance to PI3K inhibitors and future studies investigating the combined use of EGFR/PI3K and LPA inhibitors may be promising.