Medicine (St Vincent's) - Theses
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ItemUnderstanding and manipulating epigenetic deregulations in osteosarcoma( 2016)Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer related death in young adults. Current 5-year survival rates have plateaued at ~70% for patients with localised disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS translating into the identification of effective therapeutic targets may yield new approaches to improve outcomes for OS patients. To this end, I applied previously described murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNA) and epigenetic modulators in OS. miRNA and epigenetic modulator expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross species comparison to human osteoblasts and osteosarcoma cultures. This led to the identification of miR-155-5p, miR-148a-3p and miR 335-5p as deregulated miRNA in OS. Additionally, miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, I used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. While I could not resolve the mechanism through which miR-148a-3p or miR-335-5p impact OS, I identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine-threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, siRNA or small molecule inhibition against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively I have shown that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNA was specifically toxic to tumour cells but not to normal osteoblasts, raising the possibility that these may be tractable targets for miRNA based therapies for OS. Additionally, I investigated the outcome of targeting deregulate epigenetic modulators to achieve therapeutic efficacy in OS. The serine/threonine p21-activating kinases (Pak) are dysregulated in multiple cancers; however, their role in osteosarcoma (OS) is yet unknown. Using primary and metastatic OS tumour propagated cultures from murine models of OS I identified Pak1 and Pak2 as deregulated in OS. While reduced Pak1 expression in murine OS was not found conserved in human OS, the increased expression of Pak2, consistently existed across human and mouse OS. By inhibiting Pak2 gene expression levels using siRNA or pharmacological inhibition using 1,1′-Disulfanediyldinaphthalen-2-ol (IPA-3) and FRAX486, I demonstrated Pak2 inhibition to induce apoptosis in fibroblastic and osteoblastic murine OS cells in addition to primary human OS xenograft derived OS cells. The effects of Pak2 reduction or inhibition on cell proliferation and viability were proportional to the magnitude of Pak2 overexpression. Pak2 inhibition in normal osteoblasts, which have low basal Pak2 expression, did not induce changes in cell viability or proliferation. These findings demonstrate the efficacy of targeting Pak2 activity as a potential therapeutic strategy in OS. Finally, I investigated the effects of osteoblast restricted deletion of the oncogenic miR-17-92 cluster in delaying the onset of OS in Osx-Cre+ p53fl/fl Rbfl/fl murine models. While within my timeline I was unable to resolve the effects of conditional deletion of miR-17-92 cluster on OS onset, my parallel investigation of this deletion on skeletal and haematopoietic development in Osx-Cre+ R26eYFPki/+ miR17+/+, Osx-Cre+ R26eYFPki/+ miR17fl/+ and Osx-Cre+ R26eYFPki/+ miR17fl/fl models demonstrated unaltered parameters.