Medicine (Austin & Northern Health) - Theses
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ItemPathways to phenotypes in melanoma: regulation of plasticity and treatment escape( 2016)Melanoma exhibits a wide range of biological behaviours, many of which recapitulate melanocytic developmental processes. Alterations in melanoma cell behaviour and phenotype contribute to melanoma progression, metastasis, and influence the efficacy of drug therapies. This study examines how complex regulatory molecular networks differ between melanoma phenotypes and distinct biological states. Through the use of whole-cell transcriptomic data, the relationships between gene expression and microRNA expression in melanoma were described. Coupling between seemingly distinct phenotypes such as pigmentation and migration/invasion was shown to be frequently linked to the core melanocytic transcription factor MITF, but may involve transcriptional regulatory mechanisms driven by other factors, such as SOX10. Differential expression analysis identified a preliminary tumour cell specific gene set including several inflammatory and immune-related genes, indicative of brisk lymphocytic infiltration of melanoma tumours. A novel Systems Analysis workflow was developed to integrate gene and microRNA expression data, incorporating phenotypic annotation data to identify putative regulatory interactions of relevance to the phenotype(s) under study. Validation of this workflow using the candidate microRNA miR-29b-3p confirmed the utility of this approach in directing experimental resources, and identified a novel role for miR-29b-3p in regulating melanoma invasiveness. The specific scenario of sensitivity and/or resistance to BRAF-targeted therapy in melanomas harbouring activating BRAF V600 mutations was used to explore the adaptive changes that occur under treatment. The molecular pathway alterations in drug-resistant melanoma cells at gene level, and at microRNA level were identified, demonstrating that dysregulation of genes and of microRNAs act as an additive strategy to maximise potential regulation of the proteome. Furthermore, the finding of significantly heterogeneous and non-recurrent changes in gene and, particularly, microRNA abundance led to the proposal of a multi-state model of drug resistance and plasticity-like resensitisation. Several key microRNA species, including miR-155-5p and the miR-199a~miR-214 cluster, were implicated as direct modulators of BRAF inhibitor sensitivity. Lastly, off-target effects of BRAF mutation-selective inhibitors were examined in two unique settings of paradoxical MAPK pathway activation. The first involved a KRAS mutated colon cancer, whilst the second involved a haematological malignancy – chronic myeloid leukaemia – characterised by similar MAPK pathway activation but a substantially more diverse array of activated parallel signalling pathways. The contrasting clinical outcomes in these two cases were used to highlight the sensitive dependence on cellular context when determining the outcome of an otherwise identical pharmacological manipulation. A synthetically lethal combination of BRAF and MEK inhibitors in chronic myeloid leukaemia was demonstrated both pre-clinically and clinically, arguing for further clinical evaluation of this strategy in CML and potentially other cancer types. Together, this work provides a firm basis for understanding and evaluating the complex microRNA-gene interaction networks active in melanoma cells across diverse biological states and phenotypes. Key candidate microRNAs and target genes have been identified for further study as potential therapeutic targets in melanoma. In parallel, the complexity of signal pathway manipulation has been demonstrated in the context of paradoxical MAPK pathway activation, arguing strongly for further clinical evaluation of synthetic lethality as a therapeutic strategy.
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ItemImpact of a pregnancy-associated protein (PAPPA) on melanoma: a link between pregnancy and progression of cancer( 2016)Melanoma is the most common cancer diagnosed in pregnant women, and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. This thesis reports that migration, invasiveness and progression of melanoma are promoted by pregnancy-associated plasma protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of insulin-like growth factor (IGF)-1 by cleaving it from a circulating complex formed with IGF binding protein-4. The results presented in this thesis confirm that PAPPA is widely expressed by metastatic melanoma tumours and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in the avian embryonic neural crest model in vivo. Treatment with PAPPA-enriched pregnancy serum enhanced the motility of melanoma cells in vitro. Furthermore, this thesis reports that IGF-1 can induce the phenotypic and functional effects of epithelial-mesenchymal transition (EMT) in melanoma cells. This study establishes a clear relationship between the pregnancy-associated protein PAPPA, melanoma, and functional effects mediated through IGF-1, providing a plausible mechanism for accelerated progression of melanoma during pregnancy. This opens the possibility of therapeutic targeting of the PAPPA/IGF-1 axis.
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ItemFOXP3 expression and function in melanoma( 2012)The Forkhead boxP3 (FOXP3) transcription factor is a critical regulator of regulatory T (Treg) cell differentiation and immunosuppressive function. FOXP3 has also been reported to be expressed in many tumours, including melanoma. The role of FOXP3 in tumourigenesis is conflicting, with both tumour suppressive and tumour promoting functions described. In breast and prostate cancers loss of expression and inactivating mutations in FOXP3 suggest a tumour suppressor function. However, recent studies have failed to reproduce some of these findings. In pancreatic and urinary bladder cancer, over-expression of FOXP3 has been associated with immune suppression and poor outcome respectively, suggestive of a tumour promoting function. The aim of the current study was to determine the expression status and function of FOXP3 in melanoma. FOXP3 expression was detected in 12% (18/146) of metastatic melanomas analysed, but with low basal level of expression in a minor fraction of cells in melanoma cell lines. No mutations in FOXP3 were detected in a panel of 54 cell lines generated from patients with metastatic melanoma. To determine the effect of FOXP3 overexpression on the growth of melanoma cells, we stably over-expressed FOXP3 in the SKMEL-28 melanoma cell line, which normally expresses low levels of FOXP3. FOXP3 overexpression reduced the proliferation rate and clonogenecity of SK-MEL-28 melanoma cells in vitro and reduced tumour growth when grown as xenografts in vivo. FOXP3 overexpression also increased the rate of apoptosis and pigmentation of SK-MEL-28 melanoma cells, but did not lead to suppression of T-cell proliferation in co-culture experiments. Based on its infrequent expression in melanoma tumours, and its growth inhibitory and proapoptotic effect on melanoma cells, we conclude that FOXP3 is not a major driver of melanoma progression.
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ItemOrigins of heterogeneity in melanoma( 2012)The cancer stem cell hypothesis has dominated discussions of cancer cellheterogeneity in recent years; however its relevance to melanoma is controversial. This thesis presents projects outlining sources of cellular heterogeneity in melanoma in relation to three models of cancer development: the cancer stem cell model, clonal evolution, and phenotypic plasticity. A common cancer stem cell culture method, which involves supplementing serum-free media with specific growth factors, yielded a model of melanoma that was not as representative of the original tumour as standard methods. Gene expression profiling revealed a biased towards expression of neural lineage genes, which could seriously confound therapeutic target selection. In contrast metastatic melanoma cell lines and a metastatic melanoma tissue sample were found to be heterogeneous for DNA copy number abnormalities, supporting the involvement of clonal evolution in the development of melanoma heterogeneity. Finally by segregating melanoma cells based on functional heterogeneity evidence for dynamic and reversible transitions between different phenotypes was observed. Gene expression profiling of these functionally distinct subpopulations identified genes relating to epithelial-to-mesenchymal transition and inflammation as relevant to melanoma cell invasion and drug resistance, including both cytotoxic and targeted therapies. Together these results suggest that heterogeneity between the cells of a melanoma results from ongoing genetic changes as well as cell-cell and environmental interactions, and that targeting molecules representing multiple phenotypes simultaneously might be necessary to successfully treat the disease.