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.