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Title: Mechanisms of resistance to targeted therapy in urothelial carcinoma
Author: Pettitt, Geoffrey Alasdair
ISNI:       0000 0004 7431 1127
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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Fibroblast growth factor receptor 3 (FGFR3) signalling is altered in ~80% of non-muscle-invasive and ~40% of muscle-invasive bladder cancers via activating mutations (point mutations or gene fusions), overexpression or both. FGFR inhibitors have entered clinical trials in advanced bladder cancer. As with other targeted therapies, resistance is expected to limit treatment efficacy. We have used in vitro models to explore resistance to FGFR inhibition. The urothelial cancer cell lines RT112 and RT4 express FGFR3-TACC3 fusion proteins and are sensitive to FGFR inhibition. Isogenic resistant cell lines, termed RT112 R1, R2, R3 and RT4 R1 were derived by long-term culture of parental cells in the FGFR inhibitor PD173074. RT112 R1, R2 and RT4 R1 had an altered morphology and reduced proliferation rate compared to the parental lines. These changes were reversed when the resistant cells were cultured without PD173074 for four passages. Following this ‘drug holiday’ RT112 R1 and R2 retained PD173074 resistance, whereas RT4 R1 did not. The resistance mechanism in RT112 R1, R2 and RT4 R1 appears to be epigenetic. RT112 R3 retained an epithelial morphology and a proliferation rate similar to parental RT112. Exome sequencing uncovered a HRAS G12S mutation in RT112 R3. The retroviral transduction of a HRAS G12 mutation in RT112 parental induced PD173074 resistance. Microarray analysis was conducted to examine expression changes between parental and resistant lines. MetacoreTM analysis identified the differential expression of pathways relating to cell cycle, epithelial-mesenchymal transition and Oncostatin M signalling. Microarray and immunoblot analysis identified a number of tyrosine kinases as potential resistance mediators. Viability assays showed that RT4 parental and R1 were sensitive to the EGFR inhibitor erlotinib and that RT112 parental and resistant lines were sensitive to the IGF1R inhibitor linsitinib. Investigation of PD173074-resistant derivatives suggests that genetic and epigenetic mechanisms of resistance occur following prolonged FGFR inhibition.
Supervisor: Knowles, Margaret ; Hurst, Carolyn ; Tomlinson, Darren Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available