Pathology - Theses
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ItemRegulation of E6AP and involvement of the E6AP/PML axis in human cancer( 2013)The ubiquitin-proteasome system (UPS) plays a major role in the regulation of many cellular processes. However, aberrations in the regulation of the UPS can lead to a variety of pathological conditions. A firm link has been established between UPS and the development of cancer. Velcade, a proteasome inhibitor, is in clinical use for the treatment of diseases such as multiple myeloma. This thesis is based on our recent findings of a new regulatory pathway of tumour suppression. We discovered that the HECT E3 ligase, E6-associated protein (E6AP), is a key regulator of the tumour suppressor promeylocytic protein (PML). My thesis describes 3 studies: the regulation of E6AP by c-Abl (Chapter 3); the involvement of the E6AP-PML axis in colorectal cancer (Chapter 4) and also in prostate cancer (Chapter 5). Regulation of E6AP by c-Abl: In human papillomavirus (HPV) - infected cells, p53 is degraded by the HPV-E6/E6AP complex. Our laboratory previously demonstrated that the c-Abl tyrosine kinase protects p53 from ubiquitination and degradation by the HPV-E6/E6AP complex under stress conditions. Chapter 3 explores the underlying mechanism of this protection. We demonstrated an interaction between c-Abl and E6AP in vivo and in vitro. Activation of c-Abl by DNA damage leads to phosphorylation of E6AP. Mass spectrometric analysis revealed that tyrosine (Y) 636 within the HECT catalytic domain of E6AP is phosphorylated by c-Abl. Further computational analysis of E6AP trimerisation revealed an interaction between Y636 and glutamate (E) 544 in a neighbouring E6AP molecule. Using substitution mutants we examined the effect of Y636 phosphorylation, and its interaction with E544, on the E3 ligase activity of E6AP. We demonstrated that Y636 and E544 regulate the E3 ligase activity of E6AP, in a substrate-specific manner. Our findings suggest that in response to stress, c-Abl phosphorylates E6AP on Y636, thereby reducing the interaction with E544 leading to inhibition of E6AP E3 ligase activity. This is the first description of a post-translational modification of E6AP regulating its activity. This also provides a molecular explanation for the protection of p53 in HPV-infected cells under stress. Involvement of the E6AP-PML axis in cancer: PML protein expression have been shown to be down-regulated in multiple cancer types. Since we discovered E6AP as the E3 ligase of PML, we hypothesized that E6AP may be responsible for the loss of PML expression in colon and prostate cancers. Using a panel of colorectal cancer cell lines we did not find a significant elevation of E6AP correlating with low PML, nor were we able to restore PML expression by down-regulating E6AP (Chapter 4). On the other hand, we found an inverse correlation between E6AP and PML in a panel of prostate cancer cell lines. Down-regulation of E6AP was also associated with a reduction in cell numbers and an increased sensitization of prostate cancer cells to stress-induced death. This study supports a role for the E6AP-PML axis in prostate cancer. This is consistent with our analysis of prostate cancer samples demonstrating the poorest survival for patients with high E6AP and low PML expression (Chapter 5).
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ItemAn assay to screen for mutant p53 gain of function using high content imaging( 2012)The p53 tumour suppressor gene is mutated in approximately half of all human cancers. These mutations not only inactivate the growth inhibitory functions of wild type p53 but certain mutations also confer additional oncogenic properties. These gained functions may contribute to the increased growth rate, resistance to apoptosis, reduced chemosensitivity and increased invasiveness of mutant p53 bearing tumours. For the purposes of discovering novel mediators of mutant p53 gain of function, a multi-parameter assay was developed for future use in a high throughput siRNA screen. Mutant p53 expressing cells were demonstrated to display an increased migratory phenotype in vitro compared to p53 null cells. A wound healing endpoint assay was adapted to a 96 well format using automated liquid handling and image capture. An image analysis algorithm was designed to accurately measure cell migration in a high throughput manner. Performing a low throughput pilot screen of a panel of siRNAs demonstrated that the general migration machinery was necessary for migration. However, knockdown of known components of the mutant p53 pathway failed to provide the robustness necessary for a high throughput screen. p53 protein stability is known to be regulated at multiple levels involving a complex network of feedback loops. Unlike the case with wtp53, the regulation of mutant p53 is only partially understood. A high content assay for mutant p53 stability was developed. The immunofluorescent staining protocol was adapted to a 384 well assay and an algorithm was designed to accurately measure pixel intensity in the nucleus and cytoplasm. A statistically significant and robust downregulation of mutant p53 was measured with siRNA against mutant p53. A pilot screen in endogenous mutant p53 cell lines demonstrated a sufficiently large assay window to identify siRNAs that reduce mutant p53 stability. A genome wide siRNA screen for genes that reduce mutant p53 could uncover novel therapeutic targets, which will enable the design of new molecularly targeted therapeutics. Drugs able to impede the action of mutant p53 will be relevant to a significant proportion of human cancers.