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).