The Influence of HPV16 E6 Oncoprotein on the Radiation Resistance of Cervical Cancer

Abstract

Abstract The University of Manchester, Mashael Alfaifi, October 2015 Degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences The Influence of HPV16 E6 Oncoprotein on the Radiation Resistance of Cervical Cancer Human papilloma virus (HPV) related invasive cervical cancer (ICC) is responsible for 270,000 deaths per annum. Some cases of ICC respond to radiotherapy whereas others do not. There is some evidence that the mutation status of the tumour suppressor p53 can combine with high-risk (HR) forms of HPV to influence treatment outcome. The E6 protein from HR-HPV types stimulates degradation of mutant and wild type forms of the p53 protein and the ectopic expression of HR-HPV E6 in mutant p53 expressing cells promotes the evolution of radiation resistant tumours. The p53 family member p73 can compensate for loss of p53 function by either inhibiting the cell cycle or triggering apoptosis in response to DNA damage and E6 also interacts with TAp73. Thus it is hypothesised that interaction of the E6 protein with TAp73 may compromise its ability to regulate genetic stability thereby promoting the evolution of therapy resistance. In order to investigate this, mutant p53 expressing human C33A cervical carcinoma cells transfected with E6 from either LR or HR HPV were used. Flow cytometric analysis of the cell cycle, before and after treatment with low-dose radiation, showed that expression of HR-E6 significantly enhanced the development of polyploidy (>4N). RT-PCR analysis of expression of the p73 transactivated target Bcl-2 Like 11 (or BIM) showed this was down-regulated in HR-E6 expressing cells prior to irradiation. These cells also displayed altered post-radiation kinetics of BIM expression. Since functional TAp73 is required to activate BIM and also regulate G2 checkpoint control, these results showed that the interaction of HR-E6 with p73 could contribute to enhanced genetic instability. Mammalian-two-hybrid (M2H) was carried out to define the interaction between HR-E6 and TAp73. The results showed a strong interaction between HR-E6 and the first 62 amino acids of TAp73 which contains the N-terminal transactivation domain. Attempts were made to block this interaction by delivering competitive 12AA synthetic peptides directly into cells which proved unsuccessful. Therefore, since expression of the N-terminal TAp73(62)AA fragment had shown strong binding to HR-E6 proteins, this was used to stably transfect C33A parent and C33A16-E6 cells. The effects of ectopic expression of this 62AA fragment on the cell cycle were then evaluated by flow cytometry which showed that this suppressed the development of polyploidy in HR-E6 expressing cells. These experiments support the hypothesis that interaction of HPV16 E6 with TAp73 compromises its function thus relieving G2 checkpoint control which enhances endoreduplication and enhanced genetic instability. In order to verify this, full human transcriptome microarrays were probed with mRNA extracted from C33A parental and C33A16-E6 which were then subjected to a systems biology network analysis. Novel, hitherto undefined, effects of the HR-E6 protein on upstream regulators and differentially regulated genes were identified. Notably, these findings confirmed that HPV16 E6 inactivates TAp73 and its transactivation target BIM which is consistent with the aforementioned hypothesis.

Date of Award	31 Dec 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Hampson (Supervisor) & Lynne Hampson (Supervisor)