The oncogenic interplay between Hepatitis B virus proteins and Wnt signalling

Abstract

© 2018 Dr. Bang Manh TranLiver cancer is the sixth most common cancer worldwide, and is ranked fourth for the number of cancer-related mortality. The vast majority of liver cancer cases are hepatocellular carcinoma (HCC) where viral hepatitis infection is the main cause. Alarmingly, the World Health Organisation (WHO) estimates that nearly one third of the global population has been infected by the hepatitis B virus (HBV), and 240 million people become chronic carriers of the virus. These patients carry a high risk of developing cirrhosis, liver failure and HCC. While treatments can help ameliorate the disease burden, no cure is currently available for HBV chronic infection. Cumulative risk factor for HCC development in these patients can be as high as 15%. However, the oncogenic mechanisms of HBV-related liver cancer are not well understood. Originally discovered by its link to cancer, the Wnt signalling pathway, detailed in Chapter 1, was found to be active in over 60% of HCC cases. As such, the Wnt pathway could serve as an oncogenic conduit that HBV infection utilises to drive hepatic tumourigenesis. However, HBV is highly human hepatocyte-tropic and the lack of a model system that closely resembles human primary hepatocytes has been a barrier for the scientific community to further advance our understanding. Thus, this thesis attempts to expand our knowledge on the oncogenic interplay between HBV proteins and Wnt signalling. Another aim of this thesis is to develop a novel model system that can serve as a platform that closely mimics the human liver context for future studies of early events in HBV infection and its impact on Wnt signalling. In Chapter 1, I provide an overview of the literature on HBV biology, Wnt signalling and roles of HBV on the pathogenesis of HCC. In Chapter 2, I detail all the materials, methods and techniques that I utilised to carry out the investigation in this thesis. In Chapter 3, various structural proteins of HBV are screened for their effect on the Wnt pathway in the hepatoma cell line Huh7. I identify a HBV precore protein named p22 as a novel activator of Wnt signalling. Its stimulatory role is conserved among several different genotypes and is unique to p22 as activation of Wnt signalling was not observed with other HBV precore species. In Chapter 4, I determine the Wnt-activating role of HBV p22 in the contexts of cell lines other than Huh7 and in a classical model of Wnt signalling, namely the Xenopus axis duplication assay. Upregulation of Wnt signalling by p22 was consistently observed in each non-hepatic continuous cell line examined. Interestingly, this stimulatory effect of p22 is dampened by co-transfection with a dominant-negative (dn) isoform of T-cell factor 4 (TCF4), a transcriptional activator of Wnt signalling, suggesting that p22 can activate the Wnt pathway via TCF4. In Chapter 5, I investigate the stimulatory effect of HBV p22 in vivo using the hydrodynamic tail-vein injection mouse model. The livers of mice injected with HBV p22 show a significant upregulation of Wnt target genes Fzd7 and Glul, compared to control mice. In addition, I establish the ex vivo model system of liver organoids from mouse and human, and characterise them thoroughly. I demonstrate that these liver organoids can be isolated, expanded and effectively differentiated into hepatocytes. Furthermore, they closely mimic their liver tissues of origin, thus providing a promising tool for future HBV studies. Finally, in Chapter 6, I bring all the results in this thesis together for the final discussion/overview. Collectively, the findings from this thesis demonstrate and highlight the role of HBV precore p22 as a novel potent activator of the Wnt pathway. Moreover, the ex vivo model of liver organoids can serve as a powerful tool for further investigation of the oncogenic effects of natural HBV infection as well as by HBV precore p22 in isolation. In ongoing research in the Vincan laboratory, the effect of natural HBV infection on cellular signalling pathways and processes will be defined. This might provide novel avenues for drug discovery and therapy