Investigation of Hepatocarcinogenesis Mechanisms in Transgenic Mice Carrying Hepatitis B Virus S Gene Containing the rtA181T/sW172* Truncation Mutation

In Taiwan, hepatocellular carcinoma (HCC) ranks the second common cause of cancer-related death. Despite a successful vaccination program, hepatitis B virus (HBV) remains the major etiological factor for HCC in patients older than 25 years of age. Two major therapeutic strategies are now available, the oral nucleot(s)ide analogues, reverse transcriptase inhibitors, and the interferon-based therapies. Owing to the low cost, absence of side effect, and oral route of administration, nucelot(s)ide analogues are much more widely used in Asia. Long-term usage of lamivudine resulted in drug resistance developed in a high percentage of patients. Adefovir add-on is a standard rescue treatment for these patients, indicated in several treatment guidelines. Cross resistance of the two drugs appeared in 8 to 20% of lamivudine-resistant patients in 3 years. We are the first group to identify the rtA181T/sW172* as a novel lamivudine-resistant mutation (Yeh et al. Hepatology 2000). Subsequently, this mutation was found responsible for lamivudine and adefovir cross-resistance. Further studies indicated that potent anti-HBV treatment/vaccine can lead to selection of novel S gene mutants (Hsu and Yeh et al. Hepatology 2011; Gastroenterology 2007/2012) and emergence of the rtA181T/sW172* mutation was associated with an increased risk of HCC (Lai /Yeh et al. antivir ther 2008/2009/2010; BMC cancer 2011). This problem is more severe in China because adefovir is being used as the first-line treatment owing to its very low cost. Presently, hepatitis B surface protein carrying the rtA181T/sW172* mutation is considered an oncoprotein in several international review articles. Other novel S gene truncation mutations related to antiviral drug treatment were also found recently and some of them had increased oncogenic potential. Based on these accumulated publications, we felt obliged to generate transgenic mice carrying this particular mutant S protein to validate its oncogenecity. In the past few years, we have successfully generated transgenic mice carrying the rtA181T/sW172* mutant and showed that the mice developed hepatocellular carcinoma (unpublished data). In this proposal, we aim to investigate the molecular mechanisms of hepatocarcinogenesis by use of this established transgenic mice model. Specific Aims: Two lines of transgenic mice carrying hepatitis B virus S gene containing the rtA181T/sW172* mutation has been established together with two lines carrying the wild type S gene. Of the formers, 8.3 and 23.1% (low and high expressors) developed hepatocellular carcinoma, whereas none of the wild type transgenic mice developed hepatocellular carcinoma (P = 0.002). We wish to investigate the molecular mechanisms of hepatocarcinogenesis using this mice model. 1. Examination of the hepatocarcinogenesis related signaling pathways in these transgenic mice, including ERK1/2, Akt/mTOR, GSK3beta/beta-catenin pathways. Examination of the cell proliferation/ cell death related molecules, including apoptosis assay (such as TUNEL), cell proliferation markers (such as PCNA, Ki-67, cyclin etc.). Examination of the ER stress markers (such as GRP78, XBP-1 splicing assay). 2. Search for new oncogenic pathways. This will be achieved by performing cDNA microarray and microRNA microarray assays. The data will be verified by real-time RT-PCR and western blot analysis. RNAi strategy will be used to verify the identified novel pathways. Potentially, this experiment could lead to new therapeutic strategy for truncated S gene mutant associated hepatoma patients. 3. Previous clinical analysis indicated that this mutant increased hepatoma risk in cirrhotic patients. To explore this issue, we will generate cirrhotic transgenic mice by CCL4 (preliminary data), alpha-naphthyl-isothiocyanate (ANIT), or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Incidence of hepatoma will be compared with those without drug treatment. Oncogenic pathways will be examined and compared with those without drug treatmen

Project ID:PC10501-1871
External Project ID:MOST104-2314-B182-015-MY3