Mechanisms that promote hepatocellular carcinoma due to chronic ethanol exposure

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SUMMARY Hepatocellular carcinoma (HCC) is the fifth most common cancer world-wide with over 600,000 new cases per year and a dismal five-year survival rate at less than 9%. Most HCC patients have chronic liver disease resulting mainly from HCV/HBV infection, chronic alcohol consumption or aflatoxin exposure. Although most patients that develop HCC have cirrhosis or hepatitis, the mechanisms promoting HCC differ between pathologies such that not all cases should be treated the same. This proposal is aimed at understanding the mechanisms that promote HCC due to chronic ethanol exposure, a form of HCC that is on the rise as HCV and HBV infections are being better prevented and treated. In particular, this proposal is focused on understanding chromosome 8q24 amplification that is observed more frequently in alcohol-induced HCC. Because c-Myc resides on this amplified region, we propose that at later stages of HCC, chromosome 8q24 is amplified leading to high c-Myc levels. The high Myc promotes Miz1 transcriptional repression and loss of the epithelial phenotype and acquisition of mesenchymal cell traits. Concomitantly, the high c-Myc activates Zeb1 expression driving cells through the epithelial to mesenchymal transition (EMT) further repressing the epithelial phenotype. As cells metastasize and colonize at secondary sites, epithelial traits are partially reacquired as cells progress through the mesenchymal to epithelial transition (MET). Our hypothesis is that the temporal expression patterns of c-Myc, Zeb1 and Miz1 and epithelial vs. mesenchymal traits are unique in patients with alcohol-induced HCC and can predict prognosis, drug resistance and potential treatment strategies. In Aim 1 we ask two major questions: 1) does 8q24 amplification drive EMT in alcohol-associated HCC? and 2) do metastatic lesions reacquire a more epithelial phenotype? To answer these questions, we will examine protein expression patterns using immunohistochemical (IHC) staining of paraffin-embedded human HCC resections (to answer question 1) and affected lymph nodes and metastatic lesions (to answer question 2). Staining will be correlated to cancer stage, clinicopathologic variables, alcohol consumption and patient responses to treatment. We will also confirm 8q24 amplification using FISH analysis of c-Myc expression. To directly determine the temporal relationship between c-Myc, Miz1, Zeb1 and the epithelial vs. mesenchymal phenotypes, we will perform targeted transcriptome analysis and morphological analysis of human benign and HCC tissue. We will also perform overexpression and knockdown studies to directly establish the temporal relationship between c-Myc, Miz1, Zeb1 and epithelial vs. mesenchymal phenotypes in selected cell lines and primary human normal and HCC cells in culture. Finally, due to the tremendous need to develop new strategies for effective combination therapies to treat HCC, we will examine drug sensitivity in cells to clinically relevant compounds. We have garnered the support of several others to provide their expertise to the project. We have continued access to the Imaging Facility located at nearby Johns Hopkins Medical School and are members of the Hopkins GI Center allowing us access to many services and resources. The expansive expertise of our collaborators and the access to human tissue samples and high- end resources coupled with our considerable expertise in polarized hepatic cell biology situate us perfectly to perform these important mechanistic and translational studies.