Hepatocellular carcinoma (HCC) is the 5th most common cancer worldwide, causing an estimated 600,000 deaths/yr. Alcohol abuse and hepatitis C virus (HCV) infection cause inflammation and chronic liver diseases, including cirrhosis, and synergistically increase risk for HCC. Cirrhosis is a well-defined precancerous lesion, yet its molecular underpinnings at the level of the epigenome have not been examined. Alcohol consumption is rising in many countries and results in ~1.8 million deaths/yr. Approximately 170 million people worldwide are infected with HCV, 4 million in the United States, making it the most common blood-borne disease in the U.S. The etiologic agents responsible for the majority of HCCs (>85%) are perhaps better understood than for any other cancer, and all are due to environmental agents or human behaviors (e.g. alcohol, HCV), yet there is much that we do not understand about how these agents exert their carcinogenic effects over >30 years with significant clinical variability. There is no definitive evidence for alcohol or HCV directly mediating genetic damage. There is mounting evidence, however, for early and progressive epigenetic changes during liver disease and for a role of diet and inflammation in disrupting the epigenome. Based on studies from our group and others, genes regulating development appear to be one of the preferential targets of epigenetic defects in cancers, including HCC. A comprehensive analysis of the epigenetic regulation of developmental pathways has never been performed in normal or diseased liver, limiting our knowledge of the full extent and timing of epigenetic changes in the etiology of human liver disease and how these changes may be used as prognostic markers to improve treatment. We hypothesize that epigenetic changes in developmental pathways caused by chronic alcohol exposure and/or HCV infection play a key role in the pathogenesis of liver disease, HCV-induced cellular damage, and HCC development. We propose four aims to address this hypothesis. In aim 1 we will epigenetically profile human liver tissues from patients with cirrhosis or HCC caused by alcohol and/or HCV by coupling a large liver tissue bank with a custom tiling array focused on developmental regulatory genes. In aim 2 we will epigenetically profile untreated and alcohol treated/HCV infected primary hepatocytes using a cell culture system. In aim 3 we will confirm our array data with sensitive and quantitative assays and examine the mechanisms by which ethanol/HCV modulate epigenetic control of model developmental genes and how aberrant expression of developmental genes regulates cell growth. Finally, in aim 4 we will examine how epigenetic marks/expression of developmental genes relates to clinical and pathological changes during liver disease progression. Results from these studies are expected to greatly enhance our understanding of the epigenetic etiology of HCC, provide new targets for therapy and diagnosis/prognosis, and yield novel information on how a ubiquitous recreational drug and a widespread infectious agent impact the epigenome. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinoma (HCC) is the 5th leading cause of cancer related deaths and its incidence is rising in the United States. Unlike many other tumor types, the predisposing factors for the majority of HCCs are known and all are due to external environmental agents and/or human behaviors such as chronic alcohol abuse and hepatitis C infection. While it is known that these agents alter our epigenome, a regulatory system directing gene activity and genome stability, and preferentially target certain gene pathways, the full extent of the epigenetic changes and when they occur in precancerous liver lesions (cirrhosis) remains unknown. It is therefore critical to undertake a comprehensive analysis of the epigenome in these targeted pathways in normal and diseased liver to understand how epigenetic changes lead to HCC and how they may be developed into novel treatments and diagnostic tools to improve patient survival and quality of life. Such studies are the focus of this proposal.