Mechanisms of Liver Injury by Hepatitis C and Alcohol

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染和饮酒的组合比任何一种情况都会导致更严重的肝病，并增加肝硬化和肝细胞癌的发病率。我们最近发现，丙型肝炎病毒和酒精的结合会导致转录因子FOXO_3的活性丧失，FOXO_3是叉头盒转录因子家族的成员，负责抗氧化剂应激、控制细胞增殖、细胞凋亡和胰岛素敏感性。FOXO_3 也是一种公认的肿瘤抑制分子。它的细胞定位和转录活性受蛋白质的一系列翻译后修饰(PTM)调节。我们的初步数据显示，无论是单独的丙型肝炎病毒感染还是单独的酒精暴露都会增加FOXO_3的活性，但丙型肝炎病毒和酒精的结合会导致FOXO_3的乙酰化和转录活性的丧失。我们还观察到慢性丙型肝炎患者肝活检组织中FOXO_3活性降低，85%的丙型肝炎病毒相关性肝细胞癌中FOXO_3异常定位。这一建议的中心假设是，丙型肝炎病毒引起FOXO_3的磷酸化、乙酰化和泛素化的变化。这些PTM是上游激酶、乙酰转移酶、脱乙酰酶、泛素结合酶和脱泛素酶活性改变的结果。FOXO_3中丙型肝炎病毒/酒精的改变与肝细胞癌对酒精的敏感性增强和肿瘤抑制活性丧失有关。靶向FOXO_3的治疗方法有可能改善丙型肝炎病毒-酒精相关性肝损伤，防止肝细胞癌的发展。我们将通过三个具体目标来检验这一假设。它们是：(1)建立一种新的纳米尺度等电聚焦方法，以确定丙型肝炎病毒/乙醇诱导的FOXO_3翻译后修饰的性质和机制；(2)确定FOXO_3在肝癌发生过程中细胞内异常积聚的原因及其是否影响肿瘤行为；以及(3)研究丙型肝炎病毒诱导的FOXO_3乙酰化在酒精相关肝毒性中的作用，并测试增强FOXO_3活性是否有可能减轻肝损伤。将采用的方法包括一种新的FOXO_3 PTM等电聚焦分析，检查肝细胞癌肝移植患者的肝外植体样本，以及丙型肝炎病毒-酒精相互作用的细胞和小鼠模型。这项研究的假设和方法是非常新颖的。这些研究的成功完成将确定FOX03的分子变化，这些变化具体是由于丙型肝炎病毒和酒精的相互作用造成的，确定这些变化如何导致肝炎和肝癌的发生，并利用这些信息为使用FOX03操作作为一种治疗措施来改善丙型肝炎和饮酒患者的预后奠定基础。

DESCRIPTION (provided by applicant): The combination of Hepatitis C virus (HCV) infection and alcohol consumption produces a more severe liver disease than either condition alone with increased rates of both cirrhosis and hepatocellular carcinoma. We have recently discovered that the combination of HCV and alcohol causes a loss in the activity of the transcription factor FOXO3, a member of the forkhead box family of transcription factors that is responsible for oxidant stress resistance, control of cell proliferation, apoptosis, and insulin sensitivity. FOXO3 is also an established tumor suppressor molecule. Its cellular localization and transcriptional activity are regulated by a series of post translational modifications (PTMs) of the protein. Our preliminary data has shown that either HCV infection alone or alcohol exposure alone increases FOXO3 activity, but the combination of HCV and alcohol results in acetylation of FOXO3 and loss of its transcriptional activity. We also observed decreased FOXO3 activity in liver biopsies from chronic hepatitis C patients and abnormally localized FOXO3 in 85% of HCV-associated hepatocellular carcinomas. The central hypothesis of this proposal is that HCV causes changes in FOXO3 phosphorylation, acetylation and ubiquitination. These PTMs result from altered activity of upstream kinases, acetyl transferases, deacetylases, ubiquitin conjugating enzymes and deubiquitinases. The HCV/alcohol changes in FOXO3 and are responsible for enhanced sensitivity to alcohol and the loss of tumor suppressor activity in hepatocellular carcinoma. Therapeutic approaches targeting FOXO3 have the potential to improve HCV-alcohol associated liver injury and prevent the development of hepatocellular carcinoma. We will examine this hypothesis by three specific aims. They are: (1) to develop a novel nanoscale isoelectic focusing method to determine the nature and mechanism of HCV/ethanol-induced posttranslational modifications of FOXO3; (2) to determine why there is abnormal cytosolic accumulation of FOXO3 in hepatocarcinogenesis and whether this affects tumor behavior; and (3) to examine the role of HCV-induced FOXO3 acetylation in alcohol associated hepatotoxicity and test whether augmenting FOXO3 activity has potential to reduce liver injury. Methods to be employed include a novel isoelectric focusing assay for FOXO3 PTMs, examination of liver explants samples from patients undergoing liver transplantation for hepatocellular carcinoma, and cellular and mouse models of HCV-alcohol interactions. The hypotheses and approaches in this study are highly novel. Successful completion of these studies will identify the molecular changes in FOXO3 that result specifically from the HCV-alcohol interaction, determine how these contribute to cirrhosis and liver carcinogenesis, and use this information to establish the foundation for using FOXO3 manipulation as a therapeutic measure to improve outcome of patients with Hepatitis C and alcohol consumption.