Mechanisms of Liver Injury by Hepatitis C and Alcohol

描述（由申请人提供）：丙型肝炎病毒（HCV）感染和饮酒的组合比单独的任何一种情况产生更严重的肝脏疾病，肝硬化和肝细胞癌的发生率增加。我们最近发现，HCV和酒精的组合导致转录因子FOXO 3的活性丧失，FOXO 3是负责氧化应激抗性、细胞增殖控制、凋亡和胰岛素敏感性的转录因子叉头盒家族的成员。FOXO3 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一系列翻译后修饰（PTM）的蛋白质。我们的初步数据表明，无论是单独的HCV感染或酒精暴露增加FOXO 3的活性，但HCV和酒精的组合导致FOXO 3的乙酰化和转录活性的损失。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HCV相关肝细胞癌中FOXO 3异常定位。该提议的中心假设是HCV引起FOXO 3磷酸化、乙酰化和泛素化的变化。这些PTM由上游激酶、乙酰转移酶、脱乙酰酶、泛素缀合酶和去泛素酶的活性改变引起。HCV/酒精改变FOXO 3，并负责增强对酒精的敏感性和肝细胞癌中肿瘤抑制活性的丧失。靶向FOXO 3的治疗方法有可能改善HCV-酒精相关的肝损伤并预防肝细胞癌的发展。我们将通过三个具体目标来检验这一假设。它们是：（1）开发一种新的纳米等电聚焦方法，以确定HCV/乙醇诱导的FOXO 3翻译后修饰的性质和机制;（2）确定肝癌发生过程中FOXO 3异常胞浆积聚的原因及其是否影响肿瘤行为;（3）探讨HCV在感染中的作用-在酒精相关的肝毒性中诱导FOXO 3乙酰化，并测试增加FOXO 3活性是否具有减少肝损伤的潜力。采用的方法包括一种新的等电聚焦测定FOXO 3 PTM，检查肝细胞癌肝移植患者的肝外植体样本，以及HCV-酒精相互作用的细胞和小鼠模型。本研究的假设和方法都是非常新颖的。这些研究的成功完成将确定FOXO 3中的分子变化，这些变化特别是由HCV-酒精相互作用引起的，确定这些变化如何促进肝硬化和肝癌发生，并利用这些信息为使用FOXO 3操作作为治疗措施建立基础，以改善丙型肝炎和饮酒患者的预后。

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.