HCV Strain Variation and HCC

描述（由申请人提供）：肝细胞癌（HCC）是世界上第三大癌症相关死亡原因。在美国、西欧和日本，HCV是HCC的主要原因，因此确定HCV和HCC之间关系的分子基础至关重要。已发表的数据表明，HCV核心基因密码子70和91的病毒点突变与HCC的发生、干扰素（IFN）治疗失败和胰岛素抵抗相关。这些病毒突变为HCV相关肝细胞癌发生机制的研究提供了一个令人信服的起点。我们最近发现这些突变显著改变HCV基因表达并控制两种先前未知的HCV蛋白，70和91微核蛋白的水平（Eng等，2009，J Virol）。所提出的实验检查了这些突变的影响，以及我们鉴定的另外两种HCC相关的HCV突变（Fishman等人，2009，Clin Cancer Res）。目的I的目标是确定核心基因突变是否在肝癌发生之前出现，从而可能促进肝细胞癌的发生。将进行巢式病例对照研究，以比较在丙型肝炎抗病毒长期治疗肝硬化（HALT-C）试验期间发生HCC的“病例”与未发生HCC的“对照”的核心基因序列。主要结果将是病毒RNA群体中的大多数变体（即，准种）具有密码子70突变。将进行多变量分析，以确定密码子70突变是否是HCC事件的独立预测因子。次要结果将包括其他密码子突变与临床结果之间的关联，以及准种随时间的变化。目的II的实验目标是确定在细胞培养中暴露于IFN期间，HCC相关突变是否赋予HCV存活优势。使用我们建立的方法，通过qRT/PCR和Western印迹法随时间推移测量病毒RNA和蛋白质动力学。这些研究将确定4种HCC相关突变对HCV RNA复制、蛋白质合成（包括微核合成）以及HCV耐受IFN治疗能力的影响。微核心的体内表达将通过分析肝脏标本来确认。该项目挑战了美国研究人员广泛持有的范式，即所有HCV变体都具有同等的致癌潜力。我们目标的实现将填补理解HCV相关肿瘤发生的分子基础方面的根本空白，并可能导致治疗干预的新靶点的鉴定和对携带致癌变异的患者的诊断测试的开发。 公共卫生相关性：丙型肝炎病毒（HCV）是肝细胞癌（HCC）的主要原因，肝细胞癌是世界上第三大常见的癌症相关死亡原因。临床研究表明，HCV核心基因的点突变增加了HCC的风险，并增加了干扰素治疗失败的可能性。由于它们的临床相关性，我们将定义四种HCC相关突变对病毒复制的分子效应，并确定这些突变是否在HCC之前出现，因此需要研究作为可能的生物标志物的强毒株，可用于识别具有肝癌风险升高的患者。

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality in the world. HCV is a major cause of HCC in the United States, Western Europe, and Japan and therefore it is critical to determine the molecular basis of the relationship between HCV and HCC. Published data show that viral point mutations in codons 70 and 91 of the HCV core gene are associated with the development of HCC, interferon (IFN) treatment failure, and insulin resistance. These viral mutations provide a compelling starting point for mechanistic studies of HCV-related hepatocellular carcinogenesis. We recently discovered that these mutations dramatically alter HCV gene expression and control the levels of two previously-unknown HCV proteins, 70 and 91 minicore proteins (Eng et al, 2009, J Virol). The proposed experiments examine the impact of these mutations, and two additional HCC-related HCV mutations that we identified (Fishman et al, 2009, Clin Cancer Res). The goal of Aim I is to determine whether core gene mutations arise prior to the development of liver cancer and thus could potentially promote hepatocellular carcinogenesis. A nested case-control study will be performed to compare core gene sequences of "cases" who developed HCC to those of "controls" who did not develop HCC during the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) Trial. The primary outcome will be the percentage of cases versus controls in which the majority of the variants in the viral RNA population (i.e., the quasispecies) has a codon 70 mutation. Multivariable analysis will be performed to determine whether the codon 70 mutation is an independent predictor of incident HCC. Secondary outcomes will include associations between mutations in other codons and clinical outcomes, and changes in the quasispecies over time. The experimental goal of Aim II is to determine whether the HCC-related mutations confer a survival advantage to HCV during exposure to IFN in cell culture. Viral RNA and protein kinetics will be measured over time by qRT/PCR and Western blotting, using our established methods. These studies will determine the impact of four HCC-related mutations on HCV RNA replica- tion, protein synthesis (including minicore synthesis), and on the ability of HCV to withstand IFN treatment. The in vivo expression of minicores will be confirmed through analysis of liver specimens. This project challenges the paradigm, widely-held by investigators in the United States, that all HCV variants have an equivalent oncogenic potential. Accomplishment of our Aims will fill fundamental gaps in understand- ing the molecular basis of HCV-related oncogenesis and may lead to the identification of new targets for therapeutic interventions and to the development of diagnostic tests for patients harboring oncogenic variants. PUBLIC HEALTH RELEVANCE: The hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma (HCC), the third most common cause of cancer-related mortality in the world. Clinical studies indicate that point mutations in the HCV core gene enhance HCC risk and increase the likelihood of interferon treatment failure. Because of their clinical relevance, we will define the molecular effects of four HCC-associated mutations on viral replication, and also determine whether these mutations arise prior to HCC and therefore warrant investigation as possible biomarkers of virulent strains that can be used to identify patients who have an elevated risk of developing liver cancer.