HCV Strain Variation and HCC

描述(申请人提供)：肝细胞癌是世界上与癌症相关的死亡的第三大常见原因。在美国、西欧和日本，丙型肝炎病毒是肝细胞癌的主要病因，因此确定丙型肝炎病毒与肝细胞癌之间关系的分子基础至关重要。已发表的数据显示，丙型肝炎病毒核心基因第70和91密码子的病毒点突变与肝细胞癌的发生、干扰素治疗失败和胰岛素抵抗有关。这些病毒突变为丙型肝炎病毒相关肝细胞癌变的机制研究提供了一个令人信服的起点。我们最近发现，这些突变显著改变了丙型肝炎病毒的基因表达，并控制了两种先前未知的丙型肝炎病毒蛋白--70和91微核心蛋白的水平(Eng等人，2009，J Virol)。拟议的实验检查了这些突变以及我们确定的另外两个与肝细胞癌相关的丙型肝炎病毒突变的影响(Fishman等人，2009，Clin Cancer Res)。目标I的目的是确定核心基因突变是否发生在肝癌发生之前，从而潜在地促进肝细胞癌的发生。将进行一项嵌套病例对照研究，以比较在丙型肝炎抗病毒药物长期治疗肝硬化(HALT-C)试验期间发生肝细胞癌的“病例”和未发生肝细胞癌的“对照组”的核心基因序列。主要结果将是病例与对照的百分比，在这些病例中，病毒RNA群体中的大多数变异(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突变是否是发生肝细胞癌的独立预测因子。次要结果将包括其他密码子突变和临床结果之间的关联，以及准种随时间的变化。AIM II的实验目标是确定在细胞培养中暴露于干扰素期间，与肝癌相关的突变是否赋予丙型肝炎病毒生存优势。病毒RNA和蛋白质动力学将使用我们建立的方法，通过qRT/PCR和Western blotting随时间推移进行测量。这些研究将确定四个与肝癌相关的突变对丙型肝炎病毒RNA复制、蛋白质合成(包括微核合成)以及丙型肝炎病毒耐受干扰素治疗能力的影响。通过对肝脏标本的分析，将证实微核在体内的表达。该项目挑战了美国研究人员普遍持有的范式，即所有丙型肝炎病毒变种都具有同等的致癌潜力。我们目标的实现将填补了解丙型肝炎病毒相关肿瘤发生的分子基础方面的根本空白，并可能导致为治疗干预确定新的靶点，并为携带致癌变异的患者开发诊断测试。 公共卫生相关性：丙型肝炎病毒(丙型肝炎病毒)是肝细胞癌的主要原因，肝细胞癌是世界上与癌症相关的死亡的第三大常见原因。临床研究表明，丙型肝炎病毒核心基因点突变增加了肝细胞癌的风险，增加了干扰素治疗失败的可能性。由于它们的临床相关性，我们将定义四个与肝癌相关的突变对病毒复制的分子影响，并确定这些突变是否发生在肝癌之前，因此值得研究作为毒力菌株的可能生物标记物，可用于识别罹患肝癌风险增加的患者。

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.