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的主要病因，因此确定HCV与HCC之间关系的分子基础至关重要。已发表的数据显示，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有关。这些病毒突变为hcv相关的肝细胞癌变机制研究提供了一个令人信服的起点。我们最近发现，这些突变显著地改变了HCV基因表达，并控制了两种以前未知的HCV蛋白，70和91 minicore蛋白的水平（Eng等，2009,J Virol）。提出的实验检查了这些突变的影响，以及我们发现的另外两种与丙型肝炎相关的HCV突变（Fishman等人，2009年，临床癌症研究所）。Aim I的目的是确定核心基因突变是否在肝癌发生之前发生，从而可能促进肝细胞癌的发生。将进行一项巢式病例对照研究，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“对照组”的核心基因序列。主要结果将是病毒RNA群体（即准种）中大多数变异具有密码子70突变的病例与对照的百分比。将进行多变量分析以确定密码子70突变是否是HCC发生的独立预测因子。次要结果将包括其他密码子突变与临床结果之间的关系，以及准物种随时间的变化。Aim II的实验目的是确定在细胞培养中暴露于IFN时，丙型肝炎病毒相关突变是否赋予丙型肝炎病毒生存优势。病毒RNA和蛋白质动力学将随时间通过qRT/PCR和Western blotting测定，使用我们建立的方法。这些研究将确定四种与丙型肝炎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）以及丙型肝炎病毒抗干扰素治疗能力的影响。minicores在体内的表达将通过肝脏标本的分析来证实。该项目挑战了美国研究者广泛持有的范式，即所有HCV变体具有相同的致癌潜力。实现我们的目标将填补理解hcv相关肿瘤发生的分子基础的基本空白，并可能导致确定治疗干预的新靶点，并为携带致癌变异的患者开发诊断测试。

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.