Mechanism(s) of Hepatocyte Transformation by the Hepatitis B Virus X Protein

描述（申请人提供）：慢性乙型肝炎病毒（HBV）感染是肝细胞癌（HCC）发病的主要因素。尽管有乙肝疫苗，世界卫生组织报告称，仍有4亿人慢性感染乙肝病毒。目前的治疗是无效的，从1975年到2005年，美国原发性HCC的发病率增加了两倍。为了减少肝癌，需要新的有效的治疗方法，以及用于疾病分子分类和预后的新的生物标志物。在本研究中，我们研究了有丝分裂polo样激酶1 （Plk1）在hbv介导的肝癌（HBV-HCC）发病机制中的作用。HBV- hcc的发病机制涉及慢性肝脏炎症和弱致癌性HBV X蛋白（pX）的作用。pX激活细胞有丝分裂途径，促进DNA再复制诱导的DNA损伤，激活Plk1。反过来，Plk1介导检查点适应，在未转化的表达px的肝细胞中产生部分多倍体。值得注意的是，Plk1的抑制抑制了px介导的转化。然而，Plk1在px介导的转化和HBV-HCC中的作用仍有待了解。为此，我们通过全基因组siRNA文库筛选鉴定出SUZ12和ZNF198是px介导转化的肿瘤抑制因子。我们的研究结果表明，这些蛋白受到Plk1的负调控。在人肝癌细胞系和慢性HBV合并HCC患者的组织中，Plk1蛋白水平升高，而SUZ12和ZNF198蛋白水平相对于正常对照降低。这种反比关系（Plk1蛋白水平高，SUZ12和ZNF198蛋白水平降低）也发生在HBV复制过程中，这表明Plk1蛋白的过表达和SUZ12和ZNF198蛋白的下调对HBV的转化和复制都很重要。SUZ12和ZNF198介导染色质重塑，并与调节DNA修复、细胞凋亡和病毒复制的PML核小体（NBs）相关。我们推断Plk1下调SUZ12和ZNF198会改变肝细胞基因表达，破坏PML NBs的功能。因此，我们的假设是：Plk1下调SUZ12和ZNF198，通过破坏PML NBs增强HBV复制，并通过解除肝细胞基因表达调控介导癌性转化。我们将在Aim 1中研究Plk1、SUZ12和ZNF198在px介导的转化和HBV复制中的作用；在Aim 2中，Plk1下调ZNF198和SUZ12的机制。在Aim 3中，我们将研究Plk1、SUZ12、ZNF198的蛋白水平以及已知SUZ12靶基因的表达是否与疾病进展和生存有关。影响：慢性HBV患者高水平的病毒血症是进展为HCC的危险因素。抑制Plk1可以作为一种抑制HBV复制的治疗策略，降低HCC发展的风险。Plk1抑制剂正在其他类型癌症的临床试验中，可以作为HBV-HCC的治疗方法。我们的研究有望揭示HBV-HCC的新治疗靶点和生物标志物。

DESCRIPTION (provided by applicant): Chronic Hepatitis B Virus (HBV) infection is a major factor in the pathogenesis of hepatocellular carcinoma (HCC). Despite the HBV vaccine, the World Health Organization reports 400 million people are chronically infected with HBV. Current treatments are ineffective, and rates of primary HCC have tripled in the U.S. from 1975-2005. To reduce liver cancer, new and effective therapies are needed, as well as new biomarkers for molecular classification and prognosis of the disease. In this proposal we investigate the role of the mitotic Polo-like kinase1 (Plk1) in the pathogenesis of HBV-mediated liver cancer (HBV-HCC). Pathogenesis of HBV-HCC involves chronic liver inflammation and effects of the weakly oncogenic HBV X protein (pX). pX activates cellular mitogenic pathways, promotes DNA re-replication-induced DNA damage and activates Plk1. In turn, Plk1 mediates checkpoint adaptation, generating partial polyploidy in non- transformed pX-expressing hepatocytes. Significantly, inhibition of Plk1 suppresses pX-mediated transformation. However much remains to be understood about the role of Plk1 in pX-mediated transformation and HBV-HCC. Toward this end, we have identified by a genome-wide siRNA library screen, SUZ12 and ZNF198 as tumor suppressors of pX-mediated transformation. Our results indicate that these proteins are negatively regulated by Plk1. Both in human liver cancer cell lines and tissues from chronic HBV patients with HCC, protein levels of Plk1 are increased, whereas those of SUZ12 and ZNF198 are reduced relative to normal controls. This inverse relationship (high protein levels of Plk1 and reduced levels of SUZ12 & ZNF198) also occurs during HBV replication, suggesting overexpression of Plk1 and down-regulation of SUZ12 & ZNF198 is important both for transformation and HBV replication. SUZ12 and ZNF198 mediate chromatin remodeling and associate with PML nuclear bodies (NBs) that regulate DNA repair, apoptosis and viral replication. We reason down-regulation of SUZ12 & ZNF198 by Plk1 alters hepatocyte gene expression and disrupts the function of PML NBs. Accordingly, our hypothesis is: Plk1 down-regulates SUZ12 and ZNF198, which enhances HBV replication by disrupting PML NBs, and mediates oncogenic transformation by deregulating hepatocyte gene expression. We will investigate in Aim 1, the role of Plk1, SUZ12, and ZNF198 in pX-mediated transformation and HBV replication; in Aim 2, the mechanism by which Plk1 down-regulates ZNF198 and SUZ12. In Aim 3, we will investigate whether protein levels of Plk1, SUZ12, ZNF198, and expression of known SUZ12 target genes are prognostic for disease progression and survival. Impact: High level of viremia in chronic HBV patients is a risk factor for progression to HCC. Inhibition of Plk1 could serve as a therapy strategy to suppress HBV replication, reducing the risk of HCC development. Plk1 inhibitors are in clinical trials for other types of cancer and could serve as therapy for HBV-HCC. Our studies hold promise to reveal novel therapy targets and biomarkers for HBV-HCC.