Role of Polo-like kinase (Plk-1) in Hepatitis B Virus-mediated Hepatocellular Car

描述（由申请人提供）：慢性乙型肝炎病毒（HBV）感染与肝细胞癌（HCC）有关。HBV X蛋白（pX）以一种未知的机制参与HCC的发病。本研究的长期目标是确定pX如何启动肝细胞转化并确定新的治疗靶点。我们前期研究发现，在未转化的肝细胞中，pX诱导DNA再复制，导致DNA损伤和多倍体。结果，复制应激激酶ATR被激活，已知它会激活p53。然而，尽管DNA损伤，表达px的肝细胞不会死亡，而是进行有丝分裂，DNA损伤繁殖到子细胞，产生多倍体。这个过程是如何发生的尚不清楚，对于理解px诱导的致癌转化和维持基因组完整性所涉及的细胞机制至关重要。为了鉴定与这些机制相关的分子，我们使用了慢病毒siRNA文库，并鉴定了基因，这些基因的缺失可以拯救表达px的细胞免受DNA损伤诱导的凋亡。我们确定了参与1)DNA复制和有丝分裂进程，2)DNA修复和3)p53功能的基因。因此，本提案的主要目的是确定pX如何解除这些机制，从而导致致癌转化。我们假设pX去监管化的可能候选者是polo样激酶1 (Plk1)，因为一些被耗尽的基因（例如，有丝分裂进展基因）是已知的Plk1底物。此外，Plk1终止G2/M DNA损伤检查点以启动有丝分裂，重要的是，我们发现pX诱导Plk1的表达和活性。值得注意的是，Plk1在HBV-HCC患者的肝脏肿瘤样本中过表达。由于pX诱导Plk1活性，我们的假设是，Plk1不仅终止表达pX的肝细胞的G2/M DNA损伤检查点，还抑制DNA修复和p53的促凋亡功能。因此，DNA损伤的表达px的细胞逃避凋亡，继续积累DNA损伤，遭受基因组不稳定导致恶性转化。为了解决这一假设，我们将研究表达x的细胞的G2期，并确定Plk1在终止DNA损伤检查点中的作用（Aim 1）， Plk1在终止DNA修复和p53凋亡中的作用（Aim 2），以及Plk1终止DNA修复和p53转录的机制（Aim 3）。意义：我们将研究Plk1在HBV px介导的肝细胞转化中的作用。Plk1在人类肝脏肿瘤中过度表达，但其与肝癌发病机制的直接联系尚不清楚。我们的研究将把Plk1与致癌转化联系起来，并探索Plk1在DNA修复和p53凋亡中的新功能。值得注意的是，Plk1抑制选择性地杀死肿瘤细胞系，Plk1抑制剂目前正在临床试验中。鉴于全球范围内慢性HBV感染的严重程度以及HBV- hcc通常是致命的，我们的研究有可能确定Plk1作为HBV- hcc的新诊断标志物和治疗靶点。公共卫生相关性：慢性HBV感染可导致致命性肝癌(1)。世界卫生组织报告说，有4亿人慢性感染乙肝病毒，这使他们患HCC的风险大大增加。因此，研究HBV如何导致肝癌解决了一个重要的人类健康问题，需要新的有效的治疗方法。

DESCRIPTION (provided by applicant): Chronic Hepatitis B virus (HBV) infection is linked to hepatocellular carcinoma (HCC). The HBV X protein (pX) is implicated in HCC pathogenesis by an unknown mechanism. The long term goals of this study are to determine how pX initiates hepatocyte transformation and to identify new targets for therapy. Our previous studies have found that in non-transformed hepatocytes, pX induces DNA re-replication, resulting in DNA damage and polyploidy. As a result, the replication stress kinase ATR is activated which is known to activate p53. However, despite DNA damage, pX-expressing hepatocytes do not die and instead proceed to mitosis, where DNA damage is propagated to daughter cells, giving rise to polyploidy. How this process occurs is unknown and is critical for understanding both pX-induced oncogenic transformation and the cellular mechanisms involved in maintaining genomic integrity. To identify molecules involved with these mechanisms, we employed a lentiviral siRNA library and identified genes whose depletion rescues pX-expressing cells from DNA damage-induced apoptosis. We identified genes involved in 1) DNA replication and mitotic progression, 2) DNA repair and 3) p53 function. Accordingly, the main objective of this proposal is to determine how pX deregulates these mechanisms, resulting in oncogenic transformation. We hypothesize that a likely candidate for pX deregulation is Polo-like kinase1 (Plk1) because some of the depleted genes (e.g., mitotic progression genes) are known Plk1 substrates. In addition, Plk1 terminates the G2/M DNA damage checkpoint to initiate mitosis, and importantly, we have found that pX induces both Plk1 expression and activity. Significantly, Plk1 is overexpressed in liver tumor samples from HBV-HCC patients. Since pX induces Plk1 activity, our hypothesis is that Plk1 terminates not only the G2/M DNA damage checkpoint of pX-expressing hepatocytes, but also suppresses DNA repair and the pro-apoptotic function of p53. Consequently, pX-expressing cells with DNA damage escape apoptosis continue to accrue DNA damage and suffer from genomic instability leading to malignant transformation. To address this hypothesis, we will investigate the G2 phase of X-expressing cells and establish the role of Plk1 in terminating the DNA damage checkpoint (Aim 1), the role of Plk1 in termination of DNA repair and p53 apoptosis (Aim 2), and the mechanism by which Plk1 terminates DNA repair and p53 transcription (Aim 3). Significance: We will investigate the role of Plk1 in HBV pX-mediated hepatocyte transformation. Plk1 is overexpressed in human liver tumors but its direct link to liver cancer pathogenesis is unknown. Our studies will link Plk1 to oncogenic transformation and explore novel functions of Plk1 in DNA repair and p53 apoptosis. Significantly, Plk1 inhibition selectively kills tumor cell lines and a Plk1 inhibitor is currently in clinical trials. Given the magnitude of chronic HBV infection worldwide and that HBV-HCC is usually fatal, our studies have the potential to identify Plk1 as a new diagnostic marker and a therapeutic target for HBV-HCC. PUBLIC HEALTH RELEVANCE: Chronic HBV infection leads to fatal liver cancer (1). The World Health Organization reports 400 million people are chronically infected with HBV, placing them at a greatly increased risk for HCC development. Thus, studies of how HBV causes liver cancer address a significant human health problem needing new and efficacious therapies.