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

描述（由申请方提供）：慢性B型肝炎病毒（HBV）感染与肝细胞癌（HCC）相关。HBV X蛋白（pX）通过一种未知的机制参与HCC的发病机制。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。我们以前的研究发现，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，其已知激活p53。然而，尽管DNA损伤，pX表达肝细胞不会死亡，而是进行有丝分裂，其中DNA损伤繁殖到子细胞，产生多倍性。这一过程如何发生是未知的，对于理解PX诱导的致癌转化和维持基因组完整性的细胞机制至关重要。为了鉴定与这些机制有关的分子，我们采用了慢病毒siRNA文库，并鉴定了其缺失可将pX表达细胞从DNA损伤诱导的凋亡中拯救出来的基因。我们鉴定了参与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，导致致癌转化。我们假设pX失调的可能候选者是Polo样激酶1（Plk 1），因为一些缺失的基因（例如，有丝分裂进展基因）是已知的Plk 1底物。此外，Plk 1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Plk 1表达和活性。值得注意的是，Plk 1在来自HBV-HCC患者的肝肿瘤样品中过表达。由于pX诱导Plk 1活性，我们的假设是Plk 1不仅终止了表达pX的肝细胞的G2/M DNA损伤检查点，而且还抑制了DNA修复和p53的促凋亡功能。因此，具有DNA损伤的pX表达细胞逃避细胞凋亡，继续累积DNA损伤并遭受基因组不稳定性，导致恶性转化。为了解决这一假设，我们将研究G2期的X-表达细胞，并建立Plk 1的作用，在终止DNA损伤检查点（目的1），Plk 1的作用，在终止DNA修复和p53凋亡（目的2），和Plk 1终止DNA修复和p53转录的机制（目的3）。意义：我们将研究Plk 1在HBV pX介导的肝细胞转化中的作用。Plk 1在人类肝肿瘤中过表达，但其与肝癌发病机制的直接联系尚不清楚。我们的研究将Plk 1与肿瘤转化联系起来，并探索Plk 1在DNA修复和p53凋亡中的新功能。值得注意的是，Plk 1抑制选择性地杀死肿瘤细胞系，Plk 1抑制剂目前正在临床试验中。鉴于全球慢性HBV感染的严重程度以及HBV-HCC通常是致命的，我们的研究有可能将Plk 1鉴定为HBV-HCC的新诊断标志物和治疗靶点。公共卫生相关性：慢性HBV感染导致致命的肝癌（1）。世界卫生组织报告说，有4亿人慢性感染HBV，使他们患肝癌的风险大大增加。因此，HBV如何导致肝癌的研究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.