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.
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.
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