Polo‐like kinase 1 inhibition suppresses hepatitis B virus X protein‐induced transformation in an in vitro model of liver cancer progression†

Abstract

Chronic Hepatitis B virus (HBV) infection is linked to development of hepatocellular carcinoma (HCC). The HBV X protein (pX) is implicated in HCC pathogenesis acting as a weak oncogene or a co-factor in hepatocarcinogenesis. pX induces DNA re-replication, DNA damage and partial polyploidy in a poorly differentiated, immortalized hepatocyte cell line. In this study, we employed sorted, pX-induced polyploid cells to investigate their growth and oncogenic transformation potential, over the course of 70 cell doublings. Immediately after live cell-sorting, nearly 40% of pX-induced polyploid cells undergo apoptosis, while the surviving cells exhibit proliferation sensitive to p53. After 40 cell generations, the pX-expressing polyploid cultures exhibit loss of p53 function and become growth factor-and anchorage-independent, indicative of oncogenic transformation. The pX-induced polyploid cultures in the course of 70 cell generations undergo progressively increasing DNA damage, propagate damaged DNA to daughter cells, and display increased expression of a cluster of proliferation genes shown to be elevated in human HCC, including HBV-HCC. One of these genes is the mitotic kinase Polo-like kinase 1 (Plk1). Oncogenic transformation is suppressed in the absence of pX expression, and significantly, by inhibition of Plk1. These results identify Plk1 as crucial in pX-mediated oncogenic transformation.Conclusion-Partial polyploidy induced by pX is not immediately associated with oncogenic transformation. Continued DNA damage for 40 cell generations is reproducibly associated with loss of p53 function, enhanced expression of Plk1 and oncogenic transformation. Since Plk1 expression is also elevated in HBV-HCC tumors, this in vitro cellular model simulates liver cancer progression and pathogenesis in chronic HBV patients. Inhibition of Plk1 activity suppresses pXmediated oncogenic transformation, identifying Plk1 as a promising therapeutic target for HBVmediated HCC. KeywordsHepatitis B Virus X protein; hepatocellular carcinoma; polyploidy; DNA damage; oncogenic transformation; p53; polo-like kinase1 (Plk1); BubR1 [3][4][5], that defective DNA repair causes chromosomal instability, accelerating liver carcinogenesis (6), and genomic instability is likely the primary cause of carcinogenesis, based on the slow kinetics from carcinogen exposure to cancer development (7).The mechanism of HCC pathogenesis by chronic HBV infection involves effects of chronic inflammation and regeneration of the liver (8), and effects of two HBV proteins, the X (9) and S (10) proteins. HBV DNA integrates into the host genome during early steps of tumor expansion (11), and most tumors continue to express the X protein (pX). pX is multifunctional, essential for the viral life cycle (12), and implicated in HCC pathogenesis (9,11), acting as a weak oncogene (13) or a co-factor in hepatocarcinogenesis (14). pX increases the activity of the cellular mitogenic pathways (15) . This cluster includes replication factors MCM4-6, PCNA, Thymidilate Synthase...

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

“…Plk1 also is elevated during pX-mediated hepatocyte transformation, and significantly, inhibition of Plk1 suppresses hepatocyte transformation by pX (40). However, the role of Plk1 in pX-induced oncogenic transformation is not yet understood.…”

“…Intriguingly, despite DNA re-replicationinduced DNA damage, these pX-expressing hepatocytes proceed through mitosis, propagate damaged DNA, and generate daughter cells that are partially polyploid (Ͼ4N DNA) (52). Partial polyploidy induced by pX results in oncogenic transformation (40). However, it is not understood why pX expression allows nontransformed hepatocytes with DNA damage to recover from the DNA damage checkpoint, escape apoptosis, and propagate DNA damage.…”

“…Depletion of Plk1 induces apoptosis in cancer cell lines but not normal cells (28,39). Likewise, inhibition of Plk1 suppresses hepatocyte transformation mediated by the HBV X oncoprotein (40) indicating Plk1 could serve as a novel therapeutic target for HBV-HCC and other virally induced cancers. Significantly, Plk1 inhibitors are currently in clinical trials for various types of human cancers (41,42).…”

Molecular Mechanism of DNA Damage Response Pathway During Hepatic Carcinogenesis

“…Wu et al (2008) reported that HBx-transformed cells show defective S-phase arrest and consequent G2/M arrest after DNA damage induced by mitomycin C. Importantly, they also found that HBx impairs the ATR-dependent phosphorylation of Chk1 and monoubiquitination of FANCD2, suggesting that the defect in the intra-S-phase checkpoint may be the reason for genomic instability. Studach et al (2009) reported that HBx-induced polyploid cells showed continued DNA damage after long repopulation, which was associated with loss of p53 function. They suggested that polo-like kinase 1 (Plk1), which is frequently overexpressed in human HCC, might play an important role in the acquisition of HBx-induced DNA damage.…”

Intravenous liposomal delivery of the short hairpin RNAs against Plk1 controls the growth of established human hepatocellular carcinoma

“…Our study suggested that shRNa-mediated silencing of plk1 might be a novel therapeutic approach against human hepatocellular carcinoma by inhibiting tumor cells proliferation and inducing apoptosis. Numerous reports have documented elevated expression of Plk1 in hepatocellular carcinoma 7,8 and hepatoblastomas. 9 Applications of diverse Plk1-attenuating approaches, including small molecule inhibitors, 10,11 small interfering RNAs(siRNAs) 12,13 or antisense oligonucleotides 14,15 have increased our knowledge of mitotic regulation and allowed us to assess their ability to suppress tumor growth in vivo.…”