Polo‐like‐kinase 1: A key cellular target for anti‐HBV therapy?
Authors: Michael M.C. Lai, Wen‐Chi Su
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article On Page 1750 More than 240 million individuals are estimated to be chronically infected with hepatitis B virus (HBV)1 and face a lifetime risk of developing hepatocellular carcinoma (HCC). Current treatments using nucleoside analogues and interferon are effective in inhibiting HBV replication, but unable to clear virus from all hepatocytes. Thus, an alternative strategy of targeting host factors indispensable for HBV replication began to attract serious thought. In this issue of Hepatology, Diab et al. show that Polo‐like‐kinase 1 (PLK1) fulfils the role of such a host factor and can serve as an anti‐HBV target.2 To mimic the living cell condition for HBV infection, the investigators utilized various models to highlight the role of PLK1 in HBV replication, including nontransformed primary human hepatocytes, differentiated HepaRG cells, and a humanized liver mouse model. PLK1 is a serine/threonine‐protein kinase. The investigators demonstrated that HBV infection transiently elevates PLK1 expression and activates PLK1 kinase activity, which, in turn, phosphorylates hepatitis B core protein (HBc), thereby facilitating capsid formation and DNA synthesis of HBV. HBc has long been known to be essential for multiple steps in HBV biosynthesis, including capsid assembly, encapsidation, and reverse transcription of pregenomic RNA (pgRNA), transport of DNA into the nucleus, virus maturation, and release.3 HBc performs specific functions by different domains. N‐terminal domain is sufficient to form the capsid shell, and the C‐terminal domain (CTD) is essential for viral replication. The CTD, covering residues 150‐183, of HBc is highly basic and rich in arginine. CTD contains eight potential serine/threonine phosphorylation sites, and some phosphoacceptor residues have been demonstrated to be critical for viral replication.4 PLK1 is composed of a kinase domain and a Polo‐Box domain (PBD). In the current publication, Diab et al.2 demonstrated that, upon HBV infection, PLK1 could be activated by phosphorylation at S137 and T210, which, in turn, phosphorylates HBc at S168, S176, and S178. How PLK1 phosphorylates HBc remains an interesting question. In general, the PBD of PLK1 can accommodate phosphoserine or phosphothreonine residues to mediate PLK1 interaction with targets. In other words, phosphor‐priming by other kinases is required for most PLK1 targets. Several other kinases, including cyclin‐dependent kinase 2 (CDK2), protein kinase C, SRPK, serine‐arginine protein kinase (SRPK) 1, and SRPK2, have previously been known to mediate phosphorylation on HBc.3 In this study, the investigators showed that mutation of three putative CDK2 phosphorylation sites (S155, S162, and S170) to alanine residues strikingly reduced phosphorylation of HBc by PLK1, implying that HBc may undergo hierarchical phosphorylation. How these sequential phosphorylations proceed is worthy of continued investigation. Interestingly, PLK1 is also involved in hepatitis C virus (HCV) RNA replication as well as virus production by hyperphosphorylation of viral nonstructural protein nonstructural protein 5A (NS5A).5 Intriguingly, like HBV core protein, HCV NS5A can be phosphorylated by multiple kinases to different degrees of phosphorylation, suggesting that phosphorylation of viral proteins are under exquisite control, with PLK1 providing the last piece of brick. The important roles of PLK1 in both HBV and HCV replication imply that HBV and HCV may share some common features in the mechanism of viral hepatocarcinogenesis. Moreover, the investigators demonstrated that inhibition of PLK1 activity by PLK1‐inhibitor BI‐2536 or small interfering (si)RNA reduced the formation of HBV capsid and synthesis of rcDNA (relaxed circular DNA). The decreases in rcDNA synthesis may be caused by reduced pgRNA encapsidation, preferential encapsidation of spliced RNAs, and disruption of reverse transcription. Of note, selectivity in RNA encapsidation has been shown to be regulated by the optimum amount of negative charges from phosphoserines located at the CTD of HBc and encapsidated nucleic acids.6 However, whether PLK1 plays a direct role in RNA encapsidation remains unknown. Further study to delineate the mechanism by which PLK1 affects rcDNA synthesis, for example, pgRNA encapsidation or reverse transcription, will strengthen the key role of PLK1 in regulating HBV replication. Besides its role in the HBV life cycle, PLK1 is a key player in HBV carcinogenesis.7 PLK1 plays multiple roles in the cell cycle and is essential for precisely regulating the cell division and maintaining genome stability in mitosis, spindle assembly, and DNA damage response. Homozygous loss of PLK1 resulted in early embryonic lethality.8 PLK1 expresses ubiquitously in eukaryotic cells and is highly expressed in most of human cancers, including HCC.9 PLK1 has been shown to participate in numerous cancer‐associated pathways. The role of PLK1 in HBV‐related carcinogenesis is focused here. It has been investigated that PLK1 can be activated by HBV X (HBx) protein to allow propagation of DNA damage by simultaneously attenuating the DNA damage checkpoint checkpoint kinase 1 activity and destabilizing DNA repair protein Mre11.7 In addition, PLK1, together with HOX transcript antisense RNA, induces proteasomal degradation of two transcription repression factors, suppressor of zeste 12 homolog and zinc finger protein 198, to alter the chromatin landscape, leading to epigenetic reprogramming associated with oncogenic transformation.10 Combined with the current finding by Diab et al.,2 the HBV‐infected hepatocyte appears to suffer a double whammy by PLK1 through its enhancing effect on HBV replication and its disruptive effects on cellular DNA repair, resulting in HBV‐induced carcinogenesis. The role of PLK1 in both HBV biology and carcinogenesis makes it an ideal therapeutic target for HBV infection and HBV‐induced neoplastic transformation or HCC. PLK1 has been proposed as a therapeutic target for HCC. The recent study demonstrated that Huh‐7 cells‐transplanted nude mice showed significant tumor regression in siPLK1‐treated mice as compared to control mice.9 Besides, normal human cells are largely unaffected by loss of Plk1, whereas Plk1 depletion promotes apoptosis in cancer cells. These preclinical studies suggest that Plk1 is both a feasible and relevant target for suppressing tumor cell growth. Several PLK1 inhibitors have been tested by clinical trial. However, these small‐molecule inhibitors of PLK1 have not achieved a satisfactory therapeutic effect in clinical trials.11 The monotherapy could be supplemented by combined therapy. The investigators treated HBV‐infected dHepaRG cells with the PLK1‐specific inhibitor and approved or investigational anti‐HBV drugs, and found that most of these combined treatments had additive or synergistic effects on reducing reverse transcription of pgRNA into rcDNA. It suggests the possibility of combined therapies to combat chronic hepatitis B and HBV/HCV‐induced HCC by suppression of viral replication and enhancing the immune response with immunotherapeutic interventions. This study not only revealed the function of HBc phosphorylation, but also suggested a new thinking on HBV hepatocarcinogenesis. PLK1 could serve as a promising target for HBV/HCV‐induced HCC.Figure 1: The role of PLK1 in HBV‐mediated HCC. Upon HBV infection, PLK1 is induced and activated by HBx. The phosphorylated PLK1, the active form of PLK1, then phosphorylates HBc to induce HBV replication by facilitating capsid formation and reverse transcription of pgRNA into rcDNA. In addition, the activated PLK1 allows propagation of DNA damage and epigenetic reprogramming, resulting in HBV hepatocarcinogenesis. PLK1 activity can be effectively abolished by PLK1 inhibitor. The boxes colored in blue and orange depict cellular events and viral events, respectively.Author names in bold designate shared co‐first authorship.