Tumor and Stem Cell Biology PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12 and ZNF198 during Hepatitis B Virus–Induced Liver Carcinogenesis

Elucidating mechanisms of hepatitis B virus (HBV)–mediated hepatocarcinogenesis is needed to gain insights into the etiology and treatment of liver cancer. Cells where HBV is replicating exhibit increased expression of Plk1 kinase and reduced levels of two transcription repression factors, SUZ12 and ZNF198. SUZ12 is an essential subunit of the transcription repressive complex PRC2. ZNF198 stabilizes the transcription repressive complex composed of LSD1, Co-REST, and HDAC1. These two transcription repressive complexes are held together by binding the long noncodingRNAHOTAIR. In this study, we linked these regulatory eventsmechanistically by showing that Plk1 induces proteasomal degradation of SUZ12 and ZNF198 by site-specific phosphorylation. Plk1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression ofHOTAIR, significantly reducing SUZ12 andZNF198 stability. In cells expressing theHBVXprotein (HBx), downregulation of SUZ12 and ZNF198 mediated global changes inhistonemodifications. In turn,HBx-expressing cells propagated an altered chromatin landscape after cell division, as exemplified by changes in histone modifications of the EpCAM promoter, a target of PRC2 and LSD1/Co-REST/HDAC1 complexes. Notably, liver tumors from X/c-myc bitransgenic mice exhibited downregulation of SUZ12 and ZNF198 along with elevated expression of Plk1, HOTAIR, and EpCAM. Clinically, similar effects were documented in a set of HBV-related liver tumors consistent with the likelihood that downregulation of SUZ12 and ZNF198 leads to epigenetic reprogramming of infected hepatocytes. Because both Plk1 and HOTAIR are elevated in many human cancers, we propose that their combined effects are involved in epigenetic reprogramming associated broadly with oncogenic transformation. Cancer Res; 75(11); 2363–74. 2015 AACR. Introduction Chronic infection by the hepatitis B virus (HBV) is a major etiologic factor in the development of hepatocellular carcinoma (HCC; ref. 1). The World Health Organization estimates that 250 million people globally are chronically infected with HBV. Despite the availability of the HBV vaccine, the vaccine is not always protective, and children born of infected mothers become chronically infected. Current treatments include antiviral nucleoside analogs, but eventually this treatment results in viral resistance (2). In advanced stage HCC, targeted therapies such as sorafenib (anti-MAPK) are of modest but significant benefit (3). Thus, new and effective mechanism-based therapies are needed, along with new prognostic markers for molecular staging of the disease (4). Pathogenesis of HBV-mediatedHCC involves effects of chronic inflammation of the liver (5) and effects of the HBV X protein (HBx), which acts as a weak oncogene (6) or co-factor in hepatocarcinogenesis (7).HBVDNA integrates into the host genomeat early steps of clonal tumor expansion, and themajority of tumors display continued expression ofHBx (8).HBx is amultifunctional protein (9, 10), is required for the HBV life cycle (11), and is implicated in HCC pathogenesis by a mechanism not yet understood. Regarding thismechanism, our studies (12, 13) discovered an inverse relationship between the protein levels of two transcription repressive factors, SUZ12 and ZNF198, and the mitotic polo-like kinase1 (Plk1; ref. 14). This intriguing relationship between Plk1, SUZ12, and ZNF198 was observed in cellular and animal models of HBxand HBV-mediated oncogenic transformation as well as in HBV-replicating cells (15). SUZ12 is one of the three essential subunits of the transcription repressive PRC2 complex (polycomb repressive complex 2). The other two core subunits of PRC2 include EED protein and the histone methyltransferase EZH2 (16). This chromatin-modifying complex epigenetically regulates lineage selection during embryonic development and stem cell differentiation (17, 18) by trimethylating histone3 on lysine 27 (H3K27me3), a transcription silencing modification (16). PRC2 forms multiprotein complexes with other chromatin-modifying proteins (19, 20), and associates with noncoding RNAs (21). PRC2 binds the long noncoding RNA (lncRNA) HOTAIR, which also binds another transcription repressive complex composed of lysine demethylase1 (LSD1), Co-REST, and histone deacetylase 1 (HDAC1; ref. 22). Interestingly, the LSD1/Co-REST/HDAC1 complex, Department of Basic Medical Sciences and Purdue Center for Cancer Research, Purdue University, West Lafayette Indiana. Centre de Recherche en Canc erologie de Lyon, UMR INSERM 1052, CNRS 5286, Lyon Cedex, France. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). A. Diab and H. Fan contributed equally to this article. Corresponding Author: Ourania Andrisani, Purdue University, B034 Hansen Bldg, 201 South University St, West Lafayette, IN 47907. Phone: 765-494-8131; Fax: 765-494-0781; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-14-2928 2015 American Association for Cancer Research. Cancer Research www.aacrjournals.org 2363 on July 29, 2017. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst April 8, 2015; DOI: 10.1158/0008-5472.CAN-14-2928