EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes

      Background & Aims

      Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis.


      Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients.


      EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP+/Wnt+ cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection.


      The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs.

      Lay summary

      In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions.

      Graphical abstract


      cccDNA (circular covalently closed DNA), EpCAM (epithelial cell adhesion molecule), EpICD (EpCAM intracellular domain), hCSCs (hepatic cancer stem cells), HBV (hepatitis B virus), HBc (hepatitis B virus core antigen), HCC (hepatocellular carcinoma), PRC2 (polycomb repressor complex 2), RIP (regulated intramembrane proteolysis)


      Linked Article

      • Stemness of liver cancer: From hepatitis B virus to Wnt activation
        Journal of HepatologyVol. 65Issue 5
        • Preview
          Hepatocellular carcinoma (HCC) is a heterogeneous disease in terms of etiology, morphology, biological behavior, response to treatment, and clinical outcome. Efforts have been made on classifying HCCs according to the status of gene mutations, chromosomal aberrations, gene/protein expression, and epigenetic modification in order to find hidden molecular features that can explain this heterogeneity [1,2]. The recent advances in molecular classification and the re-emergence of a cancer stem cell (CSC) hypothesis have highlighted the central role of stemness in HCC pathogenesis [3].
        • Full-Text
        • PDF
      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Beasley R.P.
        • Hwang L.Y.
        • Lin C.C.
        • Chien C.S.
        Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22,707 men in Taiwan.
        Lancet. 1981; 2: 1129-1133
        • El-Serag H.B.
        • Rudolph K.L.
        Hepatocellular carcinoma: epidemiology and molecular carcinogenesis.
        Gastroenterology. 2007; 132: 2557-2576
        • Colombo M.
        • Iavarone M.
        Role of antiviral treatment for HCC prevention.
        Best Pract Res Clin Gastroenterol. 2014; 28: 771-781
        • Zoulim F.
        • Locarnini S.
        Hepatitis B virus resistance to nucleos(t)ide analogues.
        Gastroenterology. 2009; 137 (e1591–e1592): 1593-1608
        • Llovet J.M.
        • Ricci S.
        • Mazzaferro V.
        • Hilgard P.
        • Gane E.
        • Blanc J.F.
        • et al.
        Sorafenib in advanced hepatocellular carcinoma.
        N Engl J Med. 2008; 359: 378-390
        • Xie B.
        • Wang D.H.
        • Spechler S.J.
        Sorafenib for treatment of hepatocellular carcinoma: a systematic review.
        Dig Dis Sci. 2012; 57: 1122-1129
        • Mishra L.
        • Banker T.
        • Murray J.
        • Byers S.
        • Thenappan A.
        • He A.R.
        • et al.
        Liver stem cells and hepatocellular carcinoma.
        Hepatology. 2009; 49: 318-329
        • Fernando J.
        • Malfettone A.
        • Cepeda E.B.
        • Vilarrasa-Blasi R.
        • Bertran E.
        • Raimondi G.
        • et al.
        A mesenchymal-like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells.
        Int J Cancer. 2015; 136: E161-E172
        • Yamashita T.
        • Ji J.
        • Budhu A.
        • Forgues M.
        • Yang W.
        • Wang H.Y.
        • et al.
        EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.
        Gastroenterology. 2009; 136: 1012-1024
        • Ma S.
        • Chan K.W.
        • Hu L.
        • Lee T.K.
        • Wo J.Y.
        • Ng I.O.
        • et al.
        Identification and characterization of tumorigenic liver cancer stem/progenitor cells.
        Gastroenterology. 2007; 132: 2542-2556
        • Yamashita T.
        • Honda M.
        • Nakamoto Y.
        • Baba M.
        • Nio K.
        • Hara Y.
        • et al.
        Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma.
        Hepatology. 2013; 57: 1484-1497
        • Mima K.
        • Okabe H.
        • Ishimoto T.
        • Hayashi H.
        • Nakagawa S.
        • Kuroki H.
        • et al.
        CD44s regulates the TGF-beta-mediated mesenchymal phenotype and is associated with poor prognosis in patients with hepatocellular carcinoma.
        Cancer Res. 2012; 72: 3414-3423
        • Zhao W.
        • Wang L.
        • Han H.
        • Jin K.
        • Lin N.
        • Guo T.
        • et al.
        1B50-1, a mAb raised against recurrent tumor cells, targets liver tumor-initiating cells by binding to the calcium channel alpha2delta1 subunit.
        Cancer Cell. 2013; 23: 541-556
        • Rountree C.B.
        • Mishra L.
        • Willenbring H.
        Stem cells in liver diseases and cancer: recent advances on the path to new therapies.
        Hepatology. 2012; 55: 298-306
        • Oishi N.
        • Yamashita T.
        • Kaneko S.
        Molecular biology of liver cancer stem cells.
        Liver Cancer. 2014; 3: 71-84
        • Tarlow B.D.
        • Pelz C.
        • Naugler W.E.
        • Wakefield L.
        • Wilson E.M.
        • Finegold M.J.
        • et al.
        Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
        Cell Stem Cell. 2014; 15: 605-618
        • Quasdorff M.
        • Hosel M.
        • Odenthal M.
        • Zedler U.
        • Bohne F.
        • Gripon P.
        • et al.
        A concerted action of HNF4alpha and HNF1alpha links hepatitis B virus replication to hepatocyte differentiation.
        Cell Microbiol. 2008; 10: 1478-1490
        • Wang W.H.
        • Studach L.L.
        • Andrisani O.M.
        Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication.
        Hepatology. 2011; 53: 1137-1147
        • Studach L.L.
        • Menne S.
        • Cairo S.
        • Buendia M.A.
        • Hullinger R.L.
        • Lefrancois L.
        • et al.
        Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein.
        Hepatology. 2012; 56: 1240-1251
        • Margueron R.
        • Reinberg D.
        The Polycomb complex PRC2 and its mark in life.
        Nature. 2011; 469: 343-349
        • Thornton S.R.
        • Butty V.L.
        • Levine S.S.
        • Boyer L.A.
        Polycomb Repressive Complex 2 regulates lineage fidelity during embryonic stem cell differentiation.
        PLoS One. 2014; 9 (e110498)
        • Terradillos O.
        • Billet O.
        • Renard C.A.
        • Levy R.
        • Molina T.
        • Briand P.
        • et al.
        The hepatitis B virus X gene potentiates c-myc-induced liver oncogenesis in transgenic mice.
        Oncogene. 1997; 14: 395-404
        • Madden C.R.
        • Finegold M.J.
        • Slagle B.L.
        Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions.
        J Virol. 2001; 75: 3851-3858
        • Zhang H.
        • Diab A.
        • Fan H.
        • Mani S.K.
        • Hullinger R.
        • Merle P.
        • et al.
        PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12 and ZNF198 during Hepatitis B Virus-Induced Liver Carcinogenesis.
        Cancer Res. 2015; 75: 2363-2374
        • de Boer C.J.
        • van Krieken J.H.
        • Janssen-van Rhijn C.M.
        • Litvinov S.V.
        Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver.
        J Pathol. 1999; 188: 201-206
        • Dolle L.
        • Theise N.D.
        • Schmelzer E.
        • Boulter L.
        • Gires O.
        • van Grunsven L.A.
        EpCAM and the biology of hepatic stem/progenitor cells.
        Am J Physiol Gastrointest Liver Physiol. 2015; 308: G233-G250
        • Maetzel D.
        • Denzel S.
        • Mack B.
        • Canis M.
        • Went P.
        • Benk M.
        • et al.
        Nuclear signalling by tumour-associated antigen EpCAM.
        Nat Cell Biol. 2009; 11: 162-171
        • Cole M.F.
        • Johnstone S.E.
        • Newman J.J.
        • Kagey M.H.
        • Young R.A.
        Tcf3 is an integral component of the core regulatory circuitry of embryonic stem cells.
        Genes Dev. 2008; 22: 746-755
        • Marson A.
        • Foreman R.
        • Chevalier B.
        • Bilodeau S.
        • Kahn M.
        • Young R.A.
        • et al.
        Wnt signaling promotes reprogramming of somatic cells to pluripotency.
        Cell Stem Cell. 2008; 3: 132-135
        • Pasini D.
        • Bracken A.P.
        • Hansen J.B.
        • Capillo M.
        • Helin K.
        The polycomb group protein Suz12 is required for embryonic stem cell differentiation.
        Mol Cell Biol. 2007; 27: 3769-3779
        • Chamberlain S.J.
        • Yee D.
        • Magnuson T.
        Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency.
        Stem Cells. 2008; 26: 1496-1505
        • Ladner S.K.
        • Otto M.J.
        • Barker C.S.
        • Zaifert K.
        • Wang G.H.
        • Guo J.T.
        • et al.
        Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.
        Antimicrob Agents Chemother. 1997; 41: 1715-1720
        • Ni Y.
        • Lempp F.A.
        • Mehrle S.
        • Nkongolo S.
        • Kaufman C.
        • Falth M.
        • et al.
        Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.
        Gastroenterology. 2014; 146: 1070-1083
        • Pez F.
        • Lopez A.
        • Kim M.
        • Wands J.R.
        • Caron de Fromentel C.
        • Merle P.
        Wnt signaling and hepatocarcinogenesis: molecular targets for the development of innovative anticancer drugs.
        J Hepatol. 2013; 59: 1107-1117
        • Bracken A.P.
        • Dietrich N.
        • Pasini D.
        • Hansen K.H.
        • Helin K.
        Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.
        Genes Dev. 2006; 20: 1123-1136
        • Seeger C.
        • Mason W.S.
        Molecular biology of hepatitis B virus infection.
        Virology. 2015; 479–480C: 672-686
        • Tarn C.
        • Bilodeau M.L.
        • Hullinger R.L.
        • Andrisani O.M.
        Differential immediate early gene expression in conditional hepatitis B virus pX-transforming versus nontransforming hepatocyte cell lines.
        J Biol Chem. 1999; 274: 2327-2336
        • Wu J.C.
        • Merlino G.
        • Fausto N.
        Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha.
        Proc Natl Acad Sci U S A. 1994; 91: 674-678
        • Amaddeo G.
        • Cao Q.
        • Ladeiro Y.
        • Imbeaud S.
        • Nault J.C.
        • Jaoui D.
        • et al.
        Integration of tumour and viral genomic characterisations in HBV-related hepatocellular carcinomas.
        Gut. 2015; 64: 820-829
        • Kim N.H.
        • Kim H.S.
        • Kim N.G.
        • Lee I.
        • Choi H.S.
        • Li X.Y.
        • et al.
        P53 and microRNA-34 are suppressors of canonical Wnt signaling.
        Sci Signal. 2011; 4: ra71
        • Boyault S.
        • Rickman D.S.
        • de Reynies A.
        • Balabaud C.
        • Rebouissou S.
        • Jeannot E.
        • et al.
        Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
        Hepatology. 2007; 45: 42-52
        • Li T.
        • Chen H.
        • Li W.
        • Cui J.
        • Wang G.
        • Hu X.
        • et al.
        Promoter histone H3K27 methylation in the control of IGF2 imprinting in human tumor cell lines.
        Hum Mol Genet. 2014; 23: 117-128
        • Sarma K.
        • Cifuentes-Rojas C.
        • Ergun A.
        • Del Rosario A.
        • Jeon Y.
        • White F.
        • et al.
        ATRX Directs Binding of PRC2 to Xist RNA and Polycomb Targets.
        Cell. 2014; 159: 869-883
        • Lachenmayer A.
        • Alsinet C.
        • Savic R.
        • Cabellos L.
        • Toffanin S.
        • Hoshida Y.
        • et al.
        Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.
        Clin Cancer Res. 2012; 18: 4997-5007
        • Huang H.P.
        • Chen P.H.
        • Yu C.Y.
        • Chuang C.Y.
        • Stone L.
        • Hsiao W.C.
        • et al.
        Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.
        J Biol Chem. 2011; 286: 33520-33532
        • Lin C.W.
        • Liao M.Y.
        • Lin W.W.
        • Wang Y.P.
        • Lu T.Y.
        • Wu H.C.
        Epithelial cell adhesion molecule regulates tumor initiation and tumorigenesis via activating reprogramming factors and epithelial-mesenchymal transition gene expression in colon cancer.
        J Biol Chem. 2012; 287: 39449-39459
        • Yamashita T.
        • Budhu A.
        • Forgues M.
        • Wang X.W.
        Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma.
        Cancer Res. 2007; 67: 10831-10839
        • Yamashita T.
        • Forgues M.
        • Wang W.
        • Kim J.W.
        • Ye Q.
        • Jia H.
        • et al.
        EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma.
        Cancer Res. 2008; 68: 1451-1461
        • De Jesus-Acosta A.
        • Laheru D.
        • Maitra A.
        • Arcaroli J.
        • Rudek M.A.
        • Dasari A.
        • et al.
        A phase II study of the gamma secretase inhibitor RO4929097 in patients with previously treated metastatic pancreatic adenocarcinoma.
        Invest New Drugs. 2014; 32: 739-745
        • Richter S.
        • Bedard P.L.
        • Chen E.X.
        • Clarke B.A.
        • Tran B.
        • Hotte S.J.
        • et al.
        A phase I study of the oral gamma secretase inhibitor R04929097 in combination with gemcitabine in patients with advanced solid tumors (PHL-078/CTEP 8575).
        Invest New Drugs. 2014; 32: 243-249
        • Le P.N.
        • McDermott J.D.
        • Jimeno A.
        Targeting the Wnt pathway in human cancers: therapeutic targeting with a focus on OMP-54F28.
        Pharmacol Ther. 2015; 146: 1-11