title: A CCRK-EZH2 epigenetic circuitry drives hepatocarcinogenesis and associates with tumor recurrence and poor survival of patients.
abstract: OBJECTIVE
  Aberrant chromatin modification is a key feature of hepatocellular carcinoma (HCC), which is characterized by strong sexual dimorphism. Both enhancer of zeste homolog 2 (EZH2) and cell cycle-related kinase (CCRK) contribute to hepatocarcinogenesis, yet whether the two oncogenic factors have functional crosstalk is unknown.
  
  
  METHODS
  Cellular proliferation and tumorigenicity upon transgenic expression and RNA interference were determined by colony formation and soft agar assays, xenograft, orthotopic and diethylnitrosamine-induced HCC models. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Protein levels in clinical specimens were correlated with clinicopathological parameters and patient survival rates.
  
  
  RESULTS
  Ectopic CCRK expression in immortalized human liver cells increased EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to stimulate proliferation and tumor formation. Conversely, knockdown of CCRK reduced EZH2/H3K27me3 levels and decreased HCC cell growth, which could be rescued by EZH2 over-expression. Mechanistically, GSK-3β phosphorylation by CCRK activated a β-catenin/TCF/E2F1/EZH2 transcriptional feedback loop to epigenetically enhance androgen receptor (AR) signaling. Simultaneously, the phosphorylation of AKT/EZH2 by CCRK facilitated the co-occupancy of CCRK promoter by EZH2-AR and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. Lentiviral-mediated knockdown of CCRK, which abrogated the phosphorylation-transcriptional network, prevented diethylnitrosamine-induced tumorigenicity. More importantly, the hyperactivation of the CCRK-EZH2 circuitry in human HCCs correlated with tumor recurrence and poor survival.
  
  
  CONCLUSIONS
  These findings uncover an epigenetic vicious cycle in hepatocarcinogenesis that operates through reciprocal regulation of CCRK and EZH2, providing novel therapeutic strategy for HCC.
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China; State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Yu
authors.affiliation: Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Lee
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: May S
authors.initials: MS
authors.lastname: Li
authors.affiliation: Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Minnie Y Y
authors.initials: MY
authors.lastname: Go
authors.affiliation: Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Yue-Sun
authors.initials: YS
authors.lastname: Cheung
authors.affiliation: State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Paul B S
authors.initials: PB
authors.lastname: Lai
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Andrew M L
authors.initials: AM
authors.lastname: Chan
authors.affiliation: State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Ka-Fai
authors.initials: KF
authors.lastname: To
authors.affiliation: State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China; Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Henry L Y
authors.initials: HL
authors.lastname: Chan
authors.affiliation: State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Joseph J Y
authors.initials: JJ
authors.lastname: Sung
authors.affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China; State Key Laboratory of Digestive Disease and Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong, China. Electronic address: alfredcheng@cuhk.edu.hk.
authors.forename: Alfred S L
authors.initials: AS
authors.lastname: Cheng
mesh_terms: D000818:Animals; D063646:Carcinogenesis / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D000066630:Ectopic Gene Expression; D000071221:Enhancer of Zeste Homolog 2 Protein; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009364:Neoplasm Recurrence, Local / Q000235:genetics; D063151:Polycomb Repressive Complex 2 / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics; D015996:Survival Rate; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D035683:MicroRNAs; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase
keywords: Androgen receptor; Chromatin modifications; Gender disparity; Hepatocellular carcinoma; Kinase