title: Zinc fingers and homeoboxes 2 inhibits hepatocellular carcinoma cell proliferation and represses expression of Cyclins A and E.
abstract: OBJECTIVE
  Zinc-fingers and homeoboxes 2 (ZHX2) represses transcription of several genes associated with liver cancer. However, little is known about the role of ZHX2 in the development of hepatocellular carcinoma (HCC). We investigated the mechanisms by which ZHX2 might affect proliferation of HCC cells.
  
  
  METHODS
  We overexpressed and knocked down ZHX2 in HCC cells and analyzed the effects on proliferation, colony formation, and the cell cycle. We also analyzed the effects of ZHX2 overexpression in growth of HepG2.2.15 tumor xenografts in nude mice. Chromatin immunoprecipitation and luciferase reporter assays were used to measure binding of ZHX2 target promoters. Levels of ZHX2 in HCC samples were evaluated by immunohistochemistry.
  
  
  RESULTS
  ZHX2 overexpression significantly reduced proliferation of HCC cells and growth of tumor xenografts in mice; it led to G1 arrest and reduced levels of Cyclins A and E in HCC cell lines. ZHX2 bound to promoter regions of CCNA2 (which encodes Cyclin A) and CCNE1 (which encodes Cyclin E) and inhibited their transcription. Knockdown of Cyclin A or Cyclin E reduced the increased proliferation mediated by ZHX2 knockdown. Nuclear localization of ZHX2 was required for it to inhibit proliferation of HCC cells in culture and in mice. Nuclear localization of ZHX2 was reduced in human HCC samples, even in small tumors (diameter, <5 cm), compared with adjacent nontumor tissues. Moreover, reduced nuclear levels of ZHX2 correlated with reduced survival times of patients, high levels of tumor microvascularization, and hepatocyte proliferation.
  
  
  CONCLUSIONS
  ZHX2 inhibits HCC cell proliferation by preventing expression of Cyclins A and E, and reduces growth of xenograft tumors in mice. Loss of nuclear ZHX2 might be an early step in the development of HCC.
authors.affiliation: Key Laboratory for Experimental Teratology of Ministry of Education and Institute of Immunology, Shandong University School of Medicine, Jinan, Shandong, People's Republic of China.
authors.forename: Xuetian
authors.initials: X
authors.lastname: Yue
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Zhang
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liang
authors.forename: Lifen
authors.initials: L
authors.lastname: Gao
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Zhang
authors.forename: Di
authors.initials: D
authors.lastname: Zhao
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.forename: Hongxin
authors.initials: H
authors.lastname: Ma
authors.forename: Min
authors.initials: M
authors.lastname: Guo
authors.forename: Brett T
authors.initials: BT
authors.lastname: Spear
authors.forename: Yaoqin
authors.initials: Y
authors.lastname: Gong
authors.forename: Chunhong
authors.initials: C
authors.lastname: Ma
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D055785:Gene Knockdown Techniques; D018398:Homeodomain Proteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D019925:Cyclin A; D019927:Cyclin E; D018398:Homeodomain Proteins; C481543:Zhx2 protein, mouse