title: Loss of ATOH8 Increases Stem Cell Features of Hepatocellular Carcinoma Cells.
abstract: OBJECTIVE
  Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines.
  
  
  METHODS
  HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133(+) cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses.
  
  
  RESULTS
  Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133(+) cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice.
  
  
  CONCLUSIONS
  ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Cancer Science Institute of Singapore, National University of Singapore, Singapore.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Song
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Guangjin
authors.initials: G
authors.lastname: Pan
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Anatomy, National University of Singapore, Singapore.
authors.forename: Leilei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Zeng
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore.
authors.forename: Tim Hon
authors.initials: TH
authors.lastname: Man Chan
authors.affiliation: State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Qizhou
authors.initials: Q
authors.lastname: Lian
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Raymond
authors.initials: R
authors.lastname: Chow
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Cai
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Wong
authors.affiliation: State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China.
authors.forename: Yun-Fei
authors.initials: YF
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: Duanqing
authors.initials: D
authors.lastname: Pei
authors.affiliation: Department of Clinical Oncology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Liver Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China. Electronic address: xyguan@hku.hk.
authors.forename: Xin-Yuan
authors.initials: XY
authors.lastname: Guan
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D054337:Cell Dedifferentiation; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival; D018572:Disease-Free Survival; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006023:Glycoproteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D053208:Kaplan-Meier Estimate; D008113:Liver Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D016513:Mice, SCID; D009361:Neoplasm Invasiveness; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D000067492:RNAi Therapeutics; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D000970:Antineoplastic Agents; D051792:Basic Helix-Loop-Helix Proteins; D006023:Glycoproteins; D010455:Peptides; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; C486727:ATOH8 protein, human; C000606023:PROM1 protein, human; C000606024:Prom1 protein, mouse
keywords: Cancer Stem Cells; Chemotherapy Drug Resistance; Tumor Progression; iPSCs