title: TLR4 Signaling via NANOG Cooperates With STAT3 to Activate Twist1 and Promote Formation of Tumor-Initiating Stem-Like Cells in Livers of Mice.
abstract: OBJECTIVE
  Obesity and alcohol consumption contribute to steatohepatitis, which increases the risk for hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCCs). Mouse hepatocytes that express HCV-NS5A in liver up-regulate the expression of Toll-like receptor 4 (TLR4), and develop liver tumors containing tumor-initiating stem-like cells (TICs) that express NANOG. We investigated whether the TLR4 signals to NANOG to promote the development of TICs and tumorigenesis in mice placed on a Western diet high in cholesterol and saturated fat (HCFD).
  
  
  METHODS
  We expressed HCV-NS5A from a transgene (NS5A Tg) in Tlr4-/- (C57Bl6/10ScN), and wild-type control mice. Mice were fed a HCFD for 12 months. TICs were identified and isolated based on being CD133+, CD49f+, and CD45-. We obtained 142 paraffin-embedded sections of different stage HCCs and adjacent nontumor areas from the same patients, and performed gene expression, immunofluorescence, and immunohistochemical analyses.
  
  
  RESULTS
  A higher proportion of NS5A Tg mice developed liver tumors (39%) than mice that did not express HCV NS5A after the HCFD (6%); only 9% of Tlr4-/- NS5A Tg mice fed HCFD developed liver tumors. Livers from NS5A Tg mice fed the HCFD had increased levels of TLR4, NANOG, phosphorylated signal transducer and activator of transcription (pSTAT3), and TWIST1 proteins, and increases in Tlr4, Nanog, Stat3, and Twist1 messenger RNAs. In TICs from NS5A Tg mice, NANOG and pSTAT3 directly interact to activate expression of Twist1. Levels of TLR4, NANOG, pSTAT3, and TWIST were increased in HCC compared with nontumor tissues from patients.
  
  
  CONCLUSIONS
  HCFD and HCV-NS5A together stimulated TLR4-NANOG and the leptin receptor (OB-R)-pSTAT3 signaling pathways, resulting in liver tumorigenesis through an exaggerated mesenchymal phenotype with prominent Twist1-expressing TICs.
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine of University of Southern California, Los Angeles, California.
authors.forename: Dinesh Babu
authors.initials: DB
authors.lastname: Uthaya Kumar
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine of University of Southern California, Los Angeles, California.
authors.forename: Chia-Lin
authors.initials: CL
authors.lastname: Chen
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine of University of Southern California, Los Angeles, California.
authors.forename: Jian-Chang
authors.initials: JC
authors.lastname: Liu
authors.affiliation: Department of Pathology, Keck School of Medicine of University of Southern California, Los Angeles, California.
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Feldman
authors.affiliation: Department of Surgery, Keck School of Medicine of University of Southern California, Los Angeles, California.
authors.forename: Linda S
authors.initials: LS
authors.lastname: Sher
authors.affiliation: Department of Pathology, Harbor-University California Los Angeles Medical Center, Los Angeles, California.
authors.forename: Samuel
authors.initials: S
authors.lastname: French
authors.affiliation: Department of Surgery, Keck School of Medicine of University of Southern California, Los Angeles, California.
authors.forename: Joseph
authors.initials: J
authors.lastname: DiNorcia
authors.affiliation: Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California; Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California.
authors.forename: Samuel W
authors.initials: SW
authors.lastname: French
authors.affiliation: Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: Bita V
authors.initials: BV
authors.lastname: Naini
authors.affiliation: Department of Surgery, University of California Los Angeles School of Medicine, Los Angeles, California.
authors.forename: Sunhawit
authors.initials: S
authors.lastname: Junrungsee
authors.affiliation: Department of Surgery, University of California Los Angeles School of Medicine, Los Angeles, California.
authors.forename: Vatche Garen
authors.initials: VG
authors.lastname: Agopian
authors.affiliation: Department of Surgery, University of California Los Angeles School of Medicine, Los Angeles, California.
authors.forename: Ali
authors.initials: A
authors.lastname: Zarrinpar
authors.affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine of University of Southern California, Los Angeles, California; Southern California Research Center for Alcoholic Liver and Pancreatic Disease and Cirrhosis, Los Angeles, California. Electronic address: keigo.machida@med.usc.edu.
authors.forename: Keigo
authors.initials: K
authors.lastname: Machida
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics; D002460:Cell Line; D002465:Cell Movement; D000066673:Cell Self Renewal; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D059305:Diet, High-Fat; D004195:Disease Models, Animal; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008875:Middle Aged; D000071317:Nanog Homeobox Protein; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D065626:Non-alcoholic Fatty Liver Disease / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D001057:Apolipoproteins E; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; D009687:Nuclear Proteins; D050796:STAT3 Transcription Factor; D051197:Toll-Like Receptor 4; D051879:Twist-Related Protein 1; D017361:Viral Nonstructural Proteins; C475540:Nanog protein, mouse; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; C495345:TLR4 protein, human; C103173:TWIST1 protein, human; C493487:Tlr4 protein, mouse; C067250:Twist1 protein, mouse; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: HCC; HCV; NASH; Obesity