title: TGFβ overrides HNF4α tumor suppressing activity through GSK3β inactivation: implication for hepatocellular carcinoma gene therapy.
abstract: OBJECTIVE
  The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor β (TGFβ) is a major microenvironmental factor for hepatocellular carcinoma (HCC) influencing tumor dedifferentiation, induction of epithelial-to-mesenchymal transition (EMT) and acquisition of metastatic properties. The loss of the transcriptional factor HNF4α is a predominant mechanism through which HCCs progress to a more aggressive phenotype; its re-expression, reducing tumor formation and repressing EMT program, has been suggested as a therapeutic tool for HCC gene therapy. We investigated the influence of TGFβ on the anti-EMT and tumor suppressor HNF4α activity.
  
  
  METHODS
  Cell motility and invasion were analyzed by wound healing and invasion assays. EMT was evaluated by RT-qPCR and immunofluorescence. ChIP and EMSA assays were utilized for investigation of the HNF4α DNA binding activity. HNF4α post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3β activity was modulated by chemical inhibition and constitutive active mutant expression.
  
  
  RESULTS
  We demonstrated that the presence of TGFβ impairs the efficiency of HNF4α as tumor suppressor. We found that TGFβ induces HNF4α PTMs that correlate with the early loss of HNF4α DNA binding activity on target gene promoters. Furthermore, we identified the GSK3β kinase as one of the TGFβ targets mediating HNF4α functional inactivation: GSK3β chemical inhibition results in HNF4α DNA binding impairment while a constitutively active GSK3β mutant impairs the TGFβ-induced inhibitory effect on HNF4α tumor suppressor activity.
  
  
  CONCLUSIONS
  Our data identify in the dominance of TGFβ a limit for the HNF4α-mediated gene therapy of HCC.
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, University La Sapienza, Rome, Italy.
authors.forename: Angela Maria
authors.initials: AM
authors.lastname: Cozzolino
authors.forename: Tonino
authors.initials: T
authors.lastname: Alonzi
authors.forename: Laura
authors.initials: L
authors.lastname: Santangelo
authors.forename: Carmine
authors.initials: C
authors.lastname: Mancone
authors.forename: Beatrice
authors.initials: B
authors.lastname: Conti
authors.forename: Corinna
authors.initials: C
authors.lastname: Steindler
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Musone
authors.forename: Carla
authors.initials: C
authors.lastname: Cicchini
authors.forename: Marco
authors.initials: M
authors.lastname: Tripodi
authors.forename: Alessandra
authors.initials: A
authors.lastname: Marchetti
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000556:secondary / Q000628:therapy; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D016147:Genes, Tumor Suppressor / Q000502:physiology; D015316:Genetic Therapy; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D051379:Mice; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D059016:Tumor Microenvironment / Q000235:genetics / Q000502:physiology
chemical_list: C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; D016212:Transforming Growth Factor beta; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3