3172

HNF4A

FRTS4|HNF4|HNF4a7|HNF4a8|HNF4a9|HNF4alpha|MODY|MODY1|NR2A1|NR2A21|TCF|TCF14;hepatocyte nuclear factor 4, alpha;HNF4A;hepatocyte nuclear factor 4, alpha

HNF4alpha10/11/12|TCF-14|hepatic nuclear factor 4 alpha|hepatocyte nuclear factor 4-alpha|nuclear receptor subfamily 2 group A member 1|transcription factor 14|transcription factor HNF-4

20q13.12

protein-coding

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates genes involved in lipid and bile acid synthesis, gluconeogenesis, amino acid metabolism, and blood coagulation. In addition to its metabolic role, HNF4alpha is critical for hepatocyte differentiation, and loss of HNF4alpha is associated with hepatocellular carcinoma. The hepatocyte-specific Hnf4a knock-out mouse develops severe hepatomegaly and steatosis resulting in premature death, thereby limiting studies of the role of this transcription factor in the adult animal. In addition, gene compensation may complicate analysis of the phenotype of these mice. To overcome these issues, an acute Hnf4a knock-out mouse model was generated through use of the tamoxifen-inducible ErT2cre coupled to the serum albumin gene promoter. Microarray expression analysis revealed up-regulation of genes associated with proliferation and cell cycle control only in the acute liver-specific Hnf4alpha-null mouse. BrdU and ki67 staining confirmed extensive hepatocyte proliferation in this model. Proliferation was associated with induction of the hepatomitogen Bmp7 as well as reduced basal apoptotic activity. The p53/p63 apoptosis effector gene Perp was further identified as a direct HNF4alpha target gene. These data suggest that HNF4alpha maintains hepatocyte differentiation in the adult healthy liver, and its loss may directly contribute to hepatocellular carcinoma development, thus indicating this factor as a possible liver tumor suppressor gene.

BACKGROUND & AIMS: The tumor fate derives from cell autonomous properties and niche microenvironmental cues. The transforming growth factor beta (TGFbeta) 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 HNF4alpha 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 TGFbeta on the anti-EMT and tumor suppressor HNF4alpha 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 HNF4alpha DNA binding activity. HNF4alpha post-translational modifications (PTMs) were assessed by 2-DE analysis. GSK3beta activity was modulated by chemical inhibition and constitutive active mutant expression. RESULTS: We demonstrated that the presence of TGFbeta impairs the efficiency of HNF4alpha as tumor suppressor. We found that TGFbeta induces HNF4alpha PTMs that correlate with the early loss of HNF4alpha DNA binding activity on target gene promoters. Furthermore, we identified the GSK3beta kinase as one of the TGFbeta targets mediating HNF4alpha functional inactivation: GSK3beta chemical inhibition results in HNF4alpha DNA binding impairment while a constitutively active GSK3beta mutant impairs the TGFbeta-induced inhibitory effect on HNF4alpha tumor suppressor activity. CONCLUSIONS: Our data identify in the dominance of TGFbeta a limit for the HNF4alpha-mediated gene therapy of HCC.

Hepatocyte nuclear factor 4 alpha (HNF4alpha), the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation by way of unknown mechanisms. We investigated the mechanisms of HNF4alpha-induced inhibition of hepatocyte proliferation using a novel tamoxifen (TAM)-inducible, hepatocyte-specific HNF4alpha knockdown mouse model. Hepatocyte-specific deletion of HNF4alpha in adult mice resulted in increased hepatocyte proliferation, with a significant increase in liver-to-body-weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that a significant number of up-regulated genes following deletion of HNF4alpha were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4alpha deletion. To determine whether deletion of HNF4alpha affects cancer pathogenesis, HNF4alpha knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4alpha significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4alpha-deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4alpha knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4alpha in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation.