EPS15R, TASP1, and PRPF3 are novel disease candidate genes targeted by HNF4alpha splice variants in hepatocellular carcinomas.
OBJECTIVE
The orphan nuclear receptor HNF4alpha is a member of the hepatic transcription factor network. This protein plays a pivotal role in liver development and hepatocellular differentiation. Nine splice variants have been identified, some of which are specifically regulated in disease. The role of HNF4alpha splice variants in hepatocellular carcinomas (HCC) is unknown. Here, we report an identification of novel candidate genes targeted by splice variants of HNF4alpha.


METHODS
We used chromatin immunoprecipitation followed by cloning and sequencing of DNA. Expression of HNF4alpha P1 and P2 promoter-driven isoforms and of genes targeted by HNF4alpha were analyzed by quantitative reverse-transcription polymerase chain reaction, Western blotting, electrophoretic mobility shift assay, and immunohistochemistry.


RESULTS
We observed a remarkable switch in gene and protein expression from P1 to P2 promoter-driven fetal isoforms of HNF4alpha in transgenic livers and HCCs of epidermal growth factor (EGF) overexpressing mice and in human HCCs. We further identified EGF-receptor substrate (EPS15R), related EPS15, the premessenger RNA processing factor 3 (PRPF3), and taspase 1 (TASP1) as novel HNF4alpha disease regulated genes with induced expression in mouse and in human HCCs. We suggest EPS15 and EPS15R mediated internalization of activated EGF receptor to result in receptor recycling as to reinforce the proliferative response to EGF. Regulation of the type 2 asparaginase TASP1 and of the splicing factor PRPF3 further documents a switch to fetal liver programs in HCC.


CONCLUSIONS
We report induction of P2 promoter-driven HNF4alpha splice variants and regulation of disease candidate genes in EGF-induced mouse and human HCC.
Niehof
Monika
M
Fraunhofer Institute of Toxicology and Experimental Medicine, Center of Drug Research and Medical Biotechnology, Hannover, Germany.
Borlak
Jürgen
J
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D014408:Biomarkers, Tumor; D015153:Blotting, Western; D018938:Caco-2 Cells; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D024202:Electrophoretic Mobility Shift Assay; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D051557:Hepatocyte Nuclear Factor 4 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012326:RNA Splicing; D012334:RNA, Neoplasm / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000096:biosynthesis / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; C094318:Eps15-rs protein, mouse; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C108507:PRPF3 protein, human; D010750:Phosphoproteins; D020033:Protein Isoforms; D012334:RNA, Neoplasm; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D010450:Endopeptidases; C480823:taspase1, human; C513958:taspase1, mouse