title: Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis.
abstract: Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with telomerase activation as first oncogenic hit. To elucidate critical downstream genetic events driving further transformation of immortalized liver cells, we used retroviral insertional mutagenesis as an unbiased approach to induce genetic alterations. Following isolation of hyperproliferating, provirus-bearing cell clones, we monitored cancer-associated growth properties and characterized changes toward a malignant phenotype. Three transformed clones with the ability to form colonies in soft agar were expanded. As proof-of-principle for our experimental setup, we identified a transforming insertion on chromosome 8 within the pleiomorphic adenoma gene 1 (PLAG1), resulting in a 20-fold increase in PLAG1 expression. Upregulation of PLAG1 has already been described to promote human hepatoblastoma development. In a separate clone, a transforming insertion was detected in close proximity to the receptor-interacting serine-threonine kinase 4 (RIPK4) with an approximately eightfold suppression in RIPK4 expression. As validation for this currently unknown driver in hepatocarcinogenesis, we examined RIPK4 expression in human HCC samples and confirmed a significant suppression of RIPK4 in 80% of the samples. Furthermore, overexpression of RIPK4 in transformed human fetal hepatocytes resulted in an almost complete elimination of anchorage-independent growth. On the basis of these data, we propose RIPK4 as a novel putative tumor suppressor in human hepatocarcinogenesis.
authors.affiliation: Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Heim
authors.affiliation: Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Cornils
authors.affiliation: Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Schulze
authors.affiliation: Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Fehse
authors.affiliation: Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: A W
authors.initials: AW
authors.lastname: Lohse
authors.affiliation: Department of Hematology and Oncology, University Medical Center of the Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany.
authors.forename: T H
authors.initials: TH
authors.lastname: Brümmendorf
authors.affiliation: Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Wege
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D002999:Clone Cells / Q000378:metabolism; D005333:Fetus; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; C417187:RIPK4 protein, human; D017346:Protein Serine-Threonine Kinases; D019098:Telomerase