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Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis

Oncogene volume 34, pages 364–372 (2015)Cite this article

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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.

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References

  1. Altekruse SF, McGlynn KA, Reichman ME . Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol 2009; 27: 1485–1491.

    Article  PubMed  PubMed Central  Google Scholar 

  2. El-Serag HB, Rudolph KL . Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557–2576.

    Article  CAS  PubMed  Google Scholar 

  3. Trevisani F, Cantarini MC, Wands JR, Bernardi M . Recent advances in the natural history of hepatocellular carcinoma. Carcinogenesis 2008; 29: 1299–1305.

    Article  CAS  PubMed  Google Scholar 

  4. Brechot C, Pourcel C, Louise A, Rain B, Tiollais P . Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 1980; 286: 533–535.

    Article  CAS  PubMed  Google Scholar 

  5. Forner A, Llovet JM, Bruix J . Hepatocellular carcinoma. Lancet 2012; 379: 1245–1255.

    Article  PubMed  Google Scholar 

  6. Shay JW, Bacchetti S . A survey of telomerase activity in human cancer. Eur J Cancer 1997; 33: 787–791.

    Article  CAS  PubMed  Google Scholar 

  7. Greider CW . Telomeres. Curr Opin Cell Biol 1991; 3: 444–451.

    Article  CAS  PubMed  Google Scholar 

  8. Shay JW, Pereira-Smith OM, Wright WE . A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res 1991; 196: 33–39.

    Article  CAS  PubMed  Google Scholar 

  9. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266: 2011–2015.

    Article  CAS  PubMed  Google Scholar 

  10. Oh BK, Jo CK, Park C, Kim K, Jung LW, Han KH et al. Telomere shortening and telomerase reactivation in dysplastic nodules of human hepatocarcinogenesis. J Hepatol 2003; 39: 786–792.

    Article  CAS  PubMed  Google Scholar 

  11. Oh BK, Kim YJ, Park C, Park YN . Up-regulation of telomere-binding proteins, TRF1, TRF2, and TIN2 is related to telomere shortening during human multistep hepatocarcinogenesis. Am J Pathol 2005; 166: 73–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Takaishi H, Kitamoto M, Takahashi S, Aikata H, Kawakami Y, Nakanishi T et al. Precancerous hepatic nodules had significant levels of telomerase activity determined by sensitive quantitation using a hybridization protection assay. Cancer 2000; 88: 312–317.

    Article  CAS  PubMed  Google Scholar 

  13. Hytiroglou P, Kotoula V, Thung SN, Tsokos M, Fiel MI, Papadimitriou CS . Telomerase activity in precancerous hepatic nodules. Cancer 1998; 82: 1831–1838.

    Article  CAS  PubMed  Google Scholar 

  14. Kojima H, Yokosuka O, Imazeki F, Saisho H, Omata M . Telomerase activity and telomere length in hepatocellular carcinoma and chronic liver disease. Gastroenterology 1997; 112: 493–500.

    Article  CAS  PubMed  Google Scholar 

  15. Komine F, Shimojima M, Moriyama M, Amaki S, Uchida T, Arakawa Y . Telomerase activity of needle-biopsied liver samples: its usefulness for diagnosis and judgement of efficacy of treatment of small hepatocellular carcinoma. J Hepatol 2000; 32: 235–241.

    Article  CAS  PubMed  Google Scholar 

  16. Wege H, Le HT, Chui MS, Liu L, Wu J, Giri R et al. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 2003; 124: 432–444.

    Article  CAS  PubMed  Google Scholar 

  17. Haker B, Fuchs S, Dierlamm J, Brummendorf TH, Wege H . Absence of oncogenic transformation despite acquisition of cytogenetic aberrations in long-term cultured telomerase-immortalized human fetal hepatocytes. Cancer Lett 2007; 256: 120–127.

    Article  CAS  PubMed  Google Scholar 

  18. Wege H, Heim D, Lütgehetmann M, Dierlamm J, Lohse AW, Brümmendorf TH . Forced activation of beta-catenin signaling supports the transformation of hTERT-immortalized human fetal hepatocytes. Mol Cancer Res 2011; 9: 1222–1231.

    Article  CAS  PubMed  Google Scholar 

  19. Akagi K, Suzuki T, Stephens RM, Jenkins NA, Copeland NG . RTCGD: retroviral tagged cancer gene database. Nucleic Acids Res 2004; 32: D523–D527.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hayward WS, Neel BG, Astrin SM . Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature 1981; 290: 475–480.

    Article  CAS  PubMed  Google Scholar 

  21. King W, Patel MD, Lobel LI, Goff SP, Nguyen-Huu MC . Insertion mutagenesis of embryonal carcinoma cells by retroviruses. Science 1985; 228: 554–558.

    Article  CAS  PubMed  Google Scholar 

  22. Li Z, Dullmann J, Schiedlmeier B, Schmidt M, von KC, Meyer J et al. Murine leukemia induced by retroviral gene marking. Science 2002; 296: 497.

    Article  CAS  PubMed  Google Scholar 

  23. Mikkers H, Berns A . Retroviral insertional mutagenesis: tagging cancer pathways. Adv Cancer Res 2003; 88: 53–99.

    CAS  PubMed  Google Scholar 

  24. Kustikova O, Fehse B, Modlich U, Yang M, Dullmann J, Kamino K et al. Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 2005; 308: 1171–1174.

    Article  CAS  PubMed  Google Scholar 

  25. Modlich U, Kustikova OS, Schmidt M, Rudolph C, Meyer J, Li Z et al. Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood 2005; 105: 4235–4246.

    Article  CAS  PubMed  Google Scholar 

  26. Wahlers A, Schwieger M, Li Z, Meier-Tackmann D, Lindemann C, Eckert HG et al. Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells. Gene Ther 2001; 8: 477–486.

    Article  CAS  PubMed  Google Scholar 

  27. Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA et al. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 2003; 101: 2099–2114.

    Article  CAS  PubMed  Google Scholar 

  28. Kermouni A, Van RE, Arden KC, Vermeesch JR, Weiss S, Godelaine D et al. The IL-9 receptor gene (IL9R): genomic structure, chromosomal localization in the pseudoautosomal region of the long arm of the sex chromosomes, and identification of IL9R pseudogenes at 9qter, 10pter, 16pter, and 18pter. Genomics 1995; 29: 371–382.

    Article  CAS  PubMed  Google Scholar 

  29. Weber K, Bartsch U, Stocking C, Fehse B . A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis. Mol Ther 2008; 16: 698–706.

    Article  CAS  PubMed  Google Scholar 

  30. Hoshida Y, Villanueva A, Llovet JM . Molecular profiling to predict hepatocellular carcinoma outcome. Expert Rev Gastroenterol Hepatol 2009; 3: 101–103.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Thorgeirsson SS, Lee JS, Grisham JW . Functional genomics of hepatocellular carcinoma. Hepatology 2006; 43: S145–S150.

    Article  CAS  PubMed  Google Scholar 

  32. Keng VW, Villanueva A, Chiang DY, Dupuy AJ, Ryan BJ, Matise I et al. A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol 2009; 27: 264–274.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ranzani M, Cesana D, Bartholomae CC, Sanvito F, Pala M, Benedicenti F et al. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer. Nat Methods 2013; 10: 155–161.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sawey ET, Chanrion M, Cai C, Wu G, Zhang J, Zender L et al. Identification of a therapeutic strategy targeting amplified FGF19 in liver cancer by Oncogenomic screening. Cancer Cell 2011; 19: 347–358.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gabriel R, Eckenberg R, Paruzynski A, Bartholomae CC, Nowrouzi A, Arens A et al. Comprehensive genomic access to vector integration in clinical gene therapy. Nat Med 2009; 15: 1431–1436.

    Article  CAS  PubMed  Google Scholar 

  36. Kustikova OS, Baum C, Fehse B . Retroviral integration site analysis in hematopoietic stem cells. Methods Mol Biol 2008; 430: 255–267.

    Article  CAS  PubMed  Google Scholar 

  37. Astrom A, D'Amore ES, Sainati L, Panarello C, Morerio C, Mark J et al. Evidence of involvement of the PLAG1 gene in lipoblastomas. Int J Oncol 2000; 16: 1107–1110.

    CAS  PubMed  Google Scholar 

  38. Astrom AK, Voz ML, Kas K, Roijer E, Wedell B, Mandahl N et al. Conserved mechanism of PLAG1 activation in salivary gland tumors with and without chromosome 8q12 abnormalities: identification of SII as a new fusion partner gene. Cancer Res 1999; 59: 918–923.

    CAS  PubMed  Google Scholar 

  39. Declercq J, Van DF, Braem CV, Van V IC, Voz M, Wassef M et al. Salivary gland tumors in transgenic mice with targeted PLAG1 proto-oncogene overexpression. Cancer Res 2005; 65: 4544–4553.

    Article  CAS  PubMed  Google Scholar 

  40. Hibbard MK, Kozakewich HP, Dal CP, Sciot R, Tan X, Xiao S et al. PLAG1 fusion oncogenes in lipoblastoma. Cancer Res 2000; 60: 4869–4872.

    CAS  PubMed  Google Scholar 

  41. Kas K, Voz ML, Roijer E, Astrom AK, Meyen E, Stenman G et al. Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations. Nat Genet 1997; 15: 170–174.

    Article  CAS  PubMed  Google Scholar 

  42. Landrette SF, Kuo YH, Hensen K, Barjesteh van Waalwijk van Doorn-Khosrovani S, Perrat PN, Van de Ven WJ et al. Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11. Blood 2005; 105: 2900–2907.

    Article  CAS  PubMed  Google Scholar 

  43. Zatkova A, Rouillard JM, Hartmann W, Lamb BJ, Kuick R, Eckart M et al. Amplification and overexpression of the IGF2 regulator PLAG1 in hepatoblastoma. Genes Chromosomes Cancer 2004; 39: 126–137.

    Article  CAS  PubMed  Google Scholar 

  44. Meylan E, Martinon F, Thome M, Gschwendt M, Tschopp J . RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis. EMBO Rep 2002; 3: 1201–1208.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kim SW, Oleksyn DW, Rossi RM, Jordan CT, Sanz I, Chen L et al. Protein kinase C-associated kinase is required for NF-kappaB signaling and survival in diffuse large B-cell lymphoma cells. Blood 2008; 111: 1644–1653.

    Article  CAS  PubMed  Google Scholar 

  46. Ghosh S, May MJ, Kopp EB . NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16: 225–260.

    Article  CAS  PubMed  Google Scholar 

  47. Lenardo MJ, Baltimore D . NF-kappa B a pleiotropic mediator of inducible and tissue-specific gene control. Cell 1989; 58: 227–229.

    Article  CAS  PubMed  Google Scholar 

  48. Liu ZG, Hsu H, Goeddel DV, Karin M . Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 1996; 87: 565–576.

    Article  CAS  PubMed  Google Scholar 

  49. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM . Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 1996; 274: 787–789.

    Article  CAS  PubMed  Google Scholar 

  50. Wang CY, Mayo MW, Baldwin AS Jr . TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science 1996; 274: 784–787.

    Article  CAS  PubMed  Google Scholar 

  51. Luedde T, Beraza N, Kotsikoris V, van LG, Nenci A, De VR et al. Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 2007; 11: 119–132.

    Article  CAS  PubMed  Google Scholar 

  52. Kustikova OS, Wahlers A, Kuhlcke K, Stahle B, Zander AR, Baum C et al. Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 2003; 102: 3934–3937.

    Article  CAS  PubMed  Google Scholar 

  53. Kuhlcke K, Fehse B, Schilz A, Loges S, Lindemann C, Ayuk F et al. Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels. Mol Ther 2002; 5: 473–478.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge the valuable technical assistance provided by N Knuth, J Polke, P Zemke-Trautmann and the FACS Core Unit of the UMC Hamburg-Eppendorf. We are grateful for the financial support by the Deutsche Forschungsgemeinschaft (SFB 841, projects C5 and C7).

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Authors and Affiliations

  1. Department of Gastroenterology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    D Heim, K Schulze, A W Lohse & H Wege

  2. Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    K Cornils & B Fehse

  3. Department of Hematology and Oncology, University Medical Center of the Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany

    T H Brümmendorf

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  1. D Heim
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  2. K Cornils
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Correspondence to D Heim.

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Heim, D., Cornils, K., Schulze, K. et al. Retroviral insertional mutagenesis in telomerase-immortalized hepatocytes identifies RIPK4 as novel tumor suppressor in human hepatocarcinogenesis. Oncogene 34, 364–372 (2015). https://doi.org/10.1038/onc.2013.551

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