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Combined p19Arf and interferon-beta gene transfer enhances cell death of B16 melanoma in vitro and in vivo

Cancer Gene Therapy volume 20, pages 317–325 (2013)Cite this article

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Abstract

Approximately 90% of melanomas retain wild-type p53, a characteristic that may help shape the development of novel treatment strategies. Here, we employed an adenoviral vector where transgene expression is controlled by p53 to deliver the p19 alternate reading frame (Arf) and interferon-β (IFNβ) complementary DNAs in the B16 mouse model of melanoma. In vitro, cell death was enhanced by combined gene transfer (63.82±15.30% sub-G0 cells); yet introduction of a single gene resulted in significantly fewer hypoploid cells (37.73±7.3% or 36.96±11.58%, p19Arf or IFNβ, respectively, P<0.05). Annexin V staining and caspase-3 cleavage indicate a cell death mechanism consistent with apoptosis. Using reverse transcriptase quantitative PCR, we show that key transcriptional targets of p53 were upregulated in the presence of p19Arf, although treatment with IFNβ did not alter expression of the genes studied. In situ gene therapy revealed significant inhibition of subcutaneous tumors by IFNβ (571±25 mm3) or the combination of p19Arf and IFNβ (489±124 mm3) as compared with the LacZ control (1875±33 mm3, P<0.001), whereas p19Arf yielded an intermediate result (1053±169 mm3, P<0.01 vs control). However, only the combination was associated with increased cell death and prolonged survival (P<0.01). As shown here, the combined transfer of p19Arf and IFNβ using p53-responsive vectors enhanced cell death both in vitro and in vivo.

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References

  1. Ravnan MC, Matalka MS . Vemurafenib in patients with BRAF V600E mutation-positive advanced melanoma. Clin Ther 2012; 34: 1474–1486.

    Article  CAS  PubMed  Google Scholar 

  2. Garbe C, Eigentler TK, Keilholz U, Hauschild A, Kirkwood JM . Systematic review of medical treatment in melanoma: current status and future prospects. Oncologist 2011; 16: 5–24.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Giglia-Mari G, Sarasin A . TP53 mutations in human skin cancers. Hum Mutat 2003; 21: 217–228.

    Article  CAS  PubMed  Google Scholar 

  4. Freedberg DE, Rigas SH, Russak J, Gai W, Kaplow M, Osman I et al. Frequent p16-independent inactivation of p14ARF in human melanoma. J Natl Cancer Inst 2008; 100: 784–795.

    Article  CAS  PubMed  Google Scholar 

  5. Bishop DT, Demenais F, Goldstein AM, Bergman W, Bishop JN, Bressac-de Paillerets B et al. Geographical variation in the penetrance of CDKN2A mutations for melanoma. J Natl Cancer Inst 2002; 94: 894–903.

    Article  CAS  PubMed  Google Scholar 

  6. Amos CI, Wang LE, Lee JE, Gershenwald JE, Chen WV, Fang S et al. Genome-wide association study identifies novel loci predisposing to cutaneous melanoma. Hum Mol Genet 2011; 20: 5012–5023.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Tao W, Levine AJ . P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci USA 1999; 96: 6937–6941.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tao W, Levine AJ . Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc Natl Acad Sci USA 1999; 96: 3077–3080.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 1998; 92: 713–723.

    Article  CAS  PubMed  Google Scholar 

  10. Zhang Y, Xiong Y, Yarbrough WG . ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 1998; 92: 725–734.

    Article  CAS  PubMed  Google Scholar 

  11. Sherr CJ . Divorcing ARF and p53: an unsettled case. Nat Rev Cancer 2006; 6: 663–673.

    Article  CAS  PubMed  Google Scholar 

  12. Merkel CA, da Silva Soares RB, de Carvalho AC, Zanatta DB, Bajgelman MC, Fratini P et al. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6. BMC Cancer 2010; 10: 316.

    Article  PubMed Central  PubMed  Google Scholar 

  13. Qin XQ, Runkel L, Deck C, DeDios C, Barsoum J . Interferon-beta induces S phase accumulation selectively in human transformed cells. J Interferon Cytokine Res 1997; 17: 355–367.

    Article  CAS  PubMed  Google Scholar 

  14. Ekmekcioglu S, Mumm JB, Udtha M, Chada S, Grimm EA . Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24. Cytokine 2008; 43: 34–44.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Lokshin A, Mayotte JE, Levitt ML . Mechanism of interferon beta-induced squamous differentiation and programmed cell death in human non-small-cell lung cancer cell lines. J Natl Cancer Inst 1995; 87: 206–212.

    Article  CAS  PubMed  Google Scholar 

  16. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP . Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol 1999; 17: 189–220.

    Article  CAS  PubMed  Google Scholar 

  17. Xie K, Bielenberg D, Huang S, Xu L, Salas T, Juang SH et al. Abrogation of tumorigenicity and metastasis of murine and human tumor cells by transfection with the murine IFN-beta gene: possible role of nitric oxide. Clin Cancer Res 1997; 3 (12 Pt 1): 2283–2294.

    CAS  PubMed  Google Scholar 

  18. Spear GT, Paulnock DM, Jordan RL, Meltzer DM, Merritt JA, Borden EC . Enhancement of monocyte class I and II histocompatibility antigen expression in man by in vivo beta-interferon. Clin Exp Immunol 1987; 69: 107–115.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Fujimura T, Yamasaki K, Hidaka T, Ito Y, Aiba S . A synthetic NOD2 agonist, muramyl dipeptide (MDP)-Lys (L18) and IFN-beta synergistically induce dendritic cell maturation with augmented IL-12 production and suppress melanoma growth. J Dermatol Sci 2011; 62: 107–115.

    Article  CAS  PubMed  Google Scholar 

  20. Sato M, Taniguchi T, Tanaka N . The interferon system and interferon regulatory factor transcription factors -- studies from gene knockout mice. Cytokine Growth Factor Rev 2001; 12: 133–142.

    Article  CAS  PubMed  Google Scholar 

  21. Biron CA . Interferons alpha and beta as immune regulators--a new look. Immunity 2001; 14: 661–664.

    Article  CAS  PubMed  Google Scholar 

  22. Clemens MJ . Interferons and apoptosis. J Interferon Cytokine Res 2003; 23: 277–292.

    Article  CAS  PubMed  Google Scholar 

  23. Sarna GP, Figlin RA, Pertcheck M . Phase II study of betaseron (beta ser17-interferon) as treatment of advanced malignant melanoma. J Biol Response Mod 1987; 6: 375–378.

    CAS  PubMed  Google Scholar 

  24. Kubo H, Ashida A, Matsumoto K, Kageshita T, Yamamoto A, Saida T . Interferon-beta therapy for malignant melanoma: the dose is crucial for inhibition of proliferation and induction of apoptosis of melanoma cells. Arch Dermatol Res 2008; 300: 297–301.

    Article  CAS  PubMed  Google Scholar 

  25. Salmon P, Le Cotonnec JY, Galazka A, Abdul-Ahad A, Darragh A . Pharmacokinetics and pharmacodynamics of recombinant human interferon-beta in healthy male volunteers. J Interferon Cytokine Res 1996; 16: 759–764.

    Article  CAS  PubMed  Google Scholar 

  26. Schiller JH, Storer B, Bittner G, Willson JK, Borden EC . Phase II trial of a combination of interferon-beta ser and interferon-gamma in patients with advanced malignant melanoma. J Interferon Res 1988; 8: 581–589.

    Article  CAS  PubMed  Google Scholar 

  27. Einhorn S, Grander D . Why do so many cancer patients fail to respond to interferon therapy? J Interferon Cytokine Res 1996; 16: 275–281.

    Article  CAS  PubMed  Google Scholar 

  28. Yoshida J, Mizuno M, Fujii M, Kajita Y, Nakahara N, Hatano M et al. Human gene therapy for malignant gliomas (glioblastoma multiforme and anaplastic astrocytoma) by in vivo transduction with human interferon beta gene using cationic liposomes. Hum Gene Ther 2004; 15: 77–86.

    Article  CAS  PubMed  Google Scholar 

  29. Yoshida J, Mizuno M, Wakabayashi T . Interferon-beta gene therapy for cancer: basic research to clinical application. Cancer Sci 2004; 95: 858–865.

    Article  CAS  PubMed  Google Scholar 

  30. Wakabayashi T, Natsume A, Hashizume Y, Fujii M, Mizuno M, Yoshida J . A phase I clinical trial of interferon-beta gene therapy for high-grade glioma: novel findings from gene expression profiling and autopsy. J Gene Med 2008; 10: 329–339.

    Article  CAS  PubMed  Google Scholar 

  31. Lu W, Fidler IJ, Dong Z . Eradication of primary murine fibrosarcomas and induction of systemic immunity by adenovirus-mediated interferon beta gene therapy. Cancer Res 1999; 59: 5202–5208.

    CAS  PubMed  Google Scholar 

  32. Alizadeh H, Howard K, Mellon J, Mayhew E, Rusciano D, Niederkorn JY . Reduction of liver metastasis of intraocular melanoma by interferon-beta gene transfer. Invest Ophthalmol Vis Sci 2003; 44: 3042–3051.

    Article  PubMed  Google Scholar 

  33. Dickson PV, Hamner JB, Streck CJ, Ng CY, McCarville MB, Calabrese C et al. Continuous delivery of IFN-beta promotes sustained maturation of intratumoral vasculature. Mol Cancer Res 2007; 5: 531–542.

    Article  CAS  PubMed  Google Scholar 

  34. Xiao HB, Zhou WY, Chen XF, Mei J, Lv ZW, Ding FB et al. Interferon-beta efficiently inhibited endothelial progenitor cell-induced tumor angiogenesis. Gene Ther 2012; 19: 1030–1034.

    Article  CAS  PubMed  Google Scholar 

  35. Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H et al. Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence. Nature 2003; 424: 516–523.

    Article  CAS  PubMed  Google Scholar 

  36. Sandoval R, Xue J, Pilkinton M, Salvi D, Kiyokawa H, Colamonici OR . Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines. J Biol Chem 2004; 279: 32275–32280.

    Article  CAS  PubMed  Google Scholar 

  37. Garcia MA, Collado M, Munoz-Fontela C, Matheu A, Marcos-Villar L, Arroyo J et al. Antiviral action of the tumor suppressor ARF. EMBO J 2006; 25: 4284–4292.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Zhang F, Sriram S . Identification and characterization of the interferon-beta-mediated p53 signal pathway in human peripheral blood mononuclear cells. Immunology 2009; 128 (1 Suppl): e905–e918.

    Article  PubMed Central  PubMed  Google Scholar 

  39. Bajgelman MC, Strauss BE . Development of an adenoviral vector with robust expression driven by p53. Virology 2008; 371: 8–13.

    Article  CAS  PubMed  Google Scholar 

  40. Strauss BE, Bajgelman MC, Costanzi-Strauss E . A novel gene transfer strategy that combines promoter and transgene activities for improved tumor cell inhibition. Cancer Gene Ther 2005; 12: 935–946.

    Article  CAS  PubMed  Google Scholar 

  41. Strauss BE, Costanzi-Strauss E . pCLPG: a p53-driven retroviral system. Virology 2004; 321: 165–172.

    Article  CAS  PubMed  Google Scholar 

  42. Leon RP, Hedlund T, Meech SJ, Li S, Schaack J, Hunger SP et al. Adenoviral-mediated gene transfer in lymphocytes. Proc Natl Acad Sci USA 1998; 95: 13159–13164.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Yamashita M, Ino A, Kawabata K, Sakurai F, Mizuguchi H . Expression of coxsackie and adenovirus receptor reduces the lung metastatic potential of murine tumor cells. Int J Cancer 2007; 121: 1690–1696.

    Article  CAS  PubMed  Google Scholar 

  44. Mizuguchi H, Hayakawa T . Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes. Gene 2002; 285: 69–77.

    Article  CAS  PubMed  Google Scholar 

  45. Ghattas IR, Sanes JR, Majors JE . The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol 1991; 11: 5848–5859.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Zhang F, Lu W, Dong Z . Tumor-infiltrating macrophages are involved in suppressing growth and metastasis of human prostate cancer cells by INF-beta gene therapy in nude mice. Clin Cancer Res 2002; 8: 2942–2951.

    CAS  PubMed  Google Scholar 

  47. Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, Archambault JM et al. Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp Med 2011; 208: 1989–2003.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Wilderman MJ, Sun J, Jassar AS, Kapoor V, Khan M, Vachani A et al. Intrapulmonary IFN-beta gene therapy using an adenoviral vector is highly effective in a murine orthotopic model of bronchogenic adenocarcinoma of the lung. Cancer Res 2005; 65: 8379–8387.

    Article  CAS  PubMed  Google Scholar 

  49. Roos WP, Jost E, Belohlavek C, Nagel G, Fritz G, Kaina B . Intrinsic anticancer drug resistance of malignant melanoma cells is abrogated by IFN-beta and valproic acid. Cancer Res 2011; 71: 4150–4160.

    Article  CAS  PubMed  Google Scholar 

  50. Ambjorn M, Ejlerskov P, Liu Y, Lees M, Jaattela M, Issazadeh-Navikas S . IFNB1/interferon-beta-induced autophagy in MCF-7 breast cancer cells counteracts its proapoptotic function. Autophagy 2012; 9: 3.

    Google Scholar 

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Acknowledgements

We thank José Eduardo Krieger and lab for their support and helpful discussions, the lab of Roger Chammas for helpful discussion and for providing use of the FACS, Ana Lucia Garipo for assistance with fluorescence microscopy, Marcio Chaves for facilitating the histological analyses and Paulo Roberto Del Valle for assistance with the RT-qPCR experiment. Funding was provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) in the form of fellowships (06/57823-5, CAM; 2010/03958-2, RFVM) and grants (07/50210-0, 11/50911-0, BES).

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Author notes

  1. R F V Medrano & B E Strauss

    Present address: 4Current address: Viral Vector Laboratory, Center for Translational Investigation in Oncology/LIM 24, Cancer Institute of São Paulo, University of São Paulo School of Medicine, São Paulo, Brazil.,

Authors and Affiliations

  1. Viral Vector Laboratory, Heart Institute, University of São Paulo School of Medicine, São Paulo, Brazil

    C A Merkel, R F V Medrano & B E Strauss

  2. Laboratory of Genetics and Molecular Cardiology/LIM13, Heart Institute, University of São Paulo School of Medicine, São Paulo, Brazil

    C A Merkel, V G Barauna & B E Strauss

  3. Animal Care Facility, University of São Paulo School of Medicine, São Paulo, Brazil

    C A Merkel & B E Strauss

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Correspondence to B E Strauss.

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Merkel, C., Medrano, R., Barauna, V. et al. Combined p19Arf and interferon-beta gene transfer enhances cell death of B16 melanoma in vitro and in vivo. Cancer Gene Ther 20, 317–325 (2013). https://doi.org/10.1038/cgt.2013.23

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