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Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

Cancer Gene Therapy volume 13, pages 234–241 (2006)Cite this article

Abstract

Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.

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Abbreviations

HPV:

human papillomavirus

Rb:

retinoblastoma

siRNA:

small-interfering RNA

References

  1. de Villiers EM . Heterogeneity of the human papillomavirus group. J Virol 1989; 63: 4898–4903.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Fen J, Yoshinouchi M, Nakamura K, Kodama J, Nasu Y, Yamato K et al. Eradication of HPV post-surgical treatments, its correlation with specific types, types of surgery and the physical status. Oncol Rep 2004; 12: 375–379.

    PubMed  Google Scholar 

  3. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J Natl Cancer Inst 1995; 87: 796–802.

    Article  CAS  PubMed  Google Scholar 

  4. zur Hausen H . Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus. In: zur Hausen H (ed.), Human Pathogenic Papillomavirus. Springer-Verlag: Berlin, 1994, pp. 131–156.

    Chapter  Google Scholar 

  5. von Knebel Doeberitz M, Oltersdorf T, Schwarz E, Gissmann L . Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells. Cancer Res 1988; 48: 3780–3786.

    CAS  PubMed  Google Scholar 

  6. Vousden K . Interactions of human papillomavirus transforming proteins with the products of tumor suppressor genes. FASEB J 1993; 7: 872–879.

    Article  CAS  PubMed  Google Scholar 

  7. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494–498.

    Article  CAS  PubMed  Google Scholar 

  8. Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K . Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001; 114: 4557–4565.

    CAS  PubMed  Google Scholar 

  9. Filleur S, Courtin A, Ait-Si-Ali S, Guglielmi J, Merle C, Harel-Bellan A et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res 2003; 63: 3919–3922.

    CAS  PubMed  Google Scholar 

  10. Li K, Lin SY, Brunicardi FC, Seu P . Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma. Cancer Res 2003; 63: 3593–3597.

    CAS  PubMed  Google Scholar 

  11. Yang G, Thompson JA, Fang B, Liu J . Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumor growth in a model of human ovarian cancer. Oncogene 2003; 22: 5694–5701.

    Article  CAS  PubMed  Google Scholar 

  12. Verma UN, Surabhi RM, Schmaltieg A, Becerra C, Gaynor RB . Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin Cancer Res 2003; 9: 1291–1300.

    CAS  PubMed  Google Scholar 

  13. Jiang M, Milner J . Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 2002; 21: 6041–6048.

    Article  CAS  PubMed  Google Scholar 

  14. Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T, Ikeda Y et al. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther 2003; 8: 762–768.

    Article  CAS  PubMed  Google Scholar 

  15. Pim D, Massimi P, Banks L . Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth. Oncogene 1997; 15:257–264.

    Article  CAS  PubMed  Google Scholar 

  16. Mantovani F, Banks L . The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 2001; 20: 7874–7887.

    Article  CAS  PubMed  Google Scholar 

  17. Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M, Hoppe-Seyler F . siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells. Oncogene 2003; 22: 5938–5945.

    Article  CAS  PubMed  Google Scholar 

  18. Hall AH, Alexander KA . RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells. J Virol 2003; 77: 6066–6069.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K . Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001; 114: 4557–4565.

    CAS  PubMed  Google Scholar 

  20. Yamato K, Hashimoto S, Okahashi N, Ishisaki A, Nonaka K, Kizaki M et al. Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7. Exp Cell Res 2000; 257: 198–205.

    Article  CAS  PubMed  Google Scholar 

  21. Huibregtse JM, Scheffner M, Howley PM . Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol Cell Biol 1993; 13: 775–784.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Huibregtse JM, Scheffner M, Howley PM . Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins. Mol Cell Biol 1993; 13: 4918–4927.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003; 9: 347–351.

    Article  CAS  PubMed  Google Scholar 

  24. Zender L, Hutker S, Liedtke C, Tillmann HL, Zender S, Mundt B et al. Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci USA 2003; 100: 7797–7802.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sorensen DR, Leirdal M, Sioud M . Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol 2003; 327: 761–766.

    Article  CAS  PubMed  Google Scholar 

  26. McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 2003; 21: 639–644.

    Article  CAS  PubMed  Google Scholar 

  27. Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E . Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 2003; 8: 769–776.

    Article  CAS  PubMed  Google Scholar 

  28. Kapadia SB, Brideau-Andersen A, Chisari FV . Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA 2003; 100: 2014–2018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Takigawa Y, Nagano-Fujii M, Deng L, Hidajat R, Tanaka M, Mizuta H et al. Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol 2004; 48: 591–598.

    Article  CAS  PubMed  Google Scholar 

  30. Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK et al. siRNA-directed inhibition of HIV-1 infection. Nat Med 2002; 8: 681–686.

    Article  CAS  PubMed  Google Scholar 

  31. Song E, Lee SK, Dykxhoorn DM, Novina C, Zhang D, Crawford K et al. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J Virol 2003; 77: 7174–7181.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Scheffner M, Whitaker NJ . Human papillomavirus-induced carcinogenesis and the ubiquitin–proteasome system. Semin Cancer Biol 2003; 13: 59–67.

    Article  CAS  PubMed  Google Scholar 

  33. Song S, Liem A, Miller JA, Lambert PF . Human papillomavirus types 16 E6 and E7 contribute differently to carcinogenesis. Virology 2000; 267: 141–150.

    Article  CAS  PubMed  Google Scholar 

  34. Munger K, Werness BA, Dyson N, Phelps WC, Harlow E, Howley PM . Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J 1989; 8: 4099–4105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dyson N, Howley PM, Munger K, Harlow E . The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 1989; 243: 934–937.

    Article  CAS  PubMed  Google Scholar 

  36. Askew DS, Ashmun RA, Simmons BC, Cleveland JL . Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene 1991; 6: 1915–1922.

    CAS  PubMed  Google Scholar 

  37. Thomas M, Banks L . Human papillomavirus (HPV) E6 interactions with Bak are conserved amongst E6 proteins from high and low risk HPV types. J Gen Virol 1999; 80 (Part 6): 1513–1517.

    Article  CAS  PubMed  Google Scholar 

  38. Gross-Mesilaty S, Reinstein E, Bercovich B, Tobias KE, Schwartz AL, Kahana C et al. Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc Natl Acad Sci USA 1998; 95: 8058–8063.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Smotkin D, Prokoph H, Wettstein FO . Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. J Virol 1989; 63: 1441–1447.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Sherman L, Alloul N, Golan I, Durst M, Baram A . Expression and splicing patterns of human papillomavirus type-16 mRNAs in pre-cancerous lesions and carcinomas of the cervix, in human keratinocytes immortalized by HPV 16, and in cell lines established from cervical cancers. Int J Cancer 1992; 50: 356–364.

    Article  CAS  PubMed  Google Scholar 

  41. Schneider-Gadicke A, Schwarz E . Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes. EMBO J 1986; 5: 2285–2292.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stacey SN, Jordan D, Snijders PJ, Mackett M, Walboomers JM, Arrand JR . Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame. J Virol 1995; 69: 7023–7031.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. DeFilippis RA, Goodwin EC, Wu L, DiMaio D . Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol 2003; 77: 1551–1563.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported in part by grants-in-aid for Scientific Research (C) from Japan Society for the Promotion of Science (17591740 (MY), 15591991 (KY)).

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Authors and Affiliations

  1. Molecular Cellular Oncology and Microbiology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

    K Yamato

  2. Department of Physiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

    J Fen & M Yoshinouchi

  3. Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

    H Kobuchi

  4. Department of Urology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

    Y Nasu

  5. Divisions of Pathology and Hematology, Keio University, School of Medicine, Tokyo, Japan

    T Yamada, Y Ikeda & M Kizaki

  6. Departments of Oral Microbiology and Preventive Dentistry, Kyushu Dental College, Fukuoka, Japan

    T Nishihara

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Correspondence to M Yoshinouchi.

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Yamato, K., Fen, J., Kobuchi, H. et al. Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA. Cancer Gene Ther 13, 234–241 (2006). https://doi.org/10.1038/sj.cgt.7700891

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  • DOI: https://doi.org/10.1038/sj.cgt.7700891

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