Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf

Abstract

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Parmiani G, Castelli C, Dalerba P, Mortarini R, Rivoltini L, Marincola FM et al. Cancer immunotherapy with peptide-based vaccines: what have we achieved? Where are we going? J Natl Cancer Inst 2002; 94: 805–818.

    Article  CAS  Google Scholar 

  2. Mazda O, Germeraad WTV . Tumor immunity and immuno-gene therapy of cancer. In: Mazda O (ed). Frontiers in Immuno-Gene Therapy. Research Signpost: Trivandrum, 2004, pp 1–30.

    Google Scholar 

  3. Sosman JA, Sondak VK . Melacine: an allogeneic melanoma tumor cell lysate vaccine. Expert Rev Vaccines 2003; 2: 353–368.

    Article  CAS  Google Scholar 

  4. Nakai N, Asai J, Ueda E, Takenaka H, Katoh N, Kishimoto S . Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells. J Dermatol 2006; 33: 462–472.

    Article  CAS  Google Scholar 

  5. Kageshita T, Wang Z, Calorini L, Yoshii A, Kimura T, Ono T et al. Selective loss of human leukocyte class I allospecificities and staining of melanoma cells by monoclonal antibodies recognizing monomorphic determinants of class I human leukocyte antigens. Cancer Res 1993; 53: 3349–3354.

    CAS  PubMed  Google Scholar 

  6. Chakraborty NG, Chattopadhyay S, Mehrotra S, Chhabra A, Mukherji B . Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma. Hum Immunol 2004; 65: 794–802.

    Article  CAS  Google Scholar 

  7. Hodgkinson CA, Moore KJ, Nakayama A, Steingrimsson E, Copeland NG, Jenkins NA et al. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix–loop–helix-zipper protein. Cell 1993; 74: 395–404.

    Article  CAS  Google Scholar 

  8. Tachibana M, Perez-Jurado LA, Nakayama A, Hodgkinson CA, Li X, Schneider M et al. Cloning of MITF, the human homolog of the mouse microphthalmia gene and assignment to chromosome 3p14.1-p12.3. Hum Mol Genet 1994; 3: 553–557.

    Article  CAS  Google Scholar 

  9. Yasumoto K, Amae S, Udono T, Fuse N, Takeda K, Shibahara S . A big gene linked to small eyes encodes multiple Mitf isoforms: many promoters make light work. Pigment Cell Res 1998; 11: 329–336.

    Article  CAS  Google Scholar 

  10. Amae S, Fuse N, Yasumoto K, Sato S, Yajima I, Yamamoto H et al. Identification of a novel isoform of microphthalmia-associated transcription factor that is enriched in retinal pigment epithelium. Biochem Biophys Res Commun 1998; 247: 710–715.

    Article  CAS  Google Scholar 

  11. Udono T, Yasumoto K, Takeda K, Amae S, Watanabe K, Saito H et al. Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters. Biochim Biophys Acta 2000; 1491: 205–219.

    Article  CAS  Google Scholar 

  12. Fuse N, Yasumoto K, Takeda K, Amae S, Yoshizawa M, Udono T et al. Molecular cloning of cDNA encoding a novel microphthalmia-associated transcription factor isoform with a distinct amino-terminus. J Biochem (Tokyo) 1999; 126: 1043–1051.

    Article  CAS  Google Scholar 

  13. Takeda K, Yasumoto K, Kawaguchi N, Udono T, Watanabe K, Saito H et al. Mitf-D, a newly identified isoform, expressed in the retinal pigment epithelium and monocyte-lineage cells affected by Mitf mutations. Biochim Biophys Acta 2002; 1574: 15–23.

    Article  CAS  Google Scholar 

  14. Tachibana M . MITF: a stream flowing for pigment cells. Pigment Cell Res 2000; 13: 230–240.

    Article  CAS  Google Scholar 

  15. Yasumoto K, Yokoyama K, Shibata K, Tomita Y, Shibahara S . Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene. Mol Cell Biol 1994; 14: 8058–8070.

    Article  CAS  Google Scholar 

  16. Yasumoto K, Yokoyama K, Takahashi K, Tomita Y, Shibahara S . Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes. J Biol Chem 1997; 272: 503–509.

    Article  CAS  Google Scholar 

  17. Saito H, Yasumoto K, Takeda K, Takahashi K, Yamamoto H, Shibahara S . Microphthalmia-associated transcription factor in the Wnt signaling pathway. Pigment Cell Res 2003; 16: 261–265.

    Article  CAS  Google Scholar 

  18. Du J, Miller AJ, Widlund HR, Horstmann MA, Ramaswamy S, Fisher DE . MLANA/MART1 and SILV/PMEL17/GP100 are transcriptionally regulated by MITF in melanocytes and melanoma. Am J Pathol 2003; 163: 333–343.

    Article  CAS  Google Scholar 

  19. Steingrimsson E, Moore KJ, Lamoreux ML, Ferre-D'Amare AR, Burley SK, Zimring DC et al. Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nat Genet 1994; 8: 256–263.

    Article  CAS  Google Scholar 

  20. Tassabehji M, Newton VE, Read AP . Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene. Nat Genet 1994; 8: 251–255.

    Article  CAS  Google Scholar 

  21. Van Camp G, Van Thienen MN, Handig I, Van Roy B, Rao VS, Milunsky A et al. Chromosome 13q deletion with Waardenburg syndrome: further evidence for a gene involved in neural crest function on 13q. J Med Genet 1995; 32: 531–536.

    Article  CAS  Google Scholar 

  22. Tachibana M, Takeda K, Nobukuni Y, Urabe K, Long JE, Meyers KA et al. Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics. Nat Genet 1996; 14: 50–54.

    Article  CAS  Google Scholar 

  23. Tachibana M . Evidence to suggest that expression of MITF induces melanocyte differentiation and haploinsufficiency of MITF causes Waardenburg syndrome type 2A. Pigment Cell Res 1997; 10: 25–33.

    Article  CAS  Google Scholar 

  24. Shibahara S, Yasumoto K, Amae S, Udono T, Watanabe K, Saito H et al. Regulation of pigment cell-specific gene expression by MITF. Pigment Cell Res 2000; 13 (Suppl 8): 98–102.

    Article  Google Scholar 

  25. McGill GG, Horstmann M, Widlund HR, Du J, Motyckova G, Nishimura EK et al. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 2002; 109: 707–718.

    Article  CAS  Google Scholar 

  26. Du J, Widlund HR, Horstmann MA, Ramaswamy S, Ross K, Huber WE et al. Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF. Cancer Cell 2004; 6: 565–576.

    Article  CAS  Google Scholar 

  27. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005; 436: 117–122.

    Article  CAS  Google Scholar 

  28. Selzer E, Wacheck V, Lucas T, Heere-Ress E, Wu M, Weilbaecher KN et al. The melanocyte-specific isoform of the microphthalmia transcription factor affects the phenotype of human melanoma. Cancer Res 2002; 62: 2098–2103.

    CAS  PubMed  Google Scholar 

  29. Wellbrock C, Marais R . Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation. J Cell Biol 2005; 170: 703–708.

    Article  CAS  Google Scholar 

  30. Larribere L, Hilmi C, Khaled M, Gaggioli C, Bille K, Auberger P et al. The cleavage of microphthalmia-associated transcription factor, MITF, by caspases plays an essential role in melanocyte and melanoma cell apoptosis. Genes Dev 2005; 19: 1980–1985.

    Article  CAS  Google Scholar 

  31. Loercher AE, Tank EM, Delston RB, Harbour JW . MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A. J Cell Biol 2005; 168: 35–40.

    Article  CAS  Google Scholar 

  32. Carreira S, Goodall J, Aksan I, La Rocca SA, Galibert MD, Denat L et al. Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature 2005; 433: 764–769.

    Article  CAS  Google Scholar 

  33. Hemesath TJ, Price ER, Takemoto C, Badalian T, Fisher DE . MAP kinase links the transcription factor microphthalmia to c-Kit signalling in melanocytes. Nature 1998; 391: 298–301.

    Article  CAS  Google Scholar 

  34. Gaggioli C, Busca R, Abbe P, Ortonne JP, Ballotti R . Microphthalmia-associated transcription factor (MITF) is required but is not sufficient to induce the expression of melanogenic genes. Pigment Cell Res 2003; 16: 374–382.

    Article  CAS  Google Scholar 

  35. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC . Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806–811.

    Article  CAS  Google Scholar 

  36. Caplen NJ . Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference. Gene Therapy 2004; 11: 1241–1248.

    Article  CAS  Google Scholar 

  37. Wianny F, Zernicka-Goetz M . Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2000; 2: 70–75.

    Article  CAS  Google Scholar 

  38. 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  Google Scholar 

  39. Ryther RC, Flynt AS, Phillips III JA, Patton JG . siRNA therapeutics: big potential from small RNAs. Gene Therapy 2005; 12: 5–11.

    Article  CAS  Google Scholar 

  40. Morris KV, Rossi JJ . Lentiviral-mediated delivery of siRNAs for antiviral therapy. Gene Therapy 2006; 13: 553–558.

    Article  CAS  Google Scholar 

  41. Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med 2004; 10: 816–820.

    Article  CAS  Google Scholar 

  42. Rodriguez-Lebron E, Paulson HL . Allele-specific RNA interference for neurological disease. Gene Ther 2006; 13: 576–581.

    Article  CAS  Google Scholar 

  43. Inoue A, Takahashi KA, Mazda O, Terauchi R, Arai Y, Kishida T et al. Electro-transfer of small interfering RNA ameliorated arthritis in rats. Biochem Biophys Res Commun 2005; 336: 903–908.

    Article  CAS  Google Scholar 

  44. Karasarides M, Chiloeches A, Hayward R, Niculescu-Duvaz D, Scanlon I, Friedlos F et al. B-RAF is a therapeutic target in melanoma. Oncogene 2004; 23: 6292–6298.

    Article  CAS  Google Scholar 

  45. Sharma A, Trivedi NR, Zimmerman MA, Tuveson DA, Smith CD, Robertson GP . Mutant V599EB-Raf regulates growth and vascular development of malignant melanoma tumors. Cancer Res 2005; 65: 2412–2421.

    Article  CAS  Google Scholar 

  46. Cook AL, Smith AG, Smit DJ, Leonard JH, Sturm RA . Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro. Exp Cell Res 2005; 308: 222–235.

    Article  CAS  Google Scholar 

  47. Bektas M, Jolly PS, Muller C, Eberle J, Spiegel S, Geilen CC . Sphingosine kinase activity counteracts ceramide-mediated cell death in human melanoma cells: role of Bcl-2 expression. Oncogene 2005; 24: 178–187.

    Article  CAS  Google Scholar 

  48. Lefevre G, Glotin AL, Calipel A, Mouriaux F, Tran T, Kherrouche Z et al. Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis. J Biol Chem 2004; 279: 31769–31779.

    Article  CAS  Google Scholar 

  49. Tao J, Tu YT, Huang CZ, Feng AP, Wu Q, Lian YJ et al. Inhibiting the growth of malignant melanoma by blocking the expression of vascular endothelial growth factor using an RNA interference approach. Br J Dermatol 2005; 153: 715–724.

    Article  CAS  Google Scholar 

  50. Devroe E, Silver PA . Therapeutic potential of retroviral RNAi vectors. Expert Opin Biol Ther 2004; 4: 319–327.

    Article  CAS  Google Scholar 

  51. Kasahara H, Aoki H . Gene silencing using adenoviral RNAi vector in vascular smooth muscle cells and cardiomyocytes. Methods Mol Med 2005; 112: 155–172.

    Article  CAS  Google Scholar 

  52. Tomar RS, Matta H, Chaudhary PM . Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene 2003; 22: 5712–5715.

    Article  CAS  Google Scholar 

  53. Kishida T, Asada H, Gojo S, Ohashi S, Shin-Ya M, Yasutomi K et al. Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA. J Gene Med 2004; 6: 105–110.

    Article  CAS  Google Scholar 

  54. Lu PY, Xie F, Woodle MC . In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet 2005; 54: 117–142.

    CAS  PubMed  Google Scholar 

  55. Heller LC, Ugen K, Heller R . Electroporation for targeted gene transfer. Expert Opin Drug Deliv 2005; 2: 255–268.

    Article  CAS  Google Scholar 

  56. Andre F, Mir LM . DNA electrotransfer: its principles and an updated review of its therapeutic applications. Gene Ther 2004; 11 (Suppl 1): S33–S42.

    Article  CAS  Google Scholar 

  57. Ohashi S, Kubo T, Kishida T, Ikeda T, Takahashi K, Arai Y et al. Successful genetic transduction in vivo into synovium by means of electroporation. Biochem Biophys Res Commun 2002; 293: 1530–1535.

    Article  CAS  Google Scholar 

  58. Iida Y, Oda Y, Nakamori S, Tsunoda S, Kishida T, Gojo S et al. Transthoracic direct current shock facilitates intramyocardial transfection of naked plasmid DNA infused via coronary vessels in canines. Gene Therapy 2006; 13: 906–916.

    Article  CAS  Google Scholar 

  59. Kishida T, Asada H, Itokawa Y, Yasutomi K, Shin-Ya M, Gojo S et al. Electrochemo-gene therapy of cancer: intratumoral delivery of interleukin-12 gene and bleomycin synergistically induced therapeutic immunity and suppressed subcutaneous and metastatic melanomas in mice. Mol Ther 2003; 8: 738–745.

    Article  CAS  Google Scholar 

  60. Kishida T, Asada H, Satoh E, Tanaka S, Shinya M, Hirai H et al. In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice. Gene Therapy 2001; 8: 1234–1240.

    CAS  Google Scholar 

  61. Lucas ML, Heller L, Coppola D, Heller R . IL-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous B16.F10 melanoma. Mol Ther 2002; 5: 668–675.

    Article  CAS  Google Scholar 

  62. Pekarik V, Bourikas D, Miglino N, Joset P, Preiswerk S, Stoeckli ET . Screening for gene function in chicken embryo using RNAi and electroporation. Nat Biotechnol 2003; 21: 93–96.

    Article  CAS  Google Scholar 

  63. Domenge C, Orlowski S, Luboinski B, De Baere T, Schwaab G, Belehradek Jr J et al. Antitumor electrochemotherapy: new advances in the clinical protocol. Cancer 1996; 77: 956–963.

    Article  CAS  Google Scholar 

  64. Heller R, Jaroszeski MJ, Glass LF, Messina JL, Rapaport DP, DeConti RC et al. Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy. Cancer 1996; 77: 964–971.

    Article  CAS  Google Scholar 

  65. Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavcic D, Rudolf Z . Electrochemotherapy with cisplatin: potentiation of local cisplatin antitumour effectiveness by application of electric pulses in cancer patients. Eur J Cancer 1998; 34: 1213–1218.

    Article  CAS  Google Scholar 

  66. Glass LF, Pepine ML, Fenske NA, Jaroszeski M, Reintgen DS, Heller R . Bleomycin-mediated electrochemotherapy of metastatic melanoma. Arch Dermatol 1996; 132: 1353–1357.

    Article  CAS  Google Scholar 

  67. Tsunoda S, Mazda O, Oda Y, Iida Y, Akabame S, Kishida T et al. Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart. Biochem Biophys Res Commun 2005; 336: 118–127.

    Article  CAS  Google Scholar 

  68. Rossi JJ . RNAi therapeutics: SNALPing siRNAs in vivo. Gene Therapy 2006; 13: 583–584.

    Article  CAS  Google Scholar 

  69. Kamaraju AK, Bertolotto C, Chebath J, Revel M . Pax3 down-regulation and shut-off of melanogenesis in melanoma B16/F10.9 by interleukin-6 receptor signaling. J Biol Chem 2002; 277: 15132–15141.

    Article  CAS  Google Scholar 

  70. Nakanishi H, Mazda O, Satoh E, Asada H, Morioka H, Kishida T et al. Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo. Gene Ther 2003; 10: 434–442.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The present work was supported by a grant in aid of the Japanese Ministry of Education, Culture, Sports, Science and Technology. We thank Dr WTV Germeraad (University of Maastricht, Maastricht, The Netherlands) for critical reading of the paper.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to O Mazda.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nakai, N., Kishida, T., Shin-Ya, M. et al. Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf. Gene Ther 14, 357–365 (2007). https://doi.org/10.1038/sj.gt.3302868

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302868

Keywords

This article is cited by

Search

Quick links