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.

5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma

Nature Medicine volume 14, pages 1256–1263 (2008)Cite this article

Abstract

Genetic and epigenetic plasticity allows tumors to evade single-targeted treatments. Here we direct Bcl2-specific short interfering RNA (siRNA) with 5′-triphosphate ends (3p-siRNA) against melanoma. Recognition of 5′-triphosphate by the cytosolic antiviral helicase retinoic acid–induced protein I (Rig-I, encoded by Ddx58) activated innate immune cells such as dendritic cells and directly induced expression of interferons (IFNs) and apoptosis in tumor cells. These Rig-I–mediated activities synergized with siRNA-mediated Bcl2 silencing to provoke massive apoptosis of tumor cells in lung metastases in vivo. The therapeutic activity required natural killer cells and IFN, as well as silencing of Bcl2, as evidenced by rescue with a mutated Bcl2 target, by site-specific cleavage of Bcl2 messenger RNA in lung metastases and downregulation of Bcl-2 protein in tumor cells in vivo. Together, 3p-siRNA represents a single molecule–based approach in which Rig-I activation on both the immune- and tumor cell level corrects immune ignorance and in which gene silencing corrects key molecular events that govern tumor cell survival.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: 3p-2.2 siRNA potently silences Bcl2 expression and reduces metastatic growth of B16 melanoma cells in the lungs.
Figure 2: Activation of type I IFNs and NK cells are necessary for the antitumor activity of Bcl2-specific 3p-siRNA in vivo.
Figure 3: Bcl-2-specific 3p-siRNA induces cell type specific innate immune responses and apoptosis in vitro.
Figure 4: Bcl2-specific gene silencing and activation of the innate immune system synergistically promote tumor cell apoptosis in vivo.
Figure 5: Bcl2-specific gene silencing contributes to Bcl2-specific 3p-siRNA–induced inhibition of tumor growth and apoptosis.
Figure 6: Bcl2-specific 3p-siRNA is effective in other models of tumorigenesis and in human melanoma cells.

References

  1. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57–70 (2000).

    Article  CAS  Google Scholar 

  2. Bui, J.D. & Schreiber, R.D. Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes? Curr. Opin. Immunol. 19, 203–208 (2007).

    Article  CAS  Google Scholar 

  3. Rubin, B.P., Heinrich, M.C. & Corless, C.L. Gastrointestinal stromal tumour. Lancet 369, 1731–1741 (2007).

    Article  CAS  Google Scholar 

  4. Curiel, T.J. Tregs and rethinking cancer immunotherapy. J. Clin. Invest. 117, 1167–1174 (2007).

    Article  CAS  Google Scholar 

  5. Uno, T. et al. Eradication of established tumors in mice by a combination antibody-based therapy. Nat. Med. 12, 693–698 (2006).

    Article  CAS  Google Scholar 

  6. Obeid, M. et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13, 54–61 (2007).

    Article  CAS  Google Scholar 

  7. Schlee, M., Hornung, V. & Hartmann, G. siRNA and isRNA: two edges of one sword. Mol. Ther. 14, 463–470 (2006).

    Article  CAS  Google Scholar 

  8. Pei, Y. & Tuschl, T. On the art of identifying effective and specific siRNAs. Nat. Methods 3, 670–676 (2006).

    Article  CAS  Google Scholar 

  9. de Fougerolles, A., Vornlocher, H.P., Maraganore, J. & Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6, 443–453 (2007).

    Article  CAS  Google Scholar 

  10. Yoneyama, M. et al. The RNA helicase RIG-I has an essential function in double-stranded RNA–induced innate antiviral responses. Nat. Immunol. 5, 730–737 (2004).

    Article  CAS  Google Scholar 

  11. Hornung, V. et al. 5′-triphosphate RNA is the ligand for RIG-I. Science 314, 994–997 (2006).

    Article  Google Scholar 

  12. Pichlmair, A. et al. RIG-I–mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 997–1001 (2006).

    Article  CAS  Google Scholar 

  13. Yoneyama, M. & Fujita, T. Function of RIG-I–like receptors in antiviral innate immunity. J. Biol. Chem. 282, 15315–15318 (2007).

    Article  CAS  Google Scholar 

  14. Kim, D.H. et al. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat. Biotechnol. 22, 321–325 (2004).

    Article  CAS  Google Scholar 

  15. Miller, A.J. & Mihm, M.C. Jr. Melanoma. N. Engl. J. Med. 355, 51–65 (2006).

    Article  CAS  Google Scholar 

  16. Danial, N.N. & Korsmeyer, S.J. Cell death: critical control points. Cell 116, 205–219 (2004).

    Article  CAS  Google Scholar 

  17. McGill, G.G. et al. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 109, 707–718 (2002).

    Article  CAS  Google Scholar 

  18. Hornung, V. et al. Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263–270 (2005).

    Article  CAS  Google Scholar 

  19. Judge, A.D. et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457–462 (2005).

    Article  CAS  Google Scholar 

  20. Kawai, T. & Akira, S. Innate immune recognition of viral infection. Nat. Immunol. 7, 131–137 (2006).

    Article  CAS  Google Scholar 

  21. Kato, H. et al. Cell type-specific involvement of RIG-I in antiviral response. Immunity 23, 19–28 (2005).

    Article  CAS  Google Scholar 

  22. Melchjorsen, J. et al. Activation of innate defense against a paramyxovirus is mediated by RIG-I and TLR7 and TLR8 in a cell-type-specific manner. J. Virol. 79, 12944–12951 (2005).

    Article  CAS  Google Scholar 

  23. Kawai, T. et al. IPS-1, an adaptor triggering RIG-I– and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981–988 (2005).

    Article  CAS  Google Scholar 

  24. Meylan, E. et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172 (2005).

    Article  CAS  Google Scholar 

  25. Tormo, D. et al. Therapeutic efficacy of antigen-specific vaccination and Toll-like receptor stimulation against established transplanted and autochthonous melanoma in mice. Cancer Res. 66, 5427–5435 (2006).

    Article  CAS  Google Scholar 

  26. Stetson, D.B. & Medzhitov, R. Antiviral defense: interferons and beyond. J. Exp. Med. 203, 1837–1841 (2006).

    Article  CAS  Google Scholar 

  27. Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S. & Reis e Sousa, C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

    Article  CAS  Google Scholar 

  28. Kato, H. et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101–105 (2006).

    Article  CAS  Google Scholar 

  29. Muller, U. et al. Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921 (1994).

    Article  CAS  Google Scholar 

  30. Kamphuis, E., Junt, T., Waibler, Z., Forster, R. & Kalinke, U. Type I interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia. Blood 108, 3253–3261 (2006).

    Article  CAS  Google Scholar 

  31. Mocikat, R. et al. Natural killer cells activated by MHC class Ilow targets prime dendritic cells to induce protective CD8 T cell responses. Immunity 19, 561–569 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by grant Biofuture 0311896 of the Bundesministerium für Bildung und Forschung and by grants SFB 704, SFB 670, KFO115 and KFO177 of the Deutsche Forschungsgemeinschaft to G. Hartmann; by grants Tu90-6/1 of the Deutsche Forschungsgemeinschaft and 10741 of the Deutsche Krebshilfe to T.T.; by grant 107805 of the Deutsche Krebshilfe to R.B.; by Graduiertenkolleg 1202 to C.M., M.B. and T.S.; by LMUexcellent (CIPSM 114, research professorship), and by SFB-TR 36 and En 169/7-2 of the Deutsche Forschungsgemeinschaft to S.E. and C. Bourquin. This work is part of the theses of C.M. and M.B. at the University of Munich and of M.R. and J.L. at the University of Bonn. We thank A. Dann for excellent technical assistance, T. Maniatis (Harvard University) and J. Chen (University of Texas Southwestern Medical Center) for providing IFN-β–Luc reporter plasmids and wild-type pPME-myc NS3-4A (NS3-4A), respectively, T. Fujita Institute for Virus Research, Kyoto University for providing Rig-I and the empty control vector and C. Borner (Institute of Molecular Medicine and Cell Research, University of Freiburg) for providing wild-type mouse Bcl-2 (mBcl-2/pcDNA).

Author information

Author notes

  1. Hendrik Poeck, Robert Besch, Cornelius Maihoefer and Marcel Renn: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Clinical Chemistry and Pharmacology, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, 53127, Bonn, Germany

    Hendrik Poeck, Johannes Hellmuth, Veit Hornung & Gunther Hartmann

  2. Division of Clinical Pharmacology, Department of Internal Medicine, Ludwig-Maximilians University, Ziemssenstrasse 1, 80336, Munich, Germany

    Hendrik Poeck, Cornelius Maihoefer, Andreas Schmidt, David Anz, Michael Bscheider, Tobias Schwerd, Carole Bourquin, Simon Rothenfusser & Stefan Endres

  3. III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität München, Ismaninger Strasse 15, 81675, Munich, Germany

    Hendrik Poeck, Cornelius Maihoefer, Michael Bscheider & Jürgen Ruland

  4. Department of Dermatology and Allergology, Ludwig-Maximilians-University, Thalkirchner Strasse 48, 80337, Munich, Germany

    Robert Besch & Carola Berking

  5. Department of Dermatology and Allergology, Laboratory for Experimental Dermatology, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany

    Marcel Renn, Damia Tormo, Evelyn Gaffal, Jennifer Landsberg & Thomas Tüting

  6. Alnylam Pharmaceuticals, 300 Third Street, Cambridge, 02142, Massachusetts, USA

    Svetlana Shulga Morskaya & Rachel Meyers

  7. Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstrasse 30, 81675, Munich, Germany

    Susanne Kirschnek, Georg Häcker, Michael Neuenhahn & Dirk Busch

  8. Division of Immunology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225, Langen, Germany

    Ulrich Kalinke

  9. Institute of Molecular Immunology, Helmholtz Zentrum München, Marchioninistrasse 25, 81377, Munich, Germany

    Elisabeth Kremmer

  10. Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 565-0871, Osaka, Japan

    Hiroki Kato & Shizuo Akira

  11. Department of Neuropathology, University of Freiburg, Breisacherstrasse 64, 79106, Freiburg, Germany

    Marco Prinz

Authors
  1. Hendrik Poeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Besch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cornelius Maihoefer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcel Renn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Damia Tormo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Svetlana Shulga Morskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Susanne Kirschnek
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Evelyn Gaffal
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jennifer Landsberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Johannes Hellmuth
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Andreas Schmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. David Anz
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Michael Bscheider
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Tobias Schwerd
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Carola Berking
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Carole Bourquin
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Ulrich Kalinke
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Elisabeth Kremmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Hiroki Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Shizuo Akira
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Rachel Meyers
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Georg Häcker
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Michael Neuenhahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Dirk Busch
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Jürgen Ruland
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Simon Rothenfusser
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Marco Prinz
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Veit Hornung
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Stefan Endres
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Thomas Tüting
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Gunther Hartmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.P., R.B., T.T. and G. Hartmann designed the research; H.P., C.M., R.B., D.T., S.S.M., M.R., S.K., E.G., J.L., J.H., A.S., M.B., T.S., D.A., M.N., M.P., C. Berking and T.T. performed experiments; H.P., R.B., S.E., C. Bourquin, G. Häcker, C. Berking, R.M., V.H., S.R., T.T. and G. Hartmann conducted the data analyses; E.K., U.K., D.B., J.R., H.K. and S.A. provided genetically deficient mice and key research tools; H.P., C.M., R.B. and T.T. prepared the figures; and H.P., T.T. and G. Hartmann wrote the manuscript.

Corresponding authors

Correspondence to Thomas Tüting or Gunther Hartmann.

Ethics declarations

Competing interests

S.S.M. and R.M., as employees of Alnylam Pharmaceuticals, have options to purchase shares of Alnylam stock, and R.M. owns Alnylam stock.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Figs. 1–10 and Supplementary Methods (PDF 2137 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Poeck, H., Besch, R., Maihoefer, C. et al. 5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma. Nat Med 14, 1256–1263 (2008). https://doi.org/10.1038/nm.1887

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.1887

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing