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.

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References

  1. 1.

    & The hallmarks of cancer. Cell 100, 57–70 (2000).

  2. 2.

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

  3. 3.

    , & Gastrointestinal stromal tumour. Lancet 369, 1731–1741 (2007).

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    , & siRNA and isRNA: two edges of one sword. Mol. Ther. 14, 463–470 (2006).

  8. 8.

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

  9. 9.

    , , & Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6, 443–453 (2007).

  10. 10.

    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).

  11. 11.

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

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

    & Melanoma. N. Engl. J. Med. 355, 51–65 (2006).

  16. 16.

    & Cell death: critical control points. Cell 116, 205–219 (2004).

  17. 17.

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

  18. 18.

    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).

  19. 19.

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

  20. 20.

    & Innate immune recognition of viral infection. Nat. Immunol. 7, 131–137 (2006).

  21. 21.

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

  22. 22.

    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).

  23. 23.

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

  24. 24.

    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).

  25. 25.

    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).

  26. 26.

    & Antiviral defense: interferons and beyond. J. Exp. Med. 203, 1837–1841 (2006).

  27. 27.

    , , , & Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

  28. 28.

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

  29. 29.

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

  30. 30.

    , , , & Type I interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia. Blood 108, 3253–3261 (2006).

  31. 31.

    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).

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

    • Hendrik Poeck
    • , Robert Besch
    • , Cornelius Maihoefer
    •  & Marcel Renn

    These authors contributed equally to this work.

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. Laboratory for Experimental Dermatology, Department of Dermatology and Allergology, 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, Massachusetts 02142, 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, Osaka 565-0871, Japan.

    • Hiroki Kato
    •  & Shizuo Akira
  11. Department of Neuropathology, University of Freiburg, Breisacherstrasse 64, 79106 Freiburg, Germany.

    • Marco Prinz

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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.

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.

Corresponding authors

Correspondence to Thomas Tüting or Gunther Hartmann.

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    Supplementary Tables 1–3, Supplementary Figs. 1–10 and Supplementary Methods

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DOI

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

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