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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.

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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.

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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).

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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

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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.

Corresponding authors

Correspondence to Thomas Tüting or Gunther Hartmann.

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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.

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

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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

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