Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon–independent apoptosis in human melanoma cells
Robert Besch, … , Simon Rothenfusser, Gunther Hartmann
Robert Besch, … , Simon Rothenfusser, Gunther Hartmann
Published July 20, 2009
Citation Information: J Clin Invest. 2009;119(8):2399-2411. https://doi.org/10.1172/JCI37155.
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Research Article

Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon–independent apoptosis in human melanoma cells

Abstract

The retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated antigen 5 (MDA-5) helicases sense viral RNA in infected cells and initiate antiviral responses such as the production of type I IFNs. Here we have shown that RIG-I and MDA-5 also initiate a proapoptotic signaling pathway that is independent of type I IFNs. In human melanoma cells, this signaling pathway required the mitochondrial adapter Cardif (also known as IPS-1) and induced the proapoptotic BH3-only proteins Puma and Noxa. RIG-I– and MDA-5–initiated apoptosis required Noxa but was independent of the tumor suppressor p53. Triggering this pathway led to efficient activation of mitochondrial apoptosis, requiring caspase-9 and Apaf-1. Surprisingly, this proapoptotic signaling pathway was also active in nonmalignant cells, but these cells were much less sensitive to apoptosis than melanoma cells. Endogenous Bcl-xL rescued nonmalignant, but not melanoma, cells from RIG-I– and MDA-5–mediated apoptosis. In addition, we confirmed the results of the in vitro studies, demonstrating that RIG-I and MDA-5 ligands both reduced human tumor lung metastasis in immunodeficient NOD/SCID mice. These results identify an IFN-independent antiviral signaling pathway initiated by RIG-I and MDA-5 that activates proapoptotic signaling and, unless blocked by Bcl-xL, results in apoptosis. Due to their immunostimulatory and proapoptotic activity, RIG-I and MDA-5 ligands have therapeutic potential due to their ability to overcome the characteristic resistance of melanoma cells to apoptosis.

Authors

Robert Besch, Hendrik Poeck, Tobias Hohenauer, Daniela Senft, Georg Häcker, Carola Berking, Veit Hornung, Stefan Endres, Thomas Ruzicka, Simon Rothenfusser, Gunther Hartmann

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Figure 1

pppRNA and poly(I:C) induce apoptosis in melanoma cells.

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pppRNA and poly(I:C) induce apoptosis in melanoma cells. (A) The viabili...
(A) The viability of different melanoma cell lines was determined after transfection of in vitro–transcribed RNA (pppRNA) or poly(I:C) (20 ng/ml). Cells were analyzed 17 (pppRNA) or 24 hours [poly(I:C)] after transfection. Viability of cells treated with transfection reagent alone (mock-transfected cells; No RNA) was set to 100%. *P ≤ 0.05 compared with the same cell line treated with transfection reagent alone. (B) Fluorescence-activated cell sorting (FACS) analysis of apoptotic 1205Lu cells treated with pppRNA or poly(I:C) (200 ng/ml) for 24 hours either complexed with a liposomal transfection reagent (Transfection) or not (No transfection). Percentages indicate the portion of cells in each quadrant that defines annexin V– or propidium iodide–positive or –negative cells. Results are representative of 3 independent experiments. (C) Left: Viability of the metastatic melanoma cell line 1205Lu was measured 24 hours after transfection of different short in vitro–transcribed RNAs (pppRNA1–3) in double-stranded (ds) or single-stranded (ss) form or synthetic unconjugated RNAs (OH) with the same sequence. Sequences of pppRNAs are shown in Methods. Right: Viability of 1205Lu cells transfected with different doses of poly(I:C) (20–200 ng/ml) for 24 hours. Viability of mock-transfected cells was set to 100%. (D) FACS analysis of apoptotic 1205Lu cells treated with pppRNA or poly(I:C) as described in C. Annexin V–positive and propidium iodide–negative cells (AN+/PI–) are depicted. In A, C, and D, the mean ± SD of 3 independent experiments is shown. *P ≤ 0.05 compared with mock-transfected cells in C and D. (E) ppp­RNAs (pppRNA1–3) or poly(I:C) (5 ng/ml) were used with or without transfection reagent, and 1205Lu cells were analyzed 12 (pppRNA) or 24 hours [poly(I:C)] after treatment. Activation of caspase-3 was assessed by immunoblotting with an antibody specific for procaspase-3 (35 kDa) and its active cleaved subunits (17, 19 kDa). WM9 melanoma cells treated with 1 μM staurosporine (STS) for 5 hours served as a positive control for caspase cleavage. One representative of 3 independent experiments is shown.