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 5

RIG-I and MDA-5 induce expression of the proapoptotic BH3-only members of the Bcl-2 family Noxa, Puma, Bim, and Bik.

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RIG-I and MDA-5 induce expression of the proapoptotic BH3-only members o...
(A) 1205Lu cells were treated with pppRNA or poly(I:C) (3 ng/ml) for 24 hours, and levels of Noxa, Puma, Bim, Bad, Bik, Bid, and Hrk mRNA were measured by quantitative RT-PCR. Relative mRNA levels compared with mock-transfected cells are depicted. Mean ± SD of 3 independent experiments is shown. *P ≤ 0.05 and NS compared with mock-transfected cells. (B) The melanoma cell lines 1205Lu, WM239A, WM278, and WM793 were treated with pppRNA or poly(I:C) (5 ng/ml), and Noxa and Puma proteins were quantified by immunoblotting. β-Actin served as loading control. Blots are representative of 2 independent experiments. (C) Left: Expression of the BH3-only proteins Noxa, Puma, Bim, Bad, Bik, and Bid was inhibited by treatment with respective siRNAs for 48 hours. Thereafter, cells were treated with pppRNA (left), poly(I:C) (5 ng/ml; right), or transfection reagent alone and analyzed for apoptotic cells by FACS. Annexin V–positive and propidium iodide–negative cells are represented. Mean ± SD of 3 independent experiments is shown. *P ≤ 0.05 compared with control siRNA–transfected cells treated with pppRNA.