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RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels

Nature volume 447, pages 865–869 (2007)Cite this article

Abstract

Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs1. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS–RAF–MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)—a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS–RAF–MEK pathway.

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Figure 1: Erastin activates a rapid, oxidative, non-apoptotic cell death process.
Figure 2: Erastin lethality is dependent on the RAS–RAF–MEK pathway.
Figure 3: Erastin compounds act through VDACs.

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Acknowledgements

We thank S. Flaherty and S. Dolma for supporting experiments, H. Widlund for supplying the BRAF shRNA construct, R. Becklin and J. Savage for help with the analysis of the pull-down data, P. Robbins for help with the pull-down experiments, K. Brown for assistance with transmission electron microscopy, and M. Colombini for supplying engineered yeast and for discussions. B.R.S. is supported by a Career Award at the Scientific Interface from the Burroughs Welcome Fund and by the National Cancer Institute. S.L.L. is supported by an NCI grant, an American Cancer Society Research Scholars Grant, the Terri Anna Perine Sarcoma Fund, a Primary Children’s Medical Center Foundation Innovative Research Grant, a Hope Street Kids grant and a Catalyst Grant from the University of Utah School of Medicine.

Author Contributions N.Y. designed and performed the RNAi and VDAC overexpression, quantitative PCR, erastin analogue viability and chemical characterization experiments. E.Z. performed two-dimensional western analysis, PARP-1 and pro-caspase-3 cleavage, and cytochrome c release experiments. E.Z. and N.Y. performed transmission electron microscopy experiments. A.J.B., D.J.F. and N.Y. performed the NADH oxidation and direct binding experiments. W.S.Y. characterized sensitivity to erastin in the BJ-derived cell series. A.J.W. performed the MEK1/2 inhibitor experiment. I.S. and A.J.B. synthesized erastin analogues. R.S. and S.L.L. provided BRAF shRNAs, analysis of BRAF knockdown and the phospho-ERK western analysis. J.M.P., J.J.B. and S.S. were responsible for setting up the technology platform to pull down proteins binding to small molecule compounds. M.v.R. and J.M.P. performed the pull-down experiments. J.J.B., J.M.P. and S.S. designed, reviewed and supervised the pull-down experiments, and contributed to the analysis of the data. B.R.S. conceived of and supervised the project, designed and analysed experiments, and performed the anti-oxidant studies. B.R.S. and N.Y. prepared the manuscript.

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Author notes

  1. Nicholas Yagoda, Moritz von Rechenberg and Elma Zaganjor: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Fairchild Center, 1212 Amsterdam Avenue, MC 2406,

    Nicholas Yagoda, Elma Zaganjor, Andras J. Bauer, Wan Seok Yang, Daniel J. Fridman, Adam J. Wolpaw, Inese Smukste & Brent R. Stockwell

  2. Department of Chemistry, Columbia University, New York, New York 10027, USA,

    Brent R. Stockwell

  3. Prolexys Pharmaceuticals, 2150 West Dauntless Avenue, Salt Lake City, Utah 84116, USA,

    Moritz von Rechenberg, John M. Peltier, J. Jay Boniface & Sudhir Sahasrabudhe

  4. Center for Children, Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, Utah 84112, USA,

    Richard Smith & Stephen L. Lessnick

  5. Division of Pediatric Hematology/Oncology, the Department of Oncological Sciences,

    Stephen L. Lessnick

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Correspondence to Brent R. Stockwell.

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Supplementary Information

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Yagoda, N., von Rechenberg, M., Zaganjor, E. et al. RAS–RAF–MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature 447, 865–869 (2007). https://doi.org/10.1038/nature05859

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