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

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.

References

  1. Kaelin, W. G. The concept of synthetic lethality in the context of anticancer therapy. Nature Rev. Cancer 5, 689–698 (2005)

    CAS  Article  Google Scholar 

  2. Hahn, W. C. et al. Creation of human tumour cells with defined genetic elements. Nature 400, 464–468 (1999)

    ADS  CAS  Article  Google Scholar 

  3. Dolma, S., Lessnick, S. L., Hahn, W. C. & Stockwell, B. R. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3, 285–296 (2003)

    CAS  Article  Google Scholar 

  4. Root, D. E., Flaherty, S. P., Kelley, B. P. & Stockwell, B. R. Biological mechanism profiling using an annotated compound library. Chem. Biol. 10, 881–892 (2003)

    CAS  Article  Google Scholar 

  5. Song, Z. & Steller, H. Death by design: mechanism and control of apoptosis. Trends Cell Biol. 9, M49–M52 (1999)

    CAS  Article  Google Scholar 

  6. Majno, G. & Joris, I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146, 3–15 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556 (1980)

    ADS  CAS  Article  Google Scholar 

  8. Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 (1972)

    CAS  Article  Google Scholar 

  9. Martin, S. J. et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545–1556 (1995)

    CAS  Article  Google Scholar 

  10. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75, 641–652 (1993)

    CAS  Article  Google Scholar 

  11. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006)

    CAS  Article  Google Scholar 

  12. Downward, J. Targeting RAS signalling pathways in cancer therapy. Nature Rev. Cancer 3, 11–22 (2003)

    CAS  Article  Google Scholar 

  13. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002)

    ADS  CAS  Article  Google Scholar 

  14. Graham, B. H. & Craigen, W. J. Genetic approaches to analyzing mitochondrial outer membrane permeability. Curr. Top. Dev. Biol. 59, 87–118 (2004)

    CAS  Article  Google Scholar 

  15. Baker, M. A., Lane, D. J., Ly, J. D., De Pinto, V. & Lawen, A. VDAC1 is a transplasma membrane NADH-ferricyanide reductase. J. Biol. Chem. 279, 4811–4819 (2004)

    CAS  Article  Google Scholar 

  16. Rostovtseva, T. & Colombini, M. ATP flux is controlled by a voltage-gated channel from the mitochondrial outer membrane. J. Biol. Chem. 271, 28006–28008 (1996)

    CAS  Article  Google Scholar 

  17. Mannella, C. A. Minireview: on the structure and gating mechanism of the mitochondrial channel, VDAC. J. Bioenerg. Biomembr. 29, 525–531 (1997)

    CAS  Article  Google Scholar 

  18. Beck, W. T. & Danks, M. K. Mechanisms of resistance to drugs that inhibit DNA topoisomerases. Semin. Cancer Biol. 2, 235–244 (1991)

    CAS  PubMed  Google Scholar 

  19. Cheng, E. H., Sheiko, T. V., Fisher, J. K., Craigen, W. J. & Korsmeyer, S. J. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301, 513–517 (2003)

    ADS  CAS  Article  Google Scholar 

  20. Xu, X., Decker, W., Sampson, M. J., Craigen, W. J. & Colombini, M. Mouse VDAC isoforms expressed in yeast: channel properties and their roles in mitochondrial outer membrane permeability. J. Membr. Biol. 170, 89–102 (1999)

    CAS  Article  Google Scholar 

  21. Poyurovsky, M. V. et al. Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization. Mol. Cell 12, 875–887 (2003)

    CAS  Article  Google Scholar 

  22. Koppel, D. A. et al. Bacterial expression and characterization of the mitochondrial outer membrane channel. Effects of n-terminal modifications. J. Biol. Chem. 273, 13794–13800 (1998)

    CAS  Article  Google Scholar 

  23. Zhen, Y. et al. Development of an LC-MALDI method for the analysis of protein complexes. J. Am. Soc. Mass Spectrom. 15, 803–822 (2004)

    CAS  Article  Google Scholar 

  24. Sage, J., Miller, A. L., Perez-Mancera, P. A., Wysocki, J. M. & Jacks, T. Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature 424, 223–228 (2003)

    ADS  CAS  Article  Google Scholar 

  25. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990)

    CAS  Article  Google Scholar 

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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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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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