Resistance to apoptosis is a hallmark of cancer and deregulation of apoptosis often leads to chemoresistance. Therefore, new approaches to target apoptosis-resistant cancer cells are crucial for the development of directed cancer therapies. In the present study, we investigated the effect of cell cycle regulators on interferon (IFN)-induced necroptosis as an alternative cell death mechanism to overcome apoptosis resistance. Here, we report a novel combination treatment of IFNs with cell cycle arrest-inducing compounds that induce necroptosis in apoptosis-resistant cancer cells and elucidate the underlying molecular mechanisms. Combination treatment of IFNs (i.e. IFNβ) with inhibitors of the cell cycle (e.g. vinorelbine (VNR), nocodazole (Noc), polo-like kinase-1 (Plk-1) inhibitor BI 6727) co-operate to induce necroptotic cell death upon caspase inactivation. The mode of cell death was confirmed by pharmacological inhibition and siRNA-mediated downregulation of the key necroptotic factors receptor-interacting protein (RIP) kinase 3 (RIP3) and mixed-lineage kinase-like (MLKL) in various cell lines. Mechanistically, we show that necroptosis upon VNR/IFNβ/zVAD.fmk treatment is RIP1-independent but relies on IFNβ-induced gene expression of Z-DNA-binding protein 1 (ZBP1) as shown by quantitative RT-PCR and genetic knockdown experiments. Interestingly, we find that RIP3 is phosphorylated in response to compounds that trigger mitotic arrest, even in the absence of IFNβ signaling and necroptosis induction. Together, the identification of a novel combination treatment that triggers necroptosis has implications for the development of molecular-targeted therapies to circumvent apoptosis resistance and point to an underestimated role of cell cycle regulation in cell death signaling.
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Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell . 2011;144:646–74.
Fulda S. Tumor resistance to apoptosis. Int J Cancer. 2009;124:511–5.
Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P. Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol. 2014;15:135–47.
Pasparakis M, Vandenabeele P. Necroptosis and its role in inflammation. Nature. 2015;517:311–20.
Vanden Berghe T, Hassannia B, Vandenabeele P. An outline of necrosome triggers. Cell Mol Life Sci. 2016;73:2137–52.
Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep. 2014;7:971–81.
de Almagro MC, Vucic D. Necroptosis: pathway diversity and characteristics. Semin Cell Dev Biol. 2015;39:56–62.
Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ, et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem. 2013;288:31268–79.
Schneider WM, Chevillotte MD, Rice CM. Interferon-stimulated genes: a complex web of host defenses. Annu Rev Immunol. 2014;32:513–45.
McComb S, Cessford E, Alturki NA, Joseph J, Shutinoski B, Startek JB, et al. Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages. Proc Natl Acad Sci USA. 2014;111:E3206–3213.
Legarda D, Justus SJ, Ang RL, Rikhi N, Li W, Moran TM, et al. CYLD proteolysis protects macrophages from TNF-mediated auto-necroptosis induced by LPS and licensed by Type I IFN. Cell Rep. 2016;15:2449–61.
Thapa RJ, Nogusa S, Chen P, Maki JL, Lerro A, Andrake M, et al. Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases. Proc Natl Acad Sci USA. 2013;110:E3109–3118.
Robinson N, McComb S, Mulligan R, Dudani R, Krishnan L, Sad S. Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium. Nat Immunol. 2012;13:954–62.
Kuriakose T, Kanneganti TD. ZBP1: innate sensor regulating cell death and inflammation. Trends Immunol. 2018;39:123–34.
Kuriakose T, Man SM, Subbarao Malireddi RK, Karki R, Kesavardhana S, Place DE, et al. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways. Sci Immunol. 2016;1:aag2045.
Lin J, Kumari S, Kim C, Van TM, Wachsmuth L, Polykratis A, et al. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature. 2016;540:124–8.
Newton K, Wickliffe KE, Maltzman A, Dugger DL, Strasser A, Pham VC, et al. RIPK1 inhibits ZBP1-driven necroptosis during development. Nature. 2016;540:129–33.
Kaiser WJ, Upton JW, Mocarski ES. Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNA-dependent activator of IFN regulatory factors. J Immunol. 2008;181:6427–34.
Wood KW, Cornwell WD, Jackson JR. Past and future of the mitotic spindle as an oncology target. Curr Opin Pharmacol. 2001;1:370–7.
Matson DR, Stukenberg PT. Spindle poisons and cell fate: a tale of two pathways. Mol Interv. 2011;11:141–50.
Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis. 2003;8:413–50.
Perez EA. Microtubule inhibitors: differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther. 2009;8:2086–95.
Hugle M, Belz K, Fulda S. Identification of synthetic lethality of PLK1 inhibition and microtubule-destabilizing drugs. Cell Death Differ. 2015;22:1946–56.
Heinicke U, Haydn T, Kehr S, Vogler M, Fulda S. BCL-2 selective inhibitor ABT-199 primes rhabdomyosarcoma cells to histone deacetylase inhibitor-induced apoptosis. Oncogene. 2018;37:5325–39.
Kotredes KP, Gamero AM. Interferons as inducers of apoptosis in malignant cells. J Interferon Cytokine Res. 2013;33:162–70.
Jackson JR, Patrick DR, Dar MM, Huang PS. Targeted anti-mitotic therapies: can we improve on tubulin agents? Nat Rev Cancer. 2007;7:107–17.
He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009;137:1100–11.
Cho Y, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell. 2009;137:1112–23.
Biziota E, Briasoulis E, Mavroeidis L, Marselos M, Harris AL, Pappas P. Cellular and molecular effects of metronomic vinorelbine and 4-O-deacetylvinorelbine on human umbilical vein endothelial cells. Anticancer Drugs. 2016;27:216–24.
Haji Abdolvahab M, Mofrad MR, Schellekens H. Interferon beta: from molecular level to therapeutic effects. Int Rev Cell Mol Biol. 2016;326:343–72.
Chen D, Tong J, Yang L, Wei L, Stolz DB, Yu J, et al. PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors. Proc Natl Acad Sci USA. 2018;115:3930–5.
McArthur K, Whitehead LW, Heddleston JM, Li L, Padman BS, Oorschot V, et al. BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science. 2018;359. pii: eaao6047.
Riley JS, Quarato G, Cloix C, Lopez J, O'Prey J, Pearson M, et al. Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis. EMBO J 2018;37. pii: e99238.
Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49–63.
Wu XN, Yang ZH, Wang XK, Zhang Y, Wan H, Song Y, et al. Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis. Cell Death Differ. 2014;21:1709–20.
de Almagro MC, Goncharov T, Izrael-Tomasevic A, Duttler S, Kist M, Varfolomeev E, et al. Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death. Cell Death Differ. 2017;24:26–37.
Sun X, Yin J, Starovasnik MA, Fairbrother WJ, Dixit VM. Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J Biol Chem. 2002;277:9505–11.
Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 2012;148:213–27.
Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature. 2007;448:501–5.
Bake V, Roesler S, Eckhardt I, Belz K, Fulda S. Synergistic interaction of Smac mimetic and IFNalpha to trigger apoptosis in acute myeloid leukemia cells. Cancer Lett. 2014;355:224–31.
Reiter M, Eckhardt I, Haferkamp A, Fulda S. Smac mimetic sensitizes renal cell carcinoma cells to interferon-alpha-induced apoptosis. Cancer Lett. 2016;375:1–8.
Cekay MJ, Roesler S, Frank T, Knuth AK, Eckhardt I, Fulda S. Smac mimetics and type II interferon synergistically induce necroptosis in various cancer cell lines. Cancer Lett. 2017;410:228–37.
Marschall V, Fulda S. Smac mimetic-induced upregulation of interferon-beta sensitizes glioblastoma to temozolomide-induced cell death. Cell Death Dis. 2015;6:e1888.
Pellegrini F, Budman DR. Review: tubulin function, action of antitubulin drugs, and new drug development. Cancer Invest. 2005;23:264–73.
Schmidinger M, Steger GG, Budinsky AC, Wenzel C, Brodowicz T, Locker GJ, et al. Vinorelbine and interferon-alpha2c as second-line therapy in metastatic renal cell carcinoma. Anticancer Drugs. 2000;11:175–9.
We thank D. Bruecher for expert technical assistance and C. Hugenberg for excellent secretarial assistance.
This work has been partially supported by grants from the BMBF (to S.F.).
Conflict of interest
The authors declare that they have no conflict of interest.
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Edited by G. Kroemer