title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells.
abstract: Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 ('BAY') triggers death of BRAFV600Emelanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Farhan
authors.initials: F
authors.lastname: Basit
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Lisanne Mpe
authors.initials: LM
authors.lastname: van Oppen
authors.affiliation: Global Therapeutic Research Group Oncology II, Bayer Pharma AG, Berlin, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Schöckel
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Hasse M
authors.initials: HM
authors.lastname: Bossenbroek
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Sjenet E
authors.initials: SE
authors.lastname: van Emst-de Vries
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Johannes Cw
authors.initials: JC
authors.lastname: Hermeling
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Sander
authors.initials: S
authors.lastname: Grefte
authors.affiliation: Global Therapeutic Research Group Oncology II, Bayer Pharma AG, Berlin, Germany.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Kopitz
authors.affiliation: Bayer AG Innovation Strategy, Leverkusen, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Heroult
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Peter
authors.initials: P
authors.lastname: Hgm Willems
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Centre (Radboudumc), Nijmegen, The Netherlands.
authors.forename: Werner Jh
authors.initials: WJ
authors.lastname: Koopman
mesh_terms: D000071187:Autophagy-Related Protein 5 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D034281:Dynamins; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D063306:Mitophagy; D010069:Oxadiazoles / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000099202:PTEN-Induced Putative Kinase
chemical_list: D000071187:Autophagy-Related Protein 5; D034281:Dynamins; D042967:Electron Transport Complex I; D020558:GTP Phosphohydrolases; D018841:HSP90 Heat-Shock Proteins; D008869:Microtubule-Associated Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins; D010069:Oxadiazoles; D011494:Protein Kinases; D011720:Pyrazoles; D017382:Reactive Oxygen Species; D000099202:PTEN-Induced Putative Kinase; C000591541:1-cyclopropyl-4-(4-((5-methyl-3-(3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl)pyridin-2-yl)piperazine; C502778:ATG5 protein, human; C092306:TRAP1 protein, human; C110768:DNM1L protein, human