title: TSC2 Deficiency Unmasks a Novel Necrosis Pathway That Is Suppressed by the RIP1/RIP3/MLKL Signaling Cascade.
abstract: Tuberous sclerosis complex (TSC) is a genetic multiorgan disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart, and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in the hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The current study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using l-buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage, and necrotic cell death in TSC-deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3/MLKL-dependent signaling using chemical inhibitors necrostatin-1 (Nec-1) and necrosulfonamide (NSA) synergizes with BSO and auranofin in killing TSC-deficient cells. Expression analysis demonstrated that RIP1, RIP3, and MLKL protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction in a glutaminolysis-dependent and autophagy-independent manner. Finally, supplementation with the mitochondrial metabolite α-ketoglutarate, whose synthesis is regulated by RIP1/RIP3/MLKL, rescues cells from the sensitizing effect of Nec-1 and NSA. Together, this study identifies a previously unrecognized novel regulated necrotic death pathway that involves mitochondrial homeostasis, is suppressed by the RIP1/RIP3/MLKL signaling in TSC-deficient cells, and could be a promising therapeutic target for TSC-associated tumors. Cancer Res; 76(24); 7130-9. ©2016 AACR.
authors.affiliation: Lovelace Respiratory Research Institute, Albuquerque, New Mexico.
authors.forename: Piotr T
authors.initials: PT
authors.lastname: Filipczak
authors.affiliation: Lovelace Respiratory Research Institute, Albuquerque, New Mexico.
authors.forename: Cindy
authors.initials: C
authors.lastname: Thomas
authors.affiliation: Lovelace Respiratory Research Institute, Albuquerque, New Mexico.
authors.forename: Wenshu
authors.initials: W
authors.lastname: Chen
authors.affiliation: Radical Therapeutics, West Tisbury, Massachusetts.
authors.forename: Andrew
authors.initials: A
authors.lastname: Salzman
authors.affiliation: Lovelace Respiratory Research Institute, Albuquerque, New Mexico.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: McDonald
authors.affiliation: Lovelace Respiratory Research Institute, Albuquerque, New Mexico. YLin@lrri.org SBelinsk@lrri.org.
authors.forename: Yong
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Lovelace Respiratory Research Institute, Albuquerque, New Mexico. YLin@lrri.org SBelinsk@lrri.org.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Belinsky
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008819:Mice, Nude; D009336:Necrosis / Q000378:metabolism* / Q000473:pathology*; D018384:Oxidative Stress / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014402:Tuberous Sclerosis / Q000378:metabolism* / Q000473:pathology*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000172:deficiency
chemical_list: D020690:GTPase-Activating Proteins; C490620:Ralbp1 protein, mouse; D017382:Reactive Oxygen Species; C000624653:TSC2 protein, human; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse