A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS.

ABSTRACT

RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing VDAC1 abolishes C1-induced mitophagy, and gene knockdown of either KRAS, AKT or DNM1L rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer.Abbreviations ACTB/ß-actin actin beta; AKT AKT serine/threonine kinase; C1/merodantoin 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT catalase; CETSA cellular thermal shift assay; CHX cycloheximide; DKO double knockout; DNM1L/DRP1 dynamin 1 like; GAPDH glyceraldehyde-3-phosphate dehydrogenase; H2O2 hydrogen peroxide; HSPA1A/HSP70-1 heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90 heat shock protein 90 alpha family class A member 1; KRAS KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I unlipidated form of LC3B; LC3B-II phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1 MAPK associated protein 1; MAPK1/ERK2 mitogen-activated protein kinase 1; MAPK3/ERK1 mitogen-activated protein kinase 3; MFI mean fluorescence intensity; MiNA Mitochondrial Network Analysis; MTOR mechanistic target of rapamycin kinase; MTORC1 mechanistic target of rapamycin kinase complex 1; MTORC2 mechanistic target of rapamycin kinase complex 2; O2.- superoxide; OMA1 OMA1 zinc metallopeptidase; OPA1 OPA1 mitochondrial dynamin like GTPase; RICTOR RPTOR independent companion of MTOR complex 2; ROS reactive oxygen species; RPTOR/raptor regulatory associated protein of MTOR complex 1; SOD1 superoxide dismutase 1; SOD2 superoxide dismutase 2; SQSTM1/p62 sequestosome 1; VDAC1 voltage dependent anion channel 1; VDAC2 voltage dependent anion channel 2.