RESUMO
The Glucocorticoïd-induced leucine zipper (GILZ) protein has profound anti-inflammatory activities in haematopoietic cells. GILZ regulates numerous signal transduction pathways involved in proliferation and survival of normal and neoplastic cells. Here, we have demonstrated the potential of GILZ in alleviating apoptosis induced by ER stress inducers. Whereas the glucocorticoid, dexamethasone, protects from tunicamycin-induced cell death, silencing endogeneous GILZ in dexamethasone-treated cancer cells alter the capacity of glucocorticoids to protect from tunicamycin-mediated apoptosis. Under ER stress conditions, overexpression of GILZ significantly reduced activation of mitochondrial pathway of apoptosis by maintaining Bcl-xl level. GILZ protein affects the UPR signaling shifting the balance towards pro-survival signals as judged by down-regulation of CHOP, ATF4, XBP1s mRNA and increase in GRP78 protein level. Interestingly, GILZ sustains high mitochondrial OXPHOS during ER stress and cytoprotection mediated by GILZ is abolished in cells depleted of mitochondrial DNA, which are OXPHOS-deficient. These findings reveal a new role of GILZ, which acts as a cytoprotector against ER stress through a pathway involving mitochondrial OXPHOS.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Fatores de Transcrição/genética , Regulação para Cima , Animais , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Desdobramento de Proteína , Fatores de Transcrição/metabolismoRESUMO
Targeted therapies as BRAF and MEK inhibitor combination have been approved as first-line treatment for BRAF-mutant melanoma. However, disease progression occurs in most of the patients within few months of therapy. Metabolic adaptations have been described in the context of acquired resistance to BRAF inhibitors (BRAFi). BRAFi-resistant melanomas are characterized by an increase of mitochondrial oxidative phosphorylation and are more prone to cell death induced by mitochondrial-targeting drugs. BRAFi-resistant melanomas also exhibit an enhancement of oxidative stress due to mitochondrial oxygen consumption increase. To understand the mechanisms responsible for survival of BRAFi-resistant melanoma cells in the context of oxidative stress, we have established a preclinical murine model that accurately recapitulates in vivo the acquisition of resistance to MAPK inhibitors including several BRAF or MEK inhibitors alone and in combination. Using mice model and melanoma cell lines generated from mice tumors, we have confirmed that the acquisition of resistance is associated with an increase in mitochondrial oxidative phosphorylation as well as the importance of glutamine metabolism. Moreover, we have demonstrated that BRAFi-resistant melanoma can adapt mitochondrial metabolism to support glucose-derived glutamate synthesis leading to increase in glutathione content. Besides, BRAFi-resistant melanoma exhibits a strong activation of NRF-2 pathway leading to increase in the pentose phosphate pathway, which is involved in the regeneration of reduced glutathione, and to increase in xCT expression, a component of the xc-amino acid transporter essential for the uptake of cystine required for intracellular glutathione synthesis. All these metabolic modifications sustain glutathione level and contribute to the intracellular redox balance to allow survival of BRAFi-resistant melanoma cells.
Assuntos
Antioxidantes/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glucose/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Melanoma/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Glutamatos/biossíntese , Glutationa/biossíntese , Humanos , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Ácido Pirúvico/metabolismoRESUMO
Although MAPK pathway inhibitors are becoming a promising anticancer strategy, they are insufficient to fully eliminate cancer cells and their long-term efficacy is strikingly limited in patients with BRAF-mutant melanomas. It is well established that BRAF inhibitors (BRAFi) hamper glucose uptake before the apparition of cell death. Here, we show that BRAFi induce an extensive restructuring of mitochondria including an increase in mitochondrial activity and biogenesis associated with mitochondrial network remodeling. Furthermore, we report a close interaction between ER and mitochondria in melanoma exposed to BRAFi. This physical connection facilitates mitochondrial Ca2+ uptake after its release from the ER. Interestingly, Mfn2 silencing disrupts the ER-mitochondria interface, intensifies ER stress and exacerbates ER stress-induced apoptosis in cells exposed to BRAFi in vitro and in vivo. This mitochondrial control of ER stress-mediated cell death is similar in both BRAF- and NRAS-mutant melanoma cells exposed to MEK inhibitors. This evidence reinforces the relevance in combining MAPK pathway inhibitors with mitochondriotropic drugs to improve targeted therapies.
Assuntos
Neoplasias/enzimologia , Fosforilação Oxidativa , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Azetidinas/farmacologia , Cálcio/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , GTP Fosfo-Hidrolases/metabolismo , Inativação Gênica , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos SCID , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , Sulfonamidas/farmacologia , Resultado do Tratamento , VemurafenibRESUMO
Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition.
Assuntos
Indóis/farmacologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Melanoma/enzimologia , Melanoma/genética , Camundongos , Camundongos SCID , Mitocôndrias/genética , Fosforilação Oxidativa , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vemurafenib , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.