The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate–Lactate Exchange in BRAF-Mutant Human Melanoma Cells

Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. H-1 NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAF(WT) (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. C-13 NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAF(WT) cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized C-13-pyruvatelactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. (C) 2016 AACR

Mitochondrial oxidative metabolism can be therapeutically exploited for the treatment of metastatic melanoma

La plupart des cellules cancéreuses présentent une reprogrammation métabolique qui favorise la glycolyse et diminue la phosphorylation oxydative. Cette reprogrammation a reçu le nom d’effet Warburg et permet aux cellules tumorales de proliférer même en conditions adverses. Bien que l’effet Warburg ait été, pendant longtemps, associé à un dysfonctionnement mitochondrial, plusieurs études ont montré que dans les cellules cancéreuses les mitochondries ne sont pas dysfonctionnelles et jouent même un rôle important dans la tumorigénèse. Cependant, les mécanismes qui régulent cette reprogrammation métabolique dans le mélanome restent encore à être bien évalués. Dans ce contexte, nous avons montré que les mélanomes présentent une faible activité mitochondriale caractérisée par une diminution de la consommation d’oxygène. Ce profil métabolique est contrôlé au moins en partie par le facteur de transcription HIF-1, via l’expression de la pyruvate déshydrogénase kinase 3 (PDK3). L’inhibition pharmacologique de la PDK3, utilisant le Dichloroacetate (DCA) est suffisante pour réactiver la phosphorylation oxydative et induire la production des espèces réactives de l’oxygène (ROS). Ainsi, la combinaison du DCA avec la molécule pro-oxydant elesclomol permet de potentialiser l’effet antitumural de cette dernière, de manière synergique. Fait intéressant, cette combinaison est également efficace dans les cellules ayant développé une résistance au vemurafenib, un inhibiteur de la protéine BRAFV600E. Dans ce contexte, dans la deuxième partie de cette étude, nous avons évalué si les cellules résistantes au vemurafenib présentent une modification métabolique qui pourrait expliquer leur sensibilité à la combiniasion DCA+eleslclomol. Nous avons montré que le vemurafenib induit une diminution de la glycolyse rendant les cellules dépendantes à la phosphorylation oxydative et augmente la biogénèse mitochondriale de manière dépendante ou indépendante de la réactivation de la voie MITF/PGC1α. En accord avec ces résultats, les cellules résistantes au vemurafenib présentent une augmentation de la consommation d’oxygène et de la production de ROS par rapport aux cellules sensibles. Le nouveau profil métabolique des cellules résistantes les rende plus sensibles aux agents pro-oxydants tels que l’elesclomol Nos résultats ont permis de montré la possibilité de cibler les modifications métaboliques par une approche pro-oxydant dans le but d’éradiquer de manière efficace les cellules de mélanome.Most cancer cells undergo a metabolic rewiring from oxidative phosphorylation to glycolysis that allows them to proliferate even under stressful conditions. This phenomenon is known as the Warburg Effect and has been often associated to mitochondrial dysfunction. Although, many studies have shown that mitochondria is still active in cancer cells and seems to play a key role in tumorigenesis little is know about the mechanisms that regulate this metabolic swift. In this context, we first focused in the study of melanoma metabolism in different cell lines as in samples coming from patients. We first found that melanoma cells present low mitochondrial activity characterized by low oxidative phosphorylation. This metabolic behavior is at least partially controlled by the hypoxia-inducible factor-1α HIF-1α witch is constitutively express in melanoma cells even under nomoxic conditions. Inhibition of this factor induces a strong decrease in the expression and activity of PDK3. Pharmacological inhibition of PDK3 activity by dichloroacetate (DCA) is enough to reactivate mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production. Furthermore DCA increases in a synergistic manner elesclomol’s induced ROS production and cell death. Interestingly, BRAF V600E melanoma cells that were resistant to the BRAF inhibitor vemurafenib show were also sensible to this combination. Consequently, as a second part of this work we looked for to understand, why resistant cells were so sensible to these agents and if there were some metabolic modifications that could explain this behavior. We found that vemurafenib BRAFV600E induced inhibition causes an important decrease in glycolysis and renders melanoma cells addicted to oxidative phosphorylation by increasing mitochondria biogenesis dependently or not of MTIF/PCG1 axis. Conversely, vemurafenib resistant melanoma cell lines show higher mitochondrial activity associated with higher ROS production. Thus these cells are more sensible to elesclomol induced cell death than vemurafenib sensible cell lines. Our findings provide new insights into the metabolic pathways that allow cells to adapt to difficult microenvironment, showing that these metabolic modifications, especially in terms of ROS production, can be used to target and eradicate melanoma cells

Exploitation du métabolisme mitochondrial oxydatif dans l'éradication du mélanome métastatique

Most cancer cells undergo a metabolic rewiring from oxidative phosphorylation to glycolysis that allows them to proliferate even under stressful conditions. This phenomenon is known as the Warburg Effect and has been often associated to mitochondrial dysfunction. Although, many studies have shown that mitochondria is still active in cancer cells and seems to play a key role in tumorigenesis little is know about the mechanisms that regulate this metabolic swift. In this context, we first focused in the study of melanoma metabolism in different cell lines as in samples coming from patients. We first found that melanoma cells present low mitochondrial activity characterized by low oxidative phosphorylation. This metabolic behavior is at least partially controlled by the hypoxia-inducible factor-1α HIF-1α witch is constitutively express in melanoma cells even under nomoxic conditions. Inhibition of this factor induces a strong decrease in the expression and activity of PDK3. Pharmacological inhibition of PDK3 activity by dichloroacetate (DCA) is enough to reactivate mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production. Furthermore DCA increases in a synergistic manner elesclomol’s induced ROS production and cell death. Interestingly, BRAF V600E melanoma cells that were resistant to the BRAF inhibitor vemurafenib show were also sensible to this combination. Consequently, as a second part of this work we looked for to understand, why resistant cells were so sensible to these agents and if there were some metabolic modifications that could explain this behavior. We found that vemurafenib BRAFV600E induced inhibition causes an important decrease in glycolysis and renders melanoma cells addicted to oxidative phosphorylation by increasing mitochondria biogenesis dependently or not of MTIF/PCG1 axis. Conversely, vemurafenib resistant melanoma cell lines show higher mitochondrial activity associated with higher ROS production. Thus these cells are more sensible to elesclomol induced cell death than vemurafenib sensible cell lines. Our findings provide new insights into the metabolic pathways that allow cells to adapt to difficult microenvironment, showing that these metabolic modifications, especially in terms of ROS production, can be used to target and eradicate melanoma cells.La plupart des cellules cancéreuses présentent une reprogrammation métabolique qui favorise la glycolyse et diminue la phosphorylation oxydative. Cette reprogrammation a reçu le nom d’effet Warburg et permet aux cellules tumorales de proliférer même en conditions adverses. Bien que l’effet Warburg ait été, pendant longtemps, associé à un dysfonctionnement mitochondrial, plusieurs études ont montré que dans les cellules cancéreuses les mitochondries ne sont pas dysfonctionnelles et jouent même un rôle important dans la tumorigénèse. Cependant, les mécanismes qui régulent cette reprogrammation métabolique dans le mélanome restent encore à être bien évalués. Dans ce contexte, nous avons montré que les mélanomes présentent une faible activité mitochondriale caractérisée par une diminution de la consommation d’oxygène. Ce profil métabolique est contrôlé au moins en partie par le facteur de transcription HIF-1, via l’expression de la pyruvate déshydrogénase kinase 3 (PDK3). L’inhibition pharmacologique de la PDK3, utilisant le Dichloroacetate (DCA) est suffisante pour réactiver la phosphorylation oxydative et induire la production des espèces réactives de l’oxygène (ROS). Ainsi, la combinaison du DCA avec la molécule pro-oxydant elesclomol permet de potentialiser l’effet antitumural de cette dernière, de manière synergique. Fait intéressant, cette combinaison est également efficace dans les cellules ayant développé une résistance au vemurafenib, un inhibiteur de la protéine BRAFV600E. Dans ce contexte, dans la deuxième partie de cette étude, nous avons évalué si les cellules résistantes au vemurafenib présentent une modification métabolique qui pourrait expliquer leur sensibilité à la combiniasion DCA+eleslclomol. Nous avons montré que le vemurafenib induit une diminution de la glycolyse rendant les cellules dépendantes à la phosphorylation oxydative et augmente la biogénèse mitochondriale de manière dépendante ou indépendante de la réactivation de la voie MITF/PGC1α. En accord avec ces résultats, les cellules résistantes au vemurafenib présentent une augmentation de la consommation d’oxygène et de la production de ROS par rapport aux cellules sensibles. Le nouveau profil métabolique des cellules résistantes les rende plus sensibles aux agents pro-oxydants tels que l’elesclomol Nos résultats ont permis de montré la possibilité de cibler les modifications métaboliques par une approche pro-oxydant dans le but d’éradiquer de manière efficace les cellules de mélanome

Oncogenic KRAS suppresses store-operated Ca²⁺ entry and I_CRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca2+ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca2+ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca2+ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca2+ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca2+ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRASG13D), and have shown that reduced Ca2+ release from the ER and mitochondrial Ca2+ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca2+ Entry (SOCE) and its underlying current, ICRAC are under the influence of KRASG13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca2+ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRASG13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRASG13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca2+ entry downstream of KRASG13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis

Glucose metabolism and NRF2 coordinate the antioxidant response in melanoma resistant to MAPK inhibitors

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