Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil VSports app下载. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2018 Feb 27;9(3):325.
doi: 10.1038/s41419-018-0340-4.

V体育平台登录 - Glucose metabolism and NRF2 coordinate the antioxidant response in melanoma resistant to MAPK inhibitors

Raeeka Khamari  1 , Anne Trinh  1 , Pierre Elliott Gabert  1 , Paola Corazao-Rozas  1 , Samuel Riveros-Cruz  1 , Stephane Balayssac  2 , Myriam Malet-Martino  2 , Salim Dekiouk  1 , Marie Joncquel Chevalier Curt  3 , Patrice Maboudou  4 , Guillaume Garçon  5 , Laura Ravasi  6 , Pierre Guerreschi  1 , Laurent Mortier  7 , Bruno Quesnel  1   8 , Philippe Marchetti  1   9 , Jerome Kluza  10
Affiliations

Glucose metabolism and NRF2 coordinate the antioxidant response in melanoma resistant to MAPK inhibitors (VSports最新版本)

Raeeka Khamari et al. Cell Death Dis. .

Abstract

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. VSports手机版.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Conception and characterization of MAPK-resistant BRAFV600E melanoma model from SCID mice.
a Summary of the experimental procedure used to generate melanoma cell lines used in this study. Briefly, 25 SCID mice have been inoculated with A375 melanoma cells. Five mice were treated with vehicle only and killed when tumors have reached 1500 mm3. A375-v cells have been obtained after tumor dissociation from one of these tumors. Twenty other mice were treated with vemurafenib leading to a rapid tumor shrinkage. Forty days after the beginning of the treatment, two mice have exhibited tumor progression under vemurafenib therapy. Tumors have been extracted and have been dissociated to generate A375RIV1 and A375RIV2 cell lines. b Tumor progression of A375-injected mice treated with vehicule only (n = 5) or with vemurafenib as indicated in materials and method. c Proliferation of A375, A375-v, A375RIV1, and A375RIV2 cell lines exposed in vitro to vemurafenib at the indicated concentration for 72 h. The values represent the mean ± SD of three independent experiments. Statistical analyses were performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05 and **P < 0.01. d Tumor progression of A375, A375-v, and A375RIV1-injected mice (mean ± SD; n = 5, statistical analyses were performed compared to A375 by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05 and **P < 0.01). e Representative macroscopic views illustrating lungs and primary tumors in A375, A375-v, and A375RIV1-injected mice. Arrows indicate metastasis. f Sections from the primary tumors of A375, A375-v, and A375RIV1-injected mice or from lung metastasis of A375RIV1-injected mice. Samples were stained with PS100 to confirm immuno-histological profile of melanoma or with Ki67 antibody to assess proliferation. g Colony-forming ability of A375-v and A375RIV1 treated with indicated doses of vemurafenib, cobimetinib, trametinib, dabrafenib, or combination of vemurafenib/cobimetinib or debrafenib/trametinib for 7 days. The values represent the mean ± SD of three independent experiments. Statistical analyses were performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05 and **P < 0.01
Fig. 2
Fig. 2. MAPK-resistant melanoma exhibits OXPHOS dependency and glutamine addiction.
a Oxygen consumption rate (OCR pmol O2/min/mg tissues) from tumor biopsy obtained in A375, A375-v, and A375RIV1-injected mice (mean ± SD; n = 12, statistical analysis was performed by one-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05 and ***P < 0.005). b Analysis of relative mitochondrial DNA copy number in A375-v or A375RIV1 tumor biopsy by assessment of mtDNA-encoded ND2 RNA (left panel) or ATPase6 RNA. The transcripts level in each sample was normalized to that of ATPSynthB RNA. Mean ± SD; n = 3, statistical analyses were performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. ***P < 0.005). c Left panel: oxygen consumption rate (OCR pmol/min/20 000 cells) in A375-v or A375RIV1 cells. PDK inhibitor DCA (0.1, 0.5, 2, or 5 mM) has been injected when indicated (black arrow); right panel: immunoblotting of pyruvate dehydrogenase E1 component subunit alpha (PDHE1a), phospho-serine 293 of PDHE1a, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase kinase 3 (PDK3) in A375-v cells compared to A375RIV1. Actin served as loading control. d Oxygen consumption rate (OCR pmol/min/10 µg mitochondria) of isolated mitochondria obtained from tumor of A375-v and A375RIV1-injected mice. Mitochondria have been incubated in a pyruvate/malate buffer and the following molecules have been injected when indicated: adenosine 5′-diphosphate sodium (ADP), antimycin A (AA), tetramethyl-p-phenylenediamine/ascorbate (TMPD/Asc), and potassium cyanide (KCN) (n = 3). e Glutamine consumption of A375-v or A375RIV1 cell lines growing for 24, 48, 72, or 96 h in full medium (mean ± SD; n = 3, statistical analyses were performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05). f Colony-forming ability of A375-v and A375RIV1 growing in medium containing the indicated glutamine concentration for 7 days (mean ± SD; n = 3, statistical analyses were performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05). g Colony-forming ability of A375-v and A375RIV1 exposed to glutaminase (GLS) inhibitor BPTES in full medium for 7 days. Pictures are representative of three independent experiments
Fig. 3
Fig. 3. Mitochondrial pyruvate uptake increases in MAPK-resistant melanoma cells.
a Coronal and sagittal sections of A375-v or A375RIV1-injected mice xenograft scan 1 h after single 18F-FDG injection using microPET to show change in tumor activity and tracer distribution. As indicated, mice have been treated with vehicle or with vemurafenib for 7 days. Mean %ID/g values are shown below to tumor xenograft. b Glucose consumption of A375-v or A375RIV1 cell lines growing for 24, 48, or 72 h in full medium in the absence (Co.) or presence of 0.3 µM of vemurafenib. 2-deoxyglucose (2DG) has been used as positive control to inhibit glucose uptake in A375-v cells (mean ± SD; n = 3, statistical analyses were performed by two-way ANOVA compared to A375-v with a 95% interval of confidence followed by Bonferroni’s post-test. **P < 0.001). c Glycosis activity in A375-v, A375RIV1, and A375rho0 has been assessed by measuring ECAR (mpH min−1 20,000 cells) with Seahorse technology. All cell lines have been incubated in glutamine supplemented medium and the following molecules have been injected subsequently: glucose (glc), oligomycin (olig.), and 2-deoxyglucose (2DG) (mean ± SD; n = 3, statistical analysis were performed by two-way ANOVA compared to A375-v with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05). d Immunoblotting of hexokinase 1 (HK1), hexokinase 2 (HK2), aldolase A (ALDO A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA) expression in A375-v cells compared to A375RIV1. Actin served as loading control. e Colony-forming ability of A375-v and A375RIV1 exposed to mitochondrial pyruvate carrier (MPC) inhibitor UK5099 in full medium for 7 days. Pictures are representative of three independant experiments
Fig. 4
Fig. 4. Mitochondrial metabolism favorizes de novo glutamate synthesis and related glutathione content.
a Comparative steady-state isotopomer profiling of A375-v and A375RIV1 cells via 13C NMR analysis following 24 h incubation with [U-13C]-glucose. The percentage indicates the variation of the [U-13C6]-glucose-related metabolites of A375RIV1 compared to A375-v. b Determination of glutathione content by CPLH in A375-v and A375RIV1 as described in materials and methods (mean ± SD; n = 3, statistical analysis was performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05). c Determination of oxidative stress with hydroethidium (HE) and glutathione content with monobromobimane (mBBR) in A375-v and A375RIV1. A375-v and A375RIV1 cells have grown for 24 h in glutamine-free medium (supplemented with glucose) and cells have been treated with N-ethyl maleimide (0, 50, 100, and 200 µM) for 24 h. Fluorescences of mBBR and HE have been assessed by flow cytometry. Results are representing as dot plot divided in two gates: cells with high GSH content are observed in upper gate; cells with low GSH content are observed in lower gate. Left panel: representative cytofluorimetric profile of experiment performed with A375-v and A375RIV1 treated with NEM 50 µM for 24 h. Right panel: histogram A375-v and A375RIV1 treated with increasing concentration of NEM (50, 100, and 200 µM) for 24 h (mean ± SD; n = 3, statistical analysis was performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05). d Proliferation of A375-v (black line) or A375RIV1 (red line) cells treated with increasing concentration of vemurafenib for 72 h. When indicated, cell lines have been conjointly treated with PEITC (5 µM). Cell number has been determined using Cyquant reactif and measured with a spectrofluorimeter. e A375-v or A375RIV1 have grown for 24 h in full medium and amino acid concentration has been compared to a fresh full medium (mean ± SD; n = 3, Student’s t test). f Colony-forming ability of A375-v and A375RIV1 grown in full medium and treated with sulfasalazine as indicated for 7 days. The values represent the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05 and **P < 0.01)
Fig. 5
Fig. 5. Biochemical pathway in A375RIV1 in favor of GSH synthesis.
Gene overexpressed in A375RIV1 compared to A375-v are visualized in boxes (see Fig. 6a for results). Glucose-related metabolites that have been measured with higher concentration in A375RIV1 compared to A375-v are indicated in red and measured with lower concentrations are indicated in blue (see Fig. 4a, e for results)
Fig. 6
Fig. 6. NRF2 promotes antioxydant response in MAPK-resistant melanoma.
a mRNA expressions of genes implicated in oxidative stress status have been assessed by multiplex PCR in A375RIV1 compared to A375-v. b Immunofluorescence of NRF2 in A375-v and A375RIV1 cells. BSO has been used as positive control in A375-v to favorize NRF2 nuclear accumulation. DAPI staining has been used to visualize nucleus compartment. Left panel: representative pictures of three independent experiments. Right panel: percentage of A375-v or A375RIV1 treated or not with BSO exhibiting nuclear NRF2 labeling in nucleus. The values represent the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05). c Left panel: immunoblotting of NRF2 in isolated nuclei or in whole-cells lysates of non-specific scramble control (SiCo.) or NRF2 silencing (SiNRF2) cells. Actin or Lamin B served as loading control. Right panel: immunoblotting of anionic amino acid transporter light chain, xCT, or Slc7a11 (the specific subunit of system XC−), Heme oxygenase 1 (HMOX1), transketolase (TKT), and transaldolase (TALDO) in si-control or si-NRF2 treated-A375RIV1 cells. Actin served as loading control. d ROS production in A375-v or A375RIV1 has been evaluated by flow cytometry after hydroethidium (HE) labeling in si-control or siNRF2-treated cells. e Left panel: colony-forming ability of A375-v and A375RIV1 grown in full medium and treated with vemurafenib as indicated for 10 days. Prior drug treatment, cells have been transfected with non-specific scramble control (SiCo.) or with SiRNA NRF2 (SiNRF2) as indicated. Representative pictures of three independent experiments. Right panel: the number of colonies has been counted for each condition and was indicated in the histogram. The values represent the mean ± SD of three independent experiments. Statistical analysis was performed by two-way ANOVA with a 95% interval of confidence followed by Bonferroni’s post-test. *P < 0.05
See this image and copyright information in PMC

References

    1. Long GV, et al. Overall survival and durable responses in patients with BRAF V600-mutant metastatic melanoma receiving dabrafenib combined with trametinib. J. Clin. Oncol. 2016;34:871–878. doi: 10.1200/JCO.2015.62.9345. - "V体育平台登录" DOI - PubMed
    1. Luke JJ, Flaherty KT, Ribas A, Long GV. Targeted agents and immunotherapies: optimizing outcomes in melanoma. Nat. Rev. Clin. Oncol. 2017;14:463–482. doi: 10.1038/nrclinonc.2017.43. - "VSports最新版本" DOI - PubMed
    1. Haq R, et al. Oncogenic BRAF regulates oxidative metabolism via PGC1α and MITF. Cancer Cell. 2013;23:302–315. doi: 10.1016/j.ccr.2013.02.003. - DOI - PMC - PubMed
    1. Beck D, et al. Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells. Sci. Signal. 2013;6:ra7. doi: 10.1126/scisignal.2003057. - DOI - PMC - PubMed
    1. Corazao-Rozas P, et al. Mitochondrial oxidative phosphorylation controls cancer cell’s life and death decisions upon exposure to MAPK inhibitors. Oncotarget. 2016;7:39473–39485. doi: 10.18632/oncotarget.7790. - DOI - PMC - PubMed

Publication types

MeSH terms