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Nat Commun
2018 Mar 23;91:1208. doi: 10.1038/s41467-018-03525-0.
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Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects.
Corbet C
,
Bastien E
,
Draoui N
,
Doix B
,
Mignion L
,
Jordan BF
,
Marchand A
,
Vanherck JC
,
Chaltin P
,
Schakman O
,
Becker HM
,
Riant O
,
Feron O
.
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Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition.
Fig. 1. Inhibitors of lactate metabolism exert differential effects on cancer cell growth and respiration depending on glucose availability. a Cell growth (72 h) and (b) oxygen consumption rate (OCR) of SiHa cells after treatment with 10 µM 7ACC2 or AR-C155858 in media containing lactate (Lac) and/or glucose (Glc). c Lactate-dependent OCR in SiHa cells exposed to increasing doses of 7ACC2 or AR-C155858. d Lactate consumption by SiHa cells incubated for 24 h in a lactate-containing medium. e Lactate secretion by SiHa cells incubated for 24 h in indicated media. f Extracellular acidification rate (ECAR) of SiHa cells after treatment with 10 µM 7ACC2 or AR-C155858 for 24 h in glucose-containing medium. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by one-way (d, f) or two-way (a, b, e) analysis of variance (ANOVA) with Bonferroni multiple-comparison analysis. **p < 0.01; ***p < 0.001; NS, not significant
Fig. 2. AR-C155858 but not 7ACC2 inhibits MCT1 activity. a
14C-lactate uptake in MCT1-expressing Xenopus oocytes after treatment with increasing doses of 7ACC2 or AR-C155858 (n = 8 for each group). b Original recording of intracellular H+ concentration in MCT1-expressing Xenopus oocytes during application and after removal of 3 mM lactate in the absence and presence of 7ACC2 or AR-C155858. c
14C-lactate uptake and (d) intracellular H+ concentration in MCT4-expressing Xenopus oocytes in response to 7ACC2 or AR-C155858 treatments (n = 8 for each group). Data are represented as mean ± SEM
Fig. 3. Inhibition of lactate uptake by 7ACC2 is associated with increased glycolytic flux and reduced cell respiration. a Relative abundance of intracellular metabolites in SiHa cells after treatment with 10 µM 7ACC2 for 24 h. b Carbon atom transition map depicting oxidation of [U-13C6]glucose. c Relative abundance of glycolysis-derived pyruvate and lactate, (d) glucose consumption, (e) lactate/glucose ratio, relative abundance of indicated (f) TCA cycle intermediates and (g) serine synthesis pathway metabolites in SiHa cells treated or not with 10 µM 7ACC2 for 24 h. h Pyruvate-dependent OCR in SiHa cells after treatment with 10 µM 7ACC2. i OCR measurements using isolated SiHa cell mitochondria in response to pyruvate/malate substrates after treatment with 10 µM 7ACC2 and (j) extent of OCR increase in response to indicated substrates. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t-test (d, e, h), one-way ANOVA (a) or two-way ANOVA (c, f, g, j) with Bonferroni multiple-comparison analysis. ***p < 0.001; NS, not significant
Fig. 4. 7ACC2 specifically inhibits mitochondrial pyruvate transport. [2−14C]pyruvate uptake in isolated mitochondria from SiHa cells treated (a) with increasing doses of 7ACC2 for 2 min or (b) with 10 µM 7ACC2 for increasing periods of time. c BCECF fluorescence reflecting progressive changes in pH within isolated SiHa cell mitochondria treated with increasing doses of 7ACC2. d Effects of 10 µM 7ACC2 on OCR measurements using isolated SiHa cell mitochondria exposed to pyruvate/malate with or without methylpyruvate. e Lactate consumption and (f) secretion by SiHa cells incubated for 24 h with 10 µM UK-5099 in lactate or glucose-containing medium, respectively. g Representative 13C-MRS spectra acquired in vivo from treated and untreated SiHa tumor xenografts after hyperpolarized 13C-pyruvate injection. h Relative 13C-lactate NMR signals and (i) modifications in the 13C lactate/pyruvate ratio after treatment with 3 mg/kg 7ACC2 or AR-C155858 for 2 h (n = 3 for each group). j Tumor volume of SiHa xenografts in nude mice after 9-day treatment with 3 mg/kg 7ACC2 or AR-C155858 (n = 5 for each group). Data are represented as mean ± SEM of three independent experiments (with ≥ 6 technical replicates). Significance was determined by Student’s t-test (e, f), one-way ANOVA (h–j) or two-way ANOVA (d) with Bonferroni multiple-comparison analysis. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant
Fig. 5. 7ACC2 and AR-C155858 reduce the growth of tumor spheroids through different mechanisms. a Representative pictures and (b) time-dependent growth of FaDu spheroids upon treatment with 20 µM 7ACC2 or AR-C155858 for 9 days. Scale bars: 200 µm. c Cellular density within FaDu spheroids treated as indicated in a for 9 days. d Time-dependent accumulation of cellular debris (i.e., appearance of a brown ring surrounding spheroids) from FaDu spheroids treated with 20 µM 7ACC2. e Representative immunofluorescence pictures and quantification of 2-deoxyglucose-IRDye (2DG-IR, 3 h exposure), (f) distribution from the periphery to the core and (g) accumulation within the core of FaDu spheroids pre-exposed to 20 µM 7ACC2 or AR-C155858 for 24 h. Scale bars: 100 µm. h Representative immunofluorescent pictures and (i) corresponding quantification (at the indicated spheroid depth) of GLUT-1 staining in FaDu spheroids treated with 20 µM 7ACC2 or AR-C155858 for 5 days. Scale bars: 100 µm. j Representative immunoblotting for MPC1 in FaDu cells expressing control or MPC1-targeting shRNA sequences. k Representative immunofluorescence pictures and (l) quantification of 2DG-IR within the spheroid core (as in g) in FaDu spheroids expressing control or MPC1-targeting shRNA. Scale bars: 200 µm. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by one-way ANOVA (g, l) or two-way ANOVA (i) with Bonferroni multiple-comparison analysis. *p < 0.05; **p < 0.01; NS, not significant
Fig. 6. Inhibition of mitochondrial pyruvate transport radiosensitizes tumor cells by reducing hypoxia in vivo. a Representative immunofluorescent pictures of CA9 and pimonidazole stainings in FaDu spheroids treated with 20 µM 7ACC2 or AR-C155858 for 5 days. Scale bars: 100 µm. b Tumor pO2 measurements by EPR oximetry in SiHa xenografts treated with 3 mg/kg 7ACC2 for the indicated period of time (n = 5 for each group). c, d Tumor growth of SiHa xenografts in nude mice daily treated with 3 mg/kg 7ACC2 (for 10 and 5 days in c and d, respectively) followed (or not) by a single irradiation of (c) 16 Gy or (d) 5 fractions of 4 Gy; arrows indicate the days of irradiation (n = 7 for each group). 7ACC2 was formulated in DMSO (c) or in 5% DMA/50% HPβCD/45% sodium phosphate buffer (d). Data are represented as mean ± SEM. Significance was determined by Student’s t-test (c), or two-way ANOVA (b) with Bonferroni multiple-comparison analysis. *p < 0.05; ***p < 0.001; NS, not significant
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