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. 2018 Dec 11;25(11):3047-3058.e4.
doi: 10.1016/j.celrep.2018.11.043.

Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells

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Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells

Don Benjamin et al. Cell Rep. .

Abstract

Highly glycolytic cancer cells prevent intracellular acidification by excreting the glycolytic end-products lactate and H+ via the monocarboxylate transporters 1 (MCT1) and 4 (MCT4). We report that syrosingopine, an anti-hypertensive drug, is a dual MCT1 and MCT4 inhibitor (with 60-fold higher potency on MCT4) that prevents lactate and H+ efflux. Syrosingopine elicits synthetic lethality with metformin, an inhibitor of mitochondrial NADH dehydrogenase. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mitochondrial NADH dehydrogenase or lactate dehydrogenase. Syrosingopine treatment leads to high intracellular lactate levels and thereby end-product inhibition of lactate dehydrogenase. The loss of NAD+ regeneration capacity due to combined metformin and syrosingopine treatment results in glycolytic blockade, leading to ATP depletion and cell death. Accordingly, ATP levels can be partly restored by exogenously provided NAD+, the NAD precursor nicotinamide mononucleotide (NMN), or vitamin K2. Thus, pharmacological inhibition of MCT1 and MCT4 combined with metformin treatment is a potential cancer therapy.

Keywords: MCT1; MCT4; cancer; lactate; metformin; synthetic lethality; syrosingopine.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Syrosingopine Causes Intracellular Lactate (A) HeLa cells were treated for 3 hr with the indicated drugs, and extracellular or intracellular lactate levels were measured (syrosingopine, F3-syro, SyroD, reserpine, 10 μM; antimycin A [Ant], 0.5 μM; metformin, 5mM; oxamic acid [OMA], 20 mM; NaF, 5 mM). (B) Intracellular pH in drug-treated (10 μM, 3 hr) HeLa cells stained with pHrodo (n = 5). (C) Rate of extra-or intra-cellular lactate accumulation in HeLa cells treated with indicated drugs (10 μM). (D) Dose-dependent increase in intracellular lactate levels in response to syrosingopine and F3-syro. HeLa cells were treated for 3 hr. (E) Serum lactate levels in mice treated with syrosingopine. (F) Intracellular lactate levels in liver tumor nodules excised from vehicle and syrosingopine treated mice. Each experiment was performed twice in (A)–(D). Data are presented as mean ± SEM.
Figure 2
Figure 2
Intracellular Lactate Accumulation Is due to Inhibition of the Lactate Transporters MCT1 and MCT4 by Syrosingopine (A–C) Intracellular lactate accumulation in HAP1 cells deleted for MCT1 (A), MCT4 (B), and HAP1 wild-type cells (C) following treatment with ARC155858 (1 μM), syrosingopine (10 μM), or F3-syro (10 μM). (D–F) Dose-responsive increase in intracellular lactate in HAP1 cells deleted for MCT1 (D), MCT4 (E), and HAP1 wild-type cells (F) treated with ARC155858, syrosingopine, and F3-syro for 2 hrs. Each assay was performed twice and a representative experiment is shown. Data points performed in duplicate and presented as mean ± SEM.
Figure 3
Figure 3
Syrosingopine Inhibits Lactate Efflux by MCT1 and MCT4 Lactate export assays showing amount of radiolabeled lactate (cpm) retained in the cell pellet versus label released into the medium over a time course for HAP1 MCT4-KO cells pre-treated with indicated drugs (syrosingopine, 10 μM; F3-syro, 1 μM; and ARC155858, 10 μM) (A). Similar assays were performed for HAP1 MCT1-KO cells (B) and HL60 cells (C). Each assay was performed at least twice and a representative experiment is shown.
Figure 4
Figure 4
Syrosingopine Interacts Directly with MCT1 and MCT4 (A) Schematic representation of human MCT1 and MCT4 proteins. Epitopes detected by the antibody in red. (B) DARTS assay from HCT116 cell extracts. Cells were incubated with the indicated compounds (syrosingopine, 10 μM; F3-syro, 10 μM; SyroD, 10 μM; and ARC155858, 10 μM) and lysates subjected to limiting thermolysin digestion. Resulting fragments were probed with antibodies against MCT1, MCT4, CD147, and β1-integrin as control (asterisk indicates protected fragment). (C) Co-immunoprecipitation from HL60 cells treated with DMSO or syrosingopine (5 μM, 16 hr). The CD147 chaperone was immunoprecipitated and co-IP of MCT1 and MCT4 was determined by immunoblotting (WCL, whole cell lysate). (D) Levels of CD147, MCT1, and MCT4 proteins in HL60 cells treated for 24 hr with syrosingopine (5 μM).
Figure 5
Figure 5
Dual MCT1 and MCT4 Inhibition Is Necessary to Kill Cancer Cells (A and B) Survival curve of HAP1 MCT1-KO cells with increasing concentrations of syrosingopine (S; in A) and the MCT1 inhibitor ARC155858 (AR; in B). Both drugs were also titrated in the presence of a sub-lethal concentration of metformin (M, 4 mM) to elicit possible synthetic lethality. (C and D) Survival curve for HAP1 MCT4-KO cells treated with syrosingopine (C) and ARC155858 (D). (E–H) Human cancer cell lines comprising all combinations of MCT1 and MCT4 expression. HL60 (E), K562 (F), SkBr3 (G), and MDA-MD-453 (H) were titrated with increasing concentrations of ARC155858 in the presence of sub-lethal concentrations of metformin (4 mM) or syrosingopine (0.5 μM). Cell proliferation and survival was measured after 3 days. All data points are in triplicate and presented as mean ± SEM.
Figure 6
Figure 6
Syrosingopine-Metformin Lethality Can Be Rescued with Exogenous NAD+ (A) Schematic representation of the main branches of the glycolytic pathway. (B) Total NAD (NADt) and NADH levels in HL60 after metformin (4 mM) or syrosingopine (5 μM) treatment for 8 hr. (C) NAD+/NADH ratio of HL60 cells in (B). (D) ATP levels measured in HL60 cells after 30 hr of treatment with metformin (4 mM) or syrosingopine (5 μM) and parallel treatment in the presence of NMN or NAD+. (E) Micrographs of HL60 cells treated as in (D). Scale bar represents 100 μm. (F) ATP levels in HL60 cells treated with metformin (4 mM) or syrosingopine (5 μM) for 8 hr and with the addition of vitamin K2. All experiments were performed twice. Data points were performed in duplicate and presented as mean ± SEM. RLU, relative light units.

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