Lactate promotes glutamine uptake and metabolism in oxidative cancer cells

Abstract

Oxygenated cancer cells have a high metabolic plasticity as they can use glucose, glutamine and lactate as main substrates to support their bioenergetic and biosynthetic activities. Metabolic optimization requires integration. While glycolysis and glutaminolysis can cooperate to support cellular proliferation, oxidative lactate metabolism opposes glycolysis in oxidative cancer cells engaged in a symbiotic relation with their hypoxic/glycolytic neighbors. However, little is known concerning the relationship between oxidative lactate metabolism and glutamine metabolism. Using SiHa and HeLa human cancer cells, this study reports that intracellular lactate signaling promotes glutamine uptake and metabolism in oxidative cancer cells. It depends on the uptake of extracellular lactate by monocarboxylate transporter 1 (MCT1). Lactate first stabilizes hypoxia-inducible factor-2α (HIF-2α), and HIF-2α then transactivates c-Myc in a pathway that mimics a response to hypoxia. Consequently, lactate-induced c-Myc activation triggers the expression of glutamine transporter ASCT2 and of glutaminase 1 (GLS1), resulting in improved glutamine uptake and catabolism. Elucidation of this metabolic dependence could be of therapeutic interest. First, inhibitors of lactate uptake targeting MCT1 are currently entering clinical trials. They have the potential to indirectly repress glutaminolysis. Second, in oxidative cancer cells, resistance to glutaminolysis inhibition could arise from compensation by oxidative lactate metabolism and increased lactate signaling.

Keywords: Cancer; Glutamine; Hypoxia-inducible factor (HIF); Tumor metabolism; c-Myc; glutaminolysis; hypoxia-inducible factor-2 (HIF-2); lactate signaling; monocarboxylate transporter 1 (MCT1).

Figure 1.

SiHa and HeLa are more oxidative than MDA-MB-231 human cancer cells. Ratio between oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in SiHa, HeLa and MDA-MB-231 cells (n = 5–8, *p < 0.05, ***p < 0.001).

Figure 2.

Lactate promotes glutamine metabolism in cancer. (A) Representative immunoblots and bar graphs represent the expression of glutamine transporter ASCT2 in tumors collected 12 d after having been established in Matrigel plugs in mice using SiHa cancer cells transfected with a control shRNA (shCTR, left panel) or a shRNA targeting monocarboxylate transporter 1 (shMCT1, right panel). Matrigel plugs contained 30 mM of lactate or an equal volume of saline (n = 5; ns, not significant, *p < 0.05). (B) Same is in A but analyzing glutaminase 1 (GLS1) protein expression (n = 5; ns, not significant, *p < 0.05). (C) Wild-type SiHa cells were treated during 6-h with sodium lactate (10 mM) and incubated during 18 min in the presence of increasing doses of L-[3,4–³H(N)]-glutamine. The graph shows intracellular ³H incorporation (n = 8; ***p < 0.005). (D) Representative immunoblot showing GDH1 and β-actin protein expression in SiHa cancer cells transfected with a siRNA targeting GLUD1/GDH1 (siGDH1). (E) Same as C but using mock-transfected SiHa cells (left, n = 8; ***p < 0.005) or SiHa cells transfected with siGDH1 (right, n = 8; ***p < 0.005).

Figure 3.

MCT1-dependent lactate uptake stabilizes and activates c-Myc in oxidative cancer cells. (A-D) Cancer cells were treated ± 10 mM sodium lactate for 6-h. A, representative immunoblots and bar graphs represent c-Myc protein expression in oxidative SiHa (left) and HeLa (right) cancer cells (n = 4–8; *p < 0.05, **p < 0.01 ). (B) c-Myc mRNA expression detected using RT-qPCR in SiHa (left) and HeLa (right) cells (n = 6–9; ns, not significant). (C) c-Myc activity in wild-type HeLa cells was quantified using a dual reporter luciferase assay (n = 4; **p < 0.01). (D) Same as in C but using HeLa cells transfected with a control siRNA (siCTR) or with a siRNA targeting SLC16A1/MCT1 (siMCT1; n = 4; ***p < 0.005). The representative immunoblot shows MCT1 and β-actin protein expression in HeLa cells transfected with siCTR or siMCT1.

Figure 4.

Lactate stabilizes HIF-2α in oxidative cancer cells. A-C, SiHa and HeLa cancer cells were treated ± 10 mM sodium lactate for 6-h. (A) Representative immunoblots and bar graphs represent HIF-2α protein expression (n = 4–8; *p < 0.05). (B) The representative immunoblot shows HIF-2α and β-actin protein expression in SiHa and HeLa cells transfected with a control siRNA (siCTR) or with a siRNA targeting EPAS1/HIF-2α (siHIF-2α). (C) EPAS1/HIF-2α mRNA expression was detected using RT-qPCR (n = 6; ns, not significant). (D) HeLa cells were treated ± 10 mM sodium lactate for 6-h in the presence or not of increasing doses of α-ketoglutarate. Immunoblots are representative of n = 3 and show HIF-2α, HIF-1α, c-Myc, ASCT2, GLS1 and β-actin expression.

Figure 5.

HIF-2α controls c-Myc activation by lactate in oxidative cancer cells. (A) HIF-2α, HIF-1α, c-Myc, ASCT2, GLS1 and β-actin expression was detected in HeLa cells that were either mock-transfected, transfected with siHIF-1α, siHIF-2α or siMyc-1 and treated ± 10 mM sodium lactate for 6-h. Immunoblots are representative of n = 3. (B) c-Myc activity was quantified using a dual reporter luciferase assay in HeLa cells transfected with siCTR or siHIF-2α and cultured for 6-h ± 10 mM sodium lactate (n = 4; ***P < 0.005). (C) Representative immunoblots show c-Myc (left) and HIF-2α (right) protein expression in the nuclear (N) and cytoplasmic (C) fractions of HeLa cells treated for 24-h ± 10 mM sodium lactate or ± hypoxia (1% O₂). Graphs show protein expression in the nuclear fraction normalized by nuclear marker lamin A/C expression (n = 3; *p < 0.05). (D) HeLa cells were treated during 24-h ± 10 mM sodium lactate or ± hypoxia (1% O₂). Representative immunoblots of n = 3 show HIF-2α and c-Myc expression in whole cell lysate, in control IgG immunoprecipitate (IP) and in the c-Myc immunoprecipitate. E, HIF-2α:c-Myc heterocomplexes were detected using a proximity ligation assay in HeLa cells after a 6-h treatment ± 10 mM sodium lactate or ± hypoxia (1% O₂). Heterocomplexes appear as red dots in the representative pictures where cell nuclei are stained in blue (DAPI) and F-actin in green (phalloidin). Omission of the primary antibody against c-Myc was used as a negative control. Bar = 50 μm.

Figure 6.

Lactate promotes HIF-2α- and c-Myc-dependent ASCT2 and GLS1 protein expression in oxidative cancer cells. (A-D) Cancer cells were treated ± 10 mM sodium lactate for 6-h. (A), representative immunoblots and bar graphs show ASCT2 protein expression in wild-type SiHa (upper panel) and HeLa (lower panel) cells (n = 4–7; *p < 0.05). (B) Same as in A but using mock-transfected SiHa and HeLa cells (left panels), cells transfected with siHIF-2α (medium left), and cells transfected with 2 different siRNAs targeting c-Myc (siMyc-1 and siMyc-2, medium right and right) (n = 3–6; ns, not significant, *p < 0.05). (C) Representative immunoblots and bar graphs show GLS1 protein expression in wild-type SiHa (upper panel) and HeLa (lower panel) cells (n = 4–7; *p < 0.05). (D) Same as in C but using mock-transfected HeLa and SiHa cells (left), cells transfected with siHIF-2α (medium left), and cells transfected with siMyc-1 (medium right) or siMyc-2 (right) (n = 3–6; ns, not significant, *p < 0.05). (E) The representative immunoblot shows c-Myc and β-actin protein expression in SiHa and HeLa cells transfected with siMyc-1 or siMyc-2.

Figure 7.

Lactate stimulates oxidative glutaminolysis. A-B, the oxygen consumption rate (OCR) of SiHa and HeLa cells was measured in a glucose-free fresh solution containing glutamine (2 mM), 6-h after cell treatment ± 10 mM sodium lactate. (A) OCR of wild-type SiHa and HeLa cells (n = 3 independent experiments; ***p < 0.005). (B) As in A but using mock-transfected SiHa and HeLa cells or the cells transfected with siMyc-1 or siMyc-2 (n = 3 independent experiments; ns, not significant; *p < 0.05, **p < 0.01). (C) Model depicting lactate-induced glutamine uptake and metabolism in oxidative cancer cells. According to the model, lactate enters into oxidative cancer cells using monocarboxylate transporter 1 (MCT1)-facilitated transport and is oxidized to pyruvate in the cytosol (the lactate dehydrogenase 1 [LDH1] reaction). Lactate-derived pyruvate mediates lactate signaling by competitively inhibiting the use of α-ketoglutarate (α-KG) by prolylhydroxylases (PHD), which results in HIF-2α protein stabilization. HIF-2α then stabilizes c-Myc protein expression (most probably by stabilizing c-Myc:Max complexes) in the cell nucleus where c-Myc promotes the transcription of target genes, among which SLC1A5/ASCT2 is the major membrane-bound glutamine transporter and glutaminase 1 (GLS1) catalyzes the conversion of L-glutamine to L-glutamate at the first step of glutaminolysis in the mitochondrion. Glutamine can be used either in the cytosol or in the mitochondrion. In the mitochondrial matrix, glutamate dehydrogenase 1 (GDH1) converts L-glutamate in α-KG to fuel oxidative glutaminolysis. Because silencing MCT1 blocks lactate-induced c-Myc activation, MCT1 inhibitors have the potential to indirectly inhibit glutamine uptake and metabolism in oxidative cancer cells.