Lactate does not activate NF-κB in oxidative tumor cells

Abstract

The lactate anion is currently emerging as an oncometabolite. Lactate, produced and exported by glycolytic and glutaminolytic cells in tumors, can be recycled as an oxidative fuel by oxidative tumors cells. Independently of hypoxia, it can also activate transcription factor hypoxia-inducible factor-1 (HIF-1) in tumor and endothelial cells, promoting angiogenesis. These protumoral activities of lactate depend on lactate uptake, a process primarily facilitated by the inward, passive lactate-proton symporter monocarboxylate transporter 1 (MCT1); the conversion of lactate and NAD(+) to pyruvate, NADH and H(+) by lactate dehydrogenase-1 (LDH-1); and a competition between pyruvate and α-ketoglutarate that inhibits prolylhydroxylases (PHDs). Endothelial cells do not primarily use lactate as an oxidative fuel but, rather, as a signaling agent. In addition to HIF-1, lactate can indeed activate transcription factor nuclear factor-κB (NF-κB) in these cells, through a mechanism not only depending on PHD inhibition but also on NADH alimenting NAD(P)H oxidases to generate reactive oxygen species (ROS). While NF-κB activity in endothelial cells promotes angiogenesis, NF-κB activation in tumor cells is known to stimulate tumor progression by conferring resistance to apoptosis, stemness, pro-angiogenic and metastatic capabilities. In this study, we therefore tested whether exogenous lactate could activate NF-κB in oxidative tumor cells equipped for lactate signaling. We report that, precisely because they are oxidative, HeLa and SiHa human tumor cells do not activate NF-κB in response to lactate. Indeed, while lactate-derived pyruvate is well-known to inhibit PHDs in these cells, we found that NADH aliments oxidative phosphorylation (OXPHOS) in mitochondria rather than NAD(P)H oxidases in the cytosol. These data were confirmed using oxidative human Cal27 and MCF7 tumor cells. This new information positions the malate-aspartate shuttle as a key player in the oxidative metabolism of lactate: similar to glycolysis that aliments OXPHOS with pyruvate produced by pyruvate kinase and NADH produced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), oxidative lactate metabolism aliments OXPHOS in oxidative tumor cells with pyruvate and NADH produced by LDH1.

Keywords: NAD(P)H oxidases (Nox); NADH; cancer metabolism; lactate signaling; malate-aspartate shuttle; mitochondria; nuclear factor-κB; oxidative phosphorylation (OXPHOS).

Figure 1

Exogenous lactate does not activate NF-κB in oxidative HeLa and SiHa tumor cells. (A) The basal, maximal and OXPHOS-dependent (i.e., rotenone and olygomycin-sensitive) oxygen consumption rates of SiHa, HeLa human tumor cells and human umbilical vein endothelial cells (HUVEC) were determined using Seahorse bioanalysis (n≥6). (B) Representative western blots showing Nox1, Nox2, Nox4 and β-actin expression in HeLa and SiHa cells. (C) HeLa cells were treated with vehicle (control, C), 10 mM of sodium L-lactate (lactate, L) or 20 ng/ml of TNFα (positive control) for the indicated periods of time. The phosphorylation of p65 on serine 536 (p-Ser536-p65) and total p65 expression were detected using western blotting in the lysates of the cells. Treatment with TNFα (20 ng/ml) served as positive control. A representative blot is shown, and the graph depicts p-Ser536-p65/total p65 used as a marker of NF-κB activity (N = 3; n = 9) (Viatour et al., 2005). (D) As in (C) but using SiHa cells (N = 9; n = 9). (E) HeLa and SiHa cells were treated with vehicle (control), increasing doses of lactate or TNFα. NF-κB activity was determined using a dual luciferase reporter assay (n = 4 all). All data represent means ± SEM. ^**P < 0.01, ^***P < 0.005 vs. SiHa (A) or control (C–E); ^#P < 0.05, ^###P < 0.005 vs. HeLa (A).

Figure 2

Hydrogen peroxide activates NF-κB in oxidative tumor cells. (A) Inhibitor of NF-κB α (IκBα) was detected in the lysates of HeLa cells treated during 30 min with vehicle (control), lactate (10 mM) or TNFα (20 ng/ml). A representative western blot is shown and the graph depicts IκBα expression (N = 3; n = 9). (B) The phosphorylation of p65 on serine 536 (p-Ser536-p65) and total p65 expression were detected using western blotting in the cytosolic (left panel, N = 3; n = 9) and lamin A/C-positive nuclear (right panel, N = 3; n = 9); lysates of HeLa cells treated as in (A). Representative blot is shown, and the graph depicts p-Ser536-p65/total p65 ratios. (C) HeLa (left panel, N = 3; n = 9) and SiHa (right panel, N = 3; n = 9) cells were treated for 20 min with vehicle (control) or 10 mM of N-acetyl-L-cysteine (NAC), after which p-Ser536-p65 and total p65 were detected in cell lysates. Representative blots are shown, and graphs depict p-Ser536-p65/total p65 ratios. (D) HeLa (left panel, N = 3; n = 9) and SiHa (right panel, N = 3; n = 9) cells were treated during increasing amounts of time with vehicle (control, C), H₂O₂ (0.8 mM) or H₂O₂ (0.8 mM) and lactate (10 mM). p-Ser536-p65 and total p65 were detected in cell lysates. Representative western blots are shown, and graphs depict p-Ser536-p65/total p65 ratios. All data represent means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.005, ns P > 0.05 vs. control.

Figure 3

OXPHOS inhibition by rotenone activates NF-κB in oxidative HeLa tumor cells. (A) HeLa (left panel, N = 3; n = 9) and SiHa (right panel, N = 3; n = 9) cells were treated for 30 min with 10 mM of lactate, and NAD⁺/NADH ratios were determined. (B–D) HeLa (left panels) and SiHa (right panels) cells were treated for 30 min with vehicle (control, C), 10 mM of lactate (L), 2.5 μM of rotenone (Rot.), 10 mM of N-acetyl-L-cysteine (NAC), 20 ng/ml of TNFα or combinations thereof. (B) NAD⁺/NADH ratios were determined (N = 3; n = 9 all). (C) p-Ser536-p65 and total p65 expression were detected using western blotting. Representative blots are shown, and graphs depict p-Ser536-p65/total p65 ratios (N = 3; n = 9 all). (D) NF-κB activity was determined using a dual luciferase reporter assay (N = 3; n = 12 all). (E) HeLa (left panel, N = 3; n = 12) and SiHa (right panel N = 3; n = 12) cells were treated for the indicated time periods with vehicle (control) or rotenone (2.5 μM), after which intracellular ROS levels were measured using the fluorescent probe DCFH-DA. All data represent means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.005, ns P > 0.05 vs. control.

Figure 4

Inhibition of the malate-aspartate shuttle restores the ability of lactate to activate NF-κB in oxidative HeLa tumor cells. (A,B) NF-κB activity was determined using a dual luciferase reporter assay. HeLa (A, N = 3; n = 12), SiHa (B, N = 3; n = 12) cells were treated for the indicated amounts of time with vehicle (control), 10 mM of lactate, 1 mM of diethyl pyrocarbonate (DEPC, a pharmacological inhibitor of the mitochondrial glutamate-aspartate antiporter) (Samartsev et al., 1997), 10 mM of N-acetyl-L-cysteine (NAC) or combinations thereof. All data represent means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.005, ns P > 0.05 vs. control or as indicated.

Figure 5

Inhibition of the malate-aspartate shuttle restores the ability of lactate to activate NF-κB in oxidative Cal27 tumor cells. (A) The basal, maximal and OXPHOS-dependent oxygen consumption rates of HeLa and Cal27 human tumor cells were determined using Seahorse bioanalysis (n≥6). (B) Representative western blots showing Nox1, Nox2, Nox4 expression in Cal27 cells. (C) Cal27 cells were treated for the indicated amounts of time with vehicle (control, C), 1 mM of diethyl pyrocarbonate (DEPC, D), 10 mM Lactate (L), the combination of DEPC and lactate (DL), or TNFα (20 ng/ml), after which p-Ser536-p65 and total p65 were detected in cell lysates. Representative western blots are shown. (D) Cal27 cells were treated as in (C) except that NF-κB activity was determined using a dual luciferase reporter assay 1, 2, and 3 h after treatment (n = 4). (E) The basal, maximal and OXPHOS-dependent oxygen consumption rate of SiHa and MCF7 human tumor cells were determined using Seahorse bioanalysis (n≥6). (F) Representative western blots showing Nox1, Nox2, Nox4 expression in MCF7 cells. (G) MCF7 cells were treated for 3 h with vehicle (control, C), 1 mM of DEPC (D), 10 mM of L-Lactate (L) or combination (DL), after which p-Ser536-p65, total p65 and β-actin were detected in cell lysates. A 1-h treatment with TNFα was used as positive control. Representative western blots are shown. (H) Same as in (G) except that NF-κB activity was determined using a dual luciferase reporter assay (n = 4). All data represent means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.005, ns P > 0.05 vs. control or as indicated.

Figure 6

Model showing that the oxidative use of NADH opposes NF-κB activation by lactate in oxidative tumor cells. In tumors, endothelial cells (top) and oxidative tumor cells (bottom) take up lactate, a process facilitated by the passive lactate-proton symporter monocarboxylate transporter 1 (MCT1). Both cell types convert lactate + NAD⁺ to pyruvate + NADH + H⁺ intracellularly (the lactate dehydrogenase B [LDHB] reaction). Pyruvate competes with α-ketoglutarate (αKG) to inhibit prolylhydroxylases (PHDs), resulting e.g., in the stabilization of hypoxia-inducible factor-1 subunit α (HIF-1α) (Vegran et al., ; De Saedeleer et al., ; Sonveaux et al., 2012). Endothelial cells do not use lactate-derived pyruvate and NADH as oxidative fuels (Sonveaux et al., 2012), thus further rendering NADH available to fuel NAD(P)H oxidases (Noxs) (Vegran et al., 2011). Together with pyruvate-mediated PHD inhibition, the production of reactive oxygen species (ROS) by Nox accounts for lactate-induced activation of nuclear factor-κB (NF-κB) in these cells. Comparatively, oxidative tumor cells use lactate (Sonveaux et al., 2008) and NADH (this study) as oxidative fuels. It renders these cells insensitive to lactate-induced NF-κB activation. NADH influx in mitochondria is controlled by the malate-aspartate shuttle gated by the oxoglutarate carrier (OGC, a malate-αKG exchanger) and by the glutamate-aspartate antiporter (GAA). Consequently, inhibiting the malate-aspartate shuttle can restore the ability of lactate to activate NF-κB in oxidative tumor cells, as illustrated in this study with HeLa cells treated by GAA inhibitor diethyl pyrocarbonate (DEPC). Other abbreviations: AST, aspartate aminotransferase; ETC, electron transport chain; IκBα, inhibitor and Cal27 of NF-κB α; Ikkα/β, inhibitor of NF-κB kinase α/β; MDH, malate dehydrogenase; MPC, mitochondrial pyruvate carrier; OXPHOS, oxidative phosphorylation.