Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism

Abstract

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.

Keywords: CRISPR/Cas; LDHA; LDHB; OXPHOS; Warburg effect; cancer biology; genetic disruption; glucose metabolism; glycolysis; lactate dehydrogenase; lactic acid; metabolic plasticity; pentose phosphate pathway (PPP); tumor growth; tumor metabolism.

Figure 1.

CRISPR/Cas9-induced disruption of LDHA and LDHB protein expression and its effect on lactate secretion in LS174T and B16 cells. A–D, immunoblots of HIF-1α, LDHA, and LDHB and densitometric analysis of the protein expression of LDHA and LDHB 24 h after seeding under normoxia (Nx) and hypoxia (Hx) in LDHA-KO, LDHB-KO, LDHA^−/−LDHB^+/− (heterozygote, lanes 4 and 9), and LDHA/B-DKO cells compared with WT controls for the human LS174T (A and B) and the murine B16 cell lines (C and D), respectively. HSP60 and tubulin served as loading controls. E and F, concentrations of lactate secreted by WT and single and double LDH-KOs of LS174T (E) and B16 (F) cells grown for 24 h under Nx or 1% Hx. Data were normalized by protein content, and the mean ± S.E. is representative of four independent experiments. *, p < 0.03; **, p < 0.003; ****, p < 0.0001.

Figure 2.

LDHA and LDHB enzymatic activities in LS174T and B16 cells. A–H, enzymatic assays of LDHA (A, B, E, and F) and LDHB (C, D, G, and H) activities in WT and LDHA/B-DKO cells were performed by continuous spectrophotometric rate determination. The decrease in UV absorbance at 340 nm corresponds to NADH + H⁺ oxidation coupled to pyruvate reduction to lactate, whereas the increase in A_{340 nm} corresponds to NAD⁺ reduction coupled to lactate oxidation, as shown in the reactions. The numbers next to the absorbance versus time curves represent initial rates of reaction, corresponding to the tangent line slope at time = 1 min. The results depicted are normalized by protein content and are representative of four independent experiments.

Figure 3.

Fermentative glycolysis is significantly disrupted, whereas oxidative metabolism of glucose is activated, in LDHA/B-DKO cells. A and C, ECARs of LS174T (A) and B16 (C) WT and LDH-KOs cells under Nx, as analyzed by the Seahorse XF24 bioanalyzer. The mean ± S.E. is representative of three independent experiments performed in quadruplicate. B and D, OCRs of LS174T (B) and B16 (D) WT and LDH-KO cells measured by the Seahorse XF24 bioanalyzer. The mean ± S.E. is representative of four independent experiments performed in quadruplicate. rot, rotenone; antA, antimycin A.

Figure 4.

Maximal OXPHOS and ETS capacity are increased in LDHA/B-DKO cells. Mitochondrial respiration was determined by high-resolution respirometry. Cells were suspended at a concentration of ∼0.5 × 10⁶ cells/ml in MiR05, and a total of 2.1 ml was added to the Oxygraph chamber. A and C, maximum oxidative phosphorylation capacity was determined in permeabilized cells in the presence of malate (2 mm), glutamate (10 mm), pyruvate (5 mm), succinate (10 mm), and ADP (20 mm). B and D, maximum capacity of the ETS was measured in the decoupled state after stepwise titration of FCCP. Shown are single values and the median of at least three independent experiments. **, p < 0.004; ***, p < 0.0008; ****, p < 0.0001.

Figure 5.

Mean isotopic enrichment of metabolites in WT and LDHA/B-DKO cells. A–H, LS174T and B16 WT and LDHA/B-DKO cells were grown for 24 h with [¹³C₆]glucose (LS174T, A and C; B16, B and D) and [¹³C₅]glutamine (LS174T, E and G, B16, F and H). Intermediates of glycolysis and the TCA cycle (A, B, E, and F) were analyzed by GC/MS and amino acids by HPLC-MS/MS (C, D, G, and H). Three independent experiments were performed. *, p < 0.03; ***, p < 0.001; ****, p < 0.0001.

Figure 6.

Cell growth and cell viability of WT and LDH-KO cells under normoxia and 1% hypoxia. A–D, cell proliferation and viability of LS174T (A and B) and B16 (C and D) WT and LDH-KO cells under Nx (A and C) or Hx (B and D) for up to 6 days. The mean ± S.E. is representative of four independent experiments performed in triplicate. **, p < 0.003; ***, p < 0.0003.

Figure 7.

LDHA/B disruption sensitizes tumor cells to phenformin under both normoxia and hypoxia. A and B, clonal growth of LS174T (A) and B16 (B) WT and LDH-KO cells under Nx and Hx, untreated or treated with 100 μm phenformin. The results are representative of three independent experiments. CTR, control.

Figure 8.

GNE-140 treatment phenocopies LDHA/B double genetic disruption. A–D, clonal growth of LS174T (A) and B16 (C) WT and LDHA/B-DKO cells treated with 3 μm and 10 μm GNE-140, respectively, and the corresponding counts of the colonies (B and D). E–H, ECARs and OCRs of LS174T (E and F) and B16 (G and H) WT and LDHA/B-DKO cells and WT cells treated with 10 μm GNE-140 (blue lines). The mean ± S.E. is representative of three independent experiments performed in quadruplicate. glc, glucose; CTR, control; rot, rotenone; antA, antimycin A.

Figure 9.

Xenograft tumor growth assays with LS174T and B16 LDHA/B-DKO cells showed a delay but not abolishment of tumor growth. A and B, tumor volumes of WT and LDHA/B-DKO LS174T (A) and WT and Ldha/b-KO B16 xenografts (B). 1 × 10⁵ cells were injected in NSG immune-deficient mice as indicated under “Experimental procedures.” *, p < 0.05; **, p < 0.002; ****, p < 0.0001.