Our finding that tyrosine phosphorylation activates LDH-A may, at least in part, explain the enhanced lactate production in cancer cells. This could represent a common, short-term molecular mechanism underlying the Warburg effect in both leukemias and solid tumors, in addition to the chronic changes, including the upregulation of LDH-A gene expression, believed to be regulated by transcription factors, including HIF and Myc. Thus, tyrosine phosphorylation may provide a molecular switch upregulating LDH-A activity to provide a metabolic advantage facilitating tumor growth. Interestingly, Y10 is not evolutionarily conserved (). The occurrence of Y10 in the human LDH-A amino acid sequence is unique among mammals. This suggests that the Y10 phosphorylation-dependent regulation of LDH-A is specific for human cells.
Phylogenetic analysis of LDH-A in different species
Our findings demonstrate that tyrosine phosphorylation-dependent activation of LDH-A is important for redox homeostasis in cancer cells. The increased mitochondrial respiration in Y10F cells contributes to ATP production in a manner that appears to be independent of productive OXPHOS. These cells may still predominantly rely on cytosolic glycolysis, but they depend more on the increased mitochondrial respiration to generate NAD+ to sustain the levels of glycolysis. This explains the higher oxygen consumption rate in Y10F rescue cells compared to cells with hLDH-A WT. One concern about this model is that the slow rate of NADH shuttling from the cytosol to the mitochondrial electron transport chain, probably mediated by the malate/aspartate shuttle, may limit the supply of NADH to complex I. However, we observed that, in the stable “rescue” cells expressing LDH-A Y10F mutant, the total LDH activity is ca. 70% of that in cells expressing LDH-A WT (C). Therefore, the glycolysis in these cells may not entirely rely on NAD+ produced from the mitochondria. Thus, the slow rate of cytosolic NADH shuttling may still be sufficient to generate enough NAD+ from the mitochondria to essentially compensate the decreased supply of NAD+ in Y10F cells due to attenuated LDH-A activity.
However, such a compensatory increase in mitochondrial respiration in Y10F cells is unlikely to be sufficient to fully sustain glycolysis that is metabolically advantageous to the proliferative and tumorigenic potential of these cells, particularly under hypoxia. This may, in part, be due to the relatively slow rate of NADH shuttling from the cytosol to the mitochondrial electron transport chain (2
). These findings are consistent with and would explain previous observations that targeting LDH-A by shRNA or small-molecule inhibitor attenuates cancer cell proliferation and tumor growth (5
). In addition, the finding that individuals with a complete genetic lack of LDH-A subunit production demonstrate only modest myoglobinuria after intense anaerobic exercise (14
) identifies LDH-A as a promising therapeutic target to treat tumors that heavily rely on the Warburg effect for tumor cell survival and growth.
Our findings also suggest that oncogenic tyrosine kinase signaling may promote the Warburg effect by phosphorylating multiple metabolic enzymes, including LDH-A in the present report and previously reported PKM2 (12
). Phosphorylation of Y105 inhibits PKM2 to promote a metabolic switch to aerobic glycolysis from oxidative phosphorylation in cancer cells, while phosphorylation at Y10 activates LDH-A to sustain the aerobic glycolysis by providing NAD+
. It would be a bit difficult to reconcile the tyrosine phosphorylation-dependent enhanced lactate and NAD+
production with reduced PKM2 activity in cancer cells, since enhanced lactate production requires pyruvate produced by PKM2 but results in a net loss of carbon that could have been used for anabolic reactions. However, Vander Heiden et al. recently showed that the pyruvate kinase substrate, phosphoenolpyruvate (PEP) can transfer phosphate to the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) to phosphorylate the catalytic histidine 11 on PGAM1, producing pyruvate in the absence of PKM2 activity (28
). In addition, it is possible that lactate production is fueled by glutamine rather than glucose carbons when PKM2 activity is suppressed. Glutamine can be converted to α-ketoglutarate (α-KG) by glutamate dehydrogenase. α-KG can be used by the tricarboxylic acid (TCA) cycle to produce ATP and other precursors for anabolic reactions for cell growth and proliferation, or it can exit the TCA cycle as malate to be converted into pyruvate and then lactate (3