Tyrosine Phosphorylation of Lactate Dehydrogenase A Is Important for NADH/NAD⁺ Redox Homeostasis in Cancer Cells

Abstract

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD ؉ . LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD ؉ . However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD؉ redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.Cancer cells take up more glucose than normal tissue and favor aerobic glycolysis, generating lactate through a NADHdependent enzyme, lactate dehydrogenase A (LDH-A), which catalyzes the conversion of pyruvate to lactate during glycolysis. This is the last step of glycolysis that permits the regeneration of NAD ϩ , which is needed as an electron acceptor to maintain cytosolic glucose catabolism (2). Therefore, most tumor cells are reliant on lactate production for their survival.LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production (1,7,16,(25)(26)(27). In addition, expression of LDH-A was previously implicated to be involved in tumor initiation and growth. Targeting LDH-A by short hairpin RNA (shRNA) in several tumor cell lines is sufficient to stimulate oxidative phosphorylation in these cells, which is accompanied by an increase in the rate of oxygen consumption and a decrease in mitochondrial membrane potential (5). This provides evidence of the direct link between glycolysis and oxidative phosphorylation that involves LDH-A. Moreover, RNA interference (RNAi)-mediated reduction of LDH-A expression compromises the ability of tumor cells to proliferate under hypoxia and induce tumorigenesis (5). Recently, it was reported that targeting LDH-A by a small-molecule inhibitor, FX11, induced significant oxidative stre...

Sorbitol Dehydrogenase Overexpression and Other Aspects of Dysregulated Protein Expression in Human Precancerous Colorectal Neoplasms: A Quantitative Proteomics Study

“…Their expression levels were not measured separately, but LDHA is presumably responsible for the increased expression observed in our adenomas. LDHB expression is in most cases epigenetically silenced in colon cancer cells (44,45), whereas LDHA is overexpressed, and its activity is maintained via the oncogenic tyrosine kinase FGFR1 (46). LDHA is a key player in the reversible conversion of pyruvate to lactate during aerobic glycolysis, a typical feature of cancer cell metabolism first described by Warburg (47).…”

Tyr-94 Phosphorylation Inhibits Pyruvate Dehydrogenase Phosphatase 1 and Promotes Tumor Growth

“…Point mutations were introduced using QuikChange-XL sitedirected mutagenesis kit (Stratagene). [5-3 H]glucose and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]pyruvate were purchased from PerkinElmer, and R-[ 3 H]lipoic acid was purchased from American Radiolabeled Chemicals. Stable knockdown of endogenous PDP1 was achieved using a lentiviral vector harboring an shRNA construct (Open Biosystems; 5Ј-GTTCAGTTCAATTCTCAT-GTT-3Ј).…”

“…293T, MDA-MB231, MCF7, human foreskin fibroblast, and HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. TKI258 (generously provided by Novartis Pharma) treatment was performed by incubating cells with 1 M TKI258 for 4 h. For the cell proliferation assay, 125 nM oligomycin was added in the cell culture medium as described previously (13). Imatinib was purchased from Santa Cruz Biotechnology.…”

Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration

“…LDHA is an enzyme that converts pyruvate into lactate, and some studies suggested that phosphorylation of LDHA increases its activity and induces the Warburg effect (49,50), although this does not appear to be the case in Sirt6-deficient retina. VHL regulates protein degradation and downregulates HIF2A, which is reflected in our data by the decreased levels of VHL and the increased levels of HIF2A.…”