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. 2008 Aug 15;283(33):22700-8.
doi: 10.1074/jbc.M801765200. Epub 2008 Jun 9.

Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells

Thomas McFate  1 , Ahmed Mohyeldin, Huasheng Lu, Jay Thakar, Jeremy Henriques, Nader D Halim, Hong Wu, Michael J Schell, Tsz Mon Tsang, Orla Teahan, Shaoyu Zhou, Joseph A Califano, Nam Ho Jeoung, Robert A Harris, Ajay Verma
Affiliations

Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells

Thomas McFate et al. J Biol Chem. .

Abstract

High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies.

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Figures

FIGURE 1.
FIGURE 1.
Growth, metabolic phenotype, and PDHα phosphorylation in HNSCC cell lines. A, 22A, 22B, and O22 cells demonstrate similar anchorage-dependent growth, but only the 22B cells (B) develop significant anchorage-independent colonies (n = 3). C, 22B cells demonstrate higher lactate production than O22 and 22A cells (n = 3). D, Western blot analysis of nuclear extracts shows 22B cells have greater normoxic (N) and hypoxic (H) HIF-1α protein expression than O22 and 22A cells. E, Western blot analysis of whole cell lysate for phosphorylated PDHα (pPDHα), PDHα, and PDK-1–3 protein expression shows 22B cells to have the highest pPDHα and PDK-1 expression. F, Western blot analysis of pPDHα and PDHα protein expression in whole cell lysates from 22A and 22B cells treated with 0, 5, 10, and 20 mm DCA for 24 h shows dose-dependent decrease of PDHα phosphorylation in 22A but not 22B cells. G, densitometric ratio of immunoreactive pPDHα/PDHα from F. H and I, DCA lowers lactate production in 22A but not 22B cells. J, DCA shows dose-dependent toxicity in 22B cells but not 22A cells.
FIGURE 2.
FIGURE 2.
Dependence of Warburg metabolic phenotype on PDK expression. A, Western blot of 22B cells stably transfected with shPDK-1 show markedly reduced PDK-1 and pPDHα expression when compared with shCt cells, whereas PDK-2 and -3 expression changes little. B, ratio of active to total PDC activity is higher in shPDK-1 compared with shCt cells (n = 4, p < 0.001). Total activity was determined as activity following PDP-1 treatment of cell extracts. C, 14CO2 captured from uniformly labeled glucose is higher in shPDK-1 compared with shCt cells (n = 4, p < 0.01). D, extracellular lactate release is decreased in shPDK-1 cells compared with shCt (n = 3, p < 0.001). E, 1H NMR spectroscopic analysis of extracts from shCT cells (blue trace, n = 6) and shPDK-1 cells (red trace, n = 2) shows most prominent change in the lactate peaks.
FIGURE 3.
FIGURE 3.
HIF-1α expression is reduced in stably transfected PDK shRNA 22B cell lines. A, Western blot of nuclear extracts shows loss of normoxic (N) and decreased hypoxic (H) HIF-1α expression in shPDK-1 and shPDK-2 cells compared with shCt. B, basal HIF-1 Western blot of normoxic HIF-1α expression following treatment with the 26 S proteosome inhibitor MG132 (15 μm) shows equivalent HIF-1α expression between the cell lines. C, ascorbate treatment (100 μm for 24 h) decreases normoxic HIF-1α expression independently of PDH phosphorylation demonstrated by Western blot of nuclear (HIF-1α and β-actin) and cytoplasmic (pPDHα and PDHα) cell extracts. Cells in A–C were cultured for 24 in complete medium. D, Western blot of nuclear extracts from cells cultured in Krebs buffer shows higher glucose-dependent HIF-1α expression in shCt cells as compared with shPDK-1 and shPDK-2 cells. E, dose-response curves for pyruvate induction of HIF-1 in glucose-free Krebs buffer show higher pyruvate sensitivity in shCt cells than shPDK-1 or shPDK-2 cells. Incubation time for experiments in D and E was 4 h.
FIGURE 4.
FIGURE 4.
In vitro and in vivo reversal of malignant phenotype by PDK inhibition in 22B cells. A, shPDK-1 and shCt cells demonstrate similar anchorage-dependent growth rates. B, hypoxia treatment increases shPDK-1 cell death 2-fold greater than 22B shCt (n = 3, p < 0.01). C, shPDK-1 cells demonstrate decreased capacity to form colonies in soft agar compared with shCt (n = 3, p < 0.001). D, VEGF production, assayed by enzyme-linked immunosorbent assay, is reduced in shPDK-1 cells compared with shCt cells (n = 6, p < 0.001). E, invasiveness through Matrigel™-coated Boyden chambers is decreased in shPDK-1 cells compared with 22B shCt cells (n = 6, p < 0.001). F, left panel, xenograft tumor growth in nude mice is reduced for shPDK-1 cells compared with shCt cells (n = 9, p < 0.05). F, right panel, three representative images of shCt and shPDK-1 tumors are shown. G, Western blot analysis of cytoplasmic (pPDHα and PDHα) and nuclear (HIF-1α and β-actin) cell extracts from three separate xenograft tumors from each group shows that the decreased pPDHα and HIF-1α expression in shPDK-1 compared with shCt cells observed in vitro is maintained in vivo.
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