c-Myc activates multiple metabolic networks to generate substrates for cell-cycle entry

Abstract

Cell proliferation requires the coordinated activity of cytosolic and mitochondrial metabolic pathways to provide ATP and building blocks for DNA, RNA and protein synthesis. Many metabolic pathway genes are targets of the c-myc oncogene and cell-cycle regulator. However, the contribution of c-Myc to the activation of cytosolic and mitochondrial metabolic networks during cell-cycle entry is unknown. Here, we report the metabolic fates of [U-(13)C] glucose in serum-stimulated myc(-/-) and myc(+/+) fibroblasts by (13)C isotopomer NMR analysis. We demonstrate that endogenous c-myc increased (13)C labeling of ribose sugars, purines and amino acids, indicating partitioning of glucose carbons into C1/folate and pentose phosphate pathways, and increased tricarboxylic acid cycle turnover at the expense of anaplerotic flux. Myc expression also increased global O-linked N-acetylglucosamine protein modification, and inhibition of hexosamine biosynthesis selectively reduced growth of Myc-expressing cells, suggesting its importance in Myc-induced proliferation. These data reveal a central organizing function for the Myc oncogene in the metabolism of cycling cells. The pervasive deregulation of this oncogene in human cancers may be explained by its function in directing metabolic networks required for cell proliferation.

Figure 1. ¹³C NMR analysis of ¹³C glucose metabolism in myc^−/− and myc^+/+ cells during cell cycle entry

(A) ¹H decoupled ¹³C spectra. Key. (1) C3 alanine, (2) C3 lactate, (3) C3 glutamate, (4) C4 glutamate, (5) C2 glycine, (6) C5 proline, (7) C2 glutamate, (8) C2 lactate, (9) ribose and sugar moieties, (10) C4‘ NXP, (11) C2 Adenine, (12) C1 glutamate, (13) C5 glutamate, (14) C1 lactate. TMPSA is internal standard. NXP refers to the mono- di- or triphosphate form of any nucleotide. (B) tcaCALC-derived relative fluxes. (C) Percentage contribution of ¹³C labeled glutamate to total glutamate by LC/MS/MS mass isotopomer analysis.

Figure 2. ¹³C labeling of selected metabolites in myc^−/− and myc^+/+ cells

¹³C enrichment at individual carbons (left). Data is calculated from integrated peak areas relative to internal standard. Mean of 3 experiments. Significant differences (*=p<0.05, **=p<0.001) assessed by Student’s t test. Corresponding NMR spectra (right) for (A) lactate, (B) alanine, (C) glycine, (D) NXP ribose C4’ and adenine C2.

Figure 3. Metabolic pathways required for cell cycle progression in myc^+/+ cells

(A) Proton spectra for phosphocholine (3.2 ppm). (B) Immunoblots showing O-GlcNAcylated proteins and OGT expression with serum stimulation. Cyclin E and tubulin are positive and loading controls. Densitometry values indicated below each lane (ImageQuant). (C) Dose-response assay of cell growth by HO33342 staining for methotrexate and DON. TGR represents myc+/+ cells. Relative growth rates of untreated cell lines: myc−/− 0.69, mycER 0.83. Representative of 3 experiments in triplicate.

Figure 4

Myc regulation of glucose metabolism may provide key intermediates, energy and reducing power for cell proliferation. 13C-labeled metabolites from this study are boxed. Dashed arrows link mitochondrial metabolites with reversible (double-ended arrows) or irreversible (single arrows) reactions. Both glycolysis and the mitochondrial TCA cycle generate metabolic intermediates, in addition to ATP, providing building blocks for protein, lipid and nucleic acid synthesis. Post-translational protein modification may be subject to substrate-level control, including extra-mitochondrial acetyl-CoA, derived from intra-mitochondrial pyruvate and fatty acid metabolism, and glucosamine-6-phosphate, derived from fructose-6-phosphate and glutamine.