Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

Abstract

Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how "glutamine-addicted" cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis.

Fig. 1.

Glutaminase is required for maximal growth of glioblastoma cells in culture and in vivo. (A) Effect of GLS knockdown on nutrient utilization and metabolite secretion in LN229 cells. Control (Ctrl) or GLS-targeting shRNAmirs (Left) and shRNAs (Right) were tested. Positive values reflect utilization and negative values reflect secretion. Data were averaged over 7 h. (B) GLS flux (production of ¹⁵NH₄⁺ from L-[γ-¹⁵N]-glutamine) was determined for each cell line over 7 h. Data are the average ± SD of three independent cultures. (C) Cell growth in 2D culture. Each time point is the average ± SD of three parallel cultures. (D) Colony formation in soft agar. Data are the average ± SD of three independent wells. (E) Growth of s.c. xenografts. n = 9 tumors of each cell line for shRNAmirs (Left), and 4 tumors of each cell line for shRNAs (Right). Average volume ± SE is shown for each cell line. *P < 0.05, **P < 0.005. Gluc, glucose; Lac, lactate; Gln, glutamine; Glu, glutamate.

Fig. 2.

Glutaminase silencing increases glucose-dependent anaplerosis through pyruvate carboxylase (PC). (A) Gas chromatography/mass spectrometry was used to measure enrichment of citrate and malate in cells cultured with L-[3-¹³C]-glutamine (Left) and D-[1,6-¹³C]-glucose (Right). Results are from a representative experiment with duplicate 7-h cultures. (B) Predicted effect of increasing PC flux on the fractional contribution of the quartet (Q), 2,3 doublet (D23), and 1,2 doublet (D12) to overall labeling in glutamate C2 (GLU2), during culture with D-[U-¹³C]-glucose. The data were generated using the modeling software tcaSIM assuming an active TCA cycle, 95% enrichment of pyruvate, and active anaplerosis. The simulation was performed for 35 turns of the TCA cycle. (C) Western blots of LN229 cells expressing a control shRNAmir, an shRNAmir directed against GLS, or shRNAmirs against both GLS and PC. (D) NMR spectroscopy of metabolites extracted from the cell lines derived in C. Cells were cultured with 10 mM D-[U-¹³C]-glucose and 4 mM unlabeled glutamine for 8 h. Insets highlight the GLU2 multiplet. Splitting of individual peaks is the result of long-range coupling. (E) Labeling of citrate from [1-¹³C]-pyruvate in the same three cell lines. The percentage of citrate m+1 relative to an unlabeled standard (where m+1 = 0%) is plotted on the y axis. Each culture contained 5 mM Na-[1-¹³C]-pyruvate, 4 mM glutamine, and no glucose. Data are the average ± SD of three 7-h cultures. Glu, glutamate; Ala, alanine; Gly, glycine; Lac, lactate; S, singlet.

Fig. 3.

PC activity is induced during adaptation to low-glutamine conditions. (A and B) Viability and growth of parental SF-xL glioblastoma cells and cells adapted to low glutamine (aSF-xL), under conditions of glutamine abundance or deprivation. Data are the average ± SD of three independent cultures of 2 × 10⁵ cells grown in the presence or absence of glutamine for 72 h. (C) PC protein expression in parental SF-xL cells and aSF-xL cells. The parental cells were withdrawn from glutamine for 7 h, and the adapted cells were given glutamine-replete medium for 7 h. (D) Abundance of citrate m+2 in parental and adapted cells, in the presence and absence of glutamine and 4 mM dimethyl α-KG for 7 h. (E) Increased transfer of ¹³C from [1-¹³C]-pyruvate to citrate in adapted cells. Each culture contained 5 mM Na-[1-¹³C]-pyruvate and no glucose. The ratio shows the fold change between glutamine-deprived (0 mM) and glutamine-replete (4 mM) conditions during a 7-h experiment. The final citrate m+1 fraction in glutamine-deprived parental and aSF-xL cells were 11% and 16.5%, respectively. *P < 0.05, **P < 0.005.

Fig. 4.

PC is required for cells to escape glutamine addiction. (A) Suppression of PC protein expression in SF-xL cells adapted to growth in low glutamine, then infected with lentiviruses expressing a control shRNA (GFP) or shRNAs directed against PC (PC1, PC2). (B) Cell growth in the presence and absence of extracellular glutamine. Cells were plated at a density of 1 × 10⁵ /well (dashed line) and cultured for 72 h. Data are the average ± SD of three independent cultures. *P < 0.05.

Fig. 5.

PC activity predicts cellular independence from glutamine and glutaminase. (A, Left) Labeling of intracellular metabolites from D-[3,4-¹³C]-glucose in HepG2 and Huh-7 cells grown in complete medium, including 4 mM glutamine, for 7 h. (Right) Labeling of citrate from [1-¹³C]-pyruvate in HepG2 and Huh-7 cells grown in the presence and absence of glutamine for 7 h. (B) Viability and growth of HepG2 and Huh-7 cells in the presence and absence of glutamine for 72 h. (C) Protein expression in HepG2- and Huh-7-derived cell lines expressing shRNAs directed against GFP (control), GLS or PC (Top), and growth of each cell line in medium containing both glucose and glutamine. Data are the average ± SD of three independent cultures grown for 72 h. *P < 0.05, **P < 0.005.