Reductive carboxylation supports growth in tumour cells with defective mitochondria

Abstract

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.

Conflict of interest statement

None of the authors have competing financial interests to declare. Reprints and permissions information is available at www.nature.com/reprints.

Figures

Figure 1. A reductive pathway of glutamine metabolism in cancer cells lacking activity of ETC complex III

a, Glucose utilization, lactate secretion and glutamine utilization in 143Bwt and 143Bcytb cells. b, Mass isotopomer analysis of citrate in cells cultured with D[U-13C]glucose and unlabeled glutamine. c,d, Mass isotopomer analysis of citrate and fumarate in cells cultured with L[U-13C]glutamine and unlabeled glucose. Data are the average ± S.D. for three independent cultures. * p<0.05; ** p<0.005, Student’s t-test. e, Schematic of glutamine metabolism in 143Bwt and 143Bcytb cells. Colored arrows follow the paths of glutamine-derived carbon. Abbreviations: Ac-CoA, acetyl-CoA; OAA, oxaloacetate; Gln, glutamine; Glu, glutamate; αKG, α-ketoglutarate; Succ-CoA, succinyl-CoA; Fum, fumarate; Mal, malate; PDH, pyruvate dehydrogenase; ACL, ATP-citrate lyase; IDH, isocitrate dehydrogenase; αKGDH, α-ketoglutarate dehydrogenase.

Figure 2. NADP+/NADPH-dependent isoforms of isocitrate dehydrogenase contribute to reductive carboxylation

a, Transient silencing of isocitrate dehydrogenase (IDH) proteins in 143Bcytb cells with short interfering RNAs (siRNAs) directed against IDH1, IDH2 or IDH3. An siRNA directed against luciferase (Luc) was used as a negative control. When IDH1 and IDH2 siRNAs were transfected concurrently (IDH1+IDH2), a transfection with the same nanomolar amount of Luc siRNA (2xLuc) was used as a negative control. b, Mass isotopomer analysis of citrate and glutamate in 143Bcytb cells cultured with L[U-13C]glutamine after silencing of IDH isoforms. Data are the average ± S.D. of three independent cultures. *p<0.05, Student’s t-test.

Figure 3. Glutamine is the major lipogenic precursor in cells lacking oxidative CAC function

a, Cells were cultured in medium containing 3H2O (21% volume), or 14C tracers of acetate (ac, 5 μCi), glucose (glc, 10 μCi) or glutamine (gln, 10 μCi). Lipids were analyzed for 3H or 14C content by scintillation counting. In the 14C experiments, raw counts per thousand cells were normalized to the absolute rate of fatty acid synthesis established using 3H2O. *p<0.05, Student’s t-test. b, Schematic outlining synthesis of fatty acids (FA) from acetyl-CoA (Ac-CoA) and the source of the triplet and doublet at ω-1 in 13C NMR spectroscopy. c,d,13C NMR of lipids labeled with D[U-13C]glucose (c) or L[U-13C]glutamine (d). Insets are expansions of the ω-1 resonance. Abbreviations: s, singlet; d, doublet; t, triplet.

Figure 4. Glutamine-dependent reductive carboxylation in renal carcinoma cells lacking fumarate hydratase and during ETC inhibition in tumor cells with normal mitochondria

Growth of UOK262 renal carcinoma cells in complete medium, in medium in which glucose was replaced with galactose (a), and in medium lacking glutamine (b). c, Mass isotopomer analysis of citrate, fumarate and malate from UOK262 cells cultured in medium containing D[U-13C]glucose and unlabeled glutamine, or L[U-13C]glutamine and unlabeled glucose. d, Mass isotopomer analysis for citrate, fumarate and malate from 143Bwt cells cultured in medium containing unlabeled glucose and L[U-13C]glutamine. The labeled glutamine was introduced at the same time the cells were treated with vehicle (DMSO), metformin, rotenone or antimycin. Metabolites were extracted 6 hours later. In all experiments, data are the average ± S.D. of three independent cultures.