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Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited

Abstract

Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can be a proliferation bottleneck that is influenced by environmental conditions. However, a comprehensive quantitative understanding of metabolic processes that may be affected by NAD+ deficiency is currently missing. Here, we show that de novo lipid biosynthesis can impose a substantial NAD+ consumption cost in proliferating cancer cells. When electron acceptors are limited, environmental lipids become crucial for proliferation because NAD+ is required to generate precursors for fatty acid biosynthesis. We find that both oxidative and even net reductive pathways for lipogenic citrate synthesis are gated by reactions that depend on NAD+ availability. We also show that access to acetate can relieve lipid auxotrophy by bypassing the NAD+ consuming reactions. Gene expression analysis demonstrates that lipid biosynthesis strongly anti-correlates with expression of hypoxia markers across tumor types. Overall, our results define a requirement for oxidative metabolism to support biosynthetic reactions and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptor-limited conditions, such as hypoxia.

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Fig. 1: Increased lipid synthesis results in increased oxygen consumption and is predicted to increase cellular demand for NAD+.
Fig. 2: Electron acceptor availability dictates proliferation rate in the absence of exogenous lipids.
Fig. 3: Electron acceptor limitation suppresses oxidative and reductive citrate production.
Fig. 4: Lipid starvation induces dephosphorylation of PDHA.
Fig. 5: Reductive tricarboxylic acid cycle flux is gated by electron acceptor availability.
Fig. 6: Complementation of electron transport chain with NADH oxidase stimulates reductive tricarboxylic acid cycle flux.
Fig. 7: Bypassing oxidative steps in fatty acid synthesis rescues proliferation in electron acceptor-deficient cells.
Fig. 8: Correlations between mRNA expression of fatty acid synthesis or fatty acid uptake genes and markers of tumor hypoxia.

Data availability

Source data are provided with this paper.

Code availability

All code is available at https://github.com/kostyat/Lipid_synthesis/.

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Acknowledgements

We thank the members of the D.V. and M.G.V.H. laboratories for helpful discussions. The results published here are in part based upon data generated by the TCGA Research Network. This work was supported by the National Institutes of Health (NIH) grants R01CA201276 (to D.V. and M.G.V.H.) and T32GM007367 (to B.W.J.), the MD-PhD program at Columbia University (to B.W.J.) and NIH grants U54CA209997 (to P.D., K.T., B.W.J. and D.V.) and T32GM007287 (Z.L. and K.L.A.). E.C.L. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2299-17). A.M.H. was supported by an HHMI International Student Fellowship. J.C.R. is supported by the Harvard/MIT MD-PhD Program NIH award T32GM007753. L.B.S. acknowledges support from a Pathway to Independence award from the NIH (K99CA218679/R00CA218679). E.F.G. was supported by the MIT MSRP program. M.G.V.H. also acknowledges support from the Lustgarten Foundation, SU2C, the Ludwig Center at MIT, the NCI (R35CA242379 and P30CA014051), the MIT Center for Precision Cancer Medicine, the Emerald Foundation and a Faculty Scholar award from HHMI. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the NIH.

Author information

Authors and Affiliations

Authors

Contributions

B.W.J, Z.L., P.D.D., M.G.V.H. and D.V. conceived the study. Z.L., B.W.J., P.D.D., K.T., M.G.V.H. and D.V. wrote the manuscript. B.W.J., K.T. and P.D.D. developed and executed the computation analysis of global metabolic flux and NAD+ costs analysis. Z.L., A.M.H., E.F.G., K.L.A. and L.B.S. performed proliferation assays. Z.L. performed oxygen consumption assays. Z.L. and E.C.L. performed serum delipidation. Z.L. performed kinetic isotope tracing and lipid synthesis assays. Z.L. and J.C.R. performed immunoblot assays. Z.L and A.M.W. performed NAD+ measurement assays. Z.L. performed mass spectrometry and analysis for metabolites. Z.L. generated cell lines used for this study. B.W.J., K.T. and P.D.D. performed TCGA analysis of gene expression correlations. M.G.V.H. and D.V. supervised the project.

Corresponding authors

Correspondence to Matthew G. Vander Heiden or Dennis Vitkup.

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Competing interests

A.M.W. is a current employee of Revitope. M.G.V.H. is a consultant and scientific advisor for Agios Pharmaceuticals, iTeos Therapeutics, Droia Ventures, Faeth Therapeutics, Sage Therapeutics and Auron Therapeutics. All other authors declare no competing interests.

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Nature Metabolism thanks Navdeep Chandel and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editors: Alfredo Gimenez-Cassina and George Caputa, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Effects of lipid depletion and fatty acid synthesis inhibition on cell proliferation.

a, Cell culture media was prepared with delipidated serum, and then reconstituted with 1% Lipid Mixture (2 μg/ml arachidonic and 10 μg/ml each linoleic, linolenic, myristic, oleic, palmitic and stearic acid, and 0.22 mg/ml cholesterol) (+lipids) or vehicle (–lipids). Proliferation rates of HeLa cells cultured in media +lipids or –lipids without and with the FASN inhibitor GSK2194069 (0.3 µM) as indicated (n = 3 per condition from a representative experiment). b, Relative palmitate synthesis rates of HeLa cells cultured in media +lipids or –lipids without and with GSK2194069 (0.3 µM), or without and with phenformin (100 µM) as indicated (n = 3 per condition from a representative experiment). c, Oxygen consumption rate (OCR) of HeLa cells cultured in media +lipids or –lipids as indicated (n = 10 per condition from a representative experiment). d, OCR of H1299 cells cultured in media +lipids or –lipids as indicated (n = 10 per condition from a representative experiment). e, OCR of H1299 cells cultured in –lipid and acutely treated with 1% Lipid Mixture or Tween-80 and Pluronic F-68 equivalent to what is present in 1% Lipid Mixture (n = 10 per condition from a representative experiment). f, OCR of HeLa cell cultures in media +lipids or –lipids acutely treated with the SCD1 inhibitor A939572 (1 µM) and rotenone (1.5 µM) + antimycin A (1.5 µM) as indicated (n = 8 per condition from a representative experiment). All bar charts and line graphs show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more.

Source data

Extended Data Fig. 2 Electron acceptor availability dictates proliferation rate in the absence of exogenous lipids.

a, Proliferation rates of H1299, PANC-1, AL1376, A549, and 143B cells cultured in media +lipids or –lipids in normoxia (21% oxygen) or hypoxia (0.5% or 1% oxygen), without or with pyruvate (1 mM, P) and/or lactate (10 mM, L) as indicated (n = 3 per condition from a representative experiment). b, Proliferation rate of HeLa cells cultured in media +lipids or –lipids with a titration of phenformin (Complex I inhibitor), rotenone (Complex I inhibitor), or antimycin A (Complex III inhibitor) as indicated (n = 3 per condition from a representative experiment). c, Proliferation rate of H1299 cells cultured in media +lipids or –lipids with a titration of phenformin, rotenone, or antimycin A as indicated (n = 3 per condition from a representative experiment). d, Proliferation rates of HeLa cells cultured in media containing the indicated doses of Phenformin with dialyzed fetal bovine serum that has been untreated, delipidated and reconstituted with 1% Lipid Mixture (2 μg/ml arachidonic and 10 μg/ml each linoleic, linolenic, myristic, oleic, palmitic and stearic acid, and 0.22 mg/ml cholesterol), or delipidated and reconstituted with Tween-80 and Pluronic F-68 equivalent to what is present in 1% Lipid Mixture (n = 3 per condition from a representative experiment). e, Proliferation rates of HeLa cells cultured in media –lipids treated with phenformin (100 µM), when indicated, and supplemented with either 1% Lipid Mixture or the equivalent amounts of oleate (O) and/or mevalonate (M) found in 1% Lipid Mixture (n = 3 per condition from a representative experiment). f, Proliferation rates of H1299 cells cultured in media –lipids treated with phenformin (10 µM), when indicated, and supplemented with either 1% Lipid Mixture or the equivalent amounts of oleate (O) and/or mevalonate (M) found in 1% Lipid Mixture (n = 3 per condition from a representative experiment). All bar charts show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more.

Source data

Extended Data Fig. 3 Orthogonal mechanisms of electron acceptor regeneration restore lipid synthesis under ETC inhibition.

a, Proliferation rates of H1299 cells cultured in media +lipids or –lipids, without or with phenformin (10 µM), pyruvate (1 mM, P), and/or lactate (10 mM, L) as indicated (n = 3 per condition from a representative experiment). b, Proliferation rates of H1299 cells cultured in media +lipids or –lipids without or with antimycin A (15 nM), pyruvate (1 mM, P), and/or lactate (10 mM, L) as indicated (n = 3 per condition from a representative experiment). c, Relative NAD+ /NADH ratio in H1299 cells cultured in media +lipids or –lipids without or with phenformin (10 µM), pyruvate (1 mM, P), and/or lactate (10 mM, L) as indicated (n = 3 per condition from a representative experiment). d, Proliferation rates of HeLa cells cultured in medium –lipids without or with 1% Lipid Mixture, phenformin (100 µM), and/or pyruvate (1 mM, P), as indicated (n = 3 per condition from a representative experiment). e, Proliferation rates of H1299 cells cultured in medium –lipids without or with 1% Lipid Mixture, phenformin (10 µM), and/or pyruvate (1 mM, P), as indicated (n = 3 per condition from a representative experiment). f, Proliferation rates of HeLa cells cultured in medium –lipids without or with 1% Lipid Mixture, phenformin (100 µM), and/or α-ketobutyrate (1 mM, αKB), as indicated (n = 3 per condition from a representative experiment). g, Proliferation rates of HeLa cells cultured in media +lipids or –lipids without or with phenformin (100 µM), pyruvate (1 mM, P), and/or alpha-ketobutyrate (1 mM, Ak) as indicated (n = 3 per condition from a representative experiment). h, Relative proliferation rates of HeLa cells expressing empty vector (EV) or lbNOX cultured in –lipids with phenformin (100 µM) as indicated. Data were normalized to HeLa-EV cells (n = 3 per condition from a representative experiment). All bar charts show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more.

Source data

Extended Data Fig. 4 Effects of aspartate on proliferation in the absence of lipids.

a, Proliferation rates of HeLa cells cultured in media +lipids or –lipids, without or with phenformin (100 µM), and/or aspartic acid (10 mM or 20 mM) as indicated (n = 3 per condition from a representative experiment). b, Proliferation rates of HeLa cells cultured in media +lipids or –lipids, without or with phenformin (100 µM), and/or sodium aspartate (10 mM) as indicated (n = 3 per condition from a representative experiment). c, Proliferation rates of H1299 cells cultured in media +lipids or –lipids, without or with phenformin (10 µM), and/or sodium aspartate (10 mM) as indicated (n = 3 per condition from a representative experiment). All bar charts show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more.

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Extended Data Fig. 5 Effects of exogenous metabolites on ACC phosphorylation.

a, Representative immunoblot of total ACC and ACC serine 79 phosphorylation in HeLa cells cultured for 24 hours in media +lipids or –lipids without or with phenformin (100 µM), pyruvate (1 mM, Pyr), lactate (10 mM, Lac), and/or acetate (200 µM, Ac) as indicated. b, (top) Representative immunoblot of FASN, phosphorylated PDHA (Serine 293), total PDHA, and vinculin in HeLa or H1299 cells overexpressing eGFP or constitutively mature SREBP1a. (bottom) Representative immunoblot of phosphorylated PDHA (Serine 293) and Vinculin in HeLa cultured in –lipids for 24hrs, treated with vehicle, phenformin, antimycin, pyruvate, and/or alpha-ketobutyrate at the indicated doses. All experiments were repeated three times or more.

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Extended Data Fig. 6 Inhibition of mitochondrial electron transport decreases intracellular citrate levels.

a, Relative fractional distribution of citrate isotopomers in HeLa cells cultured for 24 hours in media +lipids or –lipids with U-13C-Glutamine, without and with phenformin (100 µM), pyruvate (1 mM, Pyr), and/or lactate (10 mM, Lac) as indicated (n = 3 per condition from a representative experiment). b, Normalized intracellular ratio of αKG to citrate in HeLa cells cultured in +lipids or –lipids with or without phenformin (100 µM). (n = 6 per condition from a representative experiment). c, Isotopomer distribution of total levels of intracellular citrate in HeLa cells cultured for 24 hours in media +lipids or –lipids with U-13C-Glutamine, without and with phenformin (100 µM), pyruvate (1 mM, Pyr), and/or lactate (10 mM, Lac) as indicated (n = 3 per condition from a representative experiment). All bar charts show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more. (n = 3 per condition from a representative experiment).

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Extended Data Fig. 7 Bypassing oxidative steps in fatty acid synthesis rescues proliferation in electron acceptor-deficient cells.

a, Proliferation rates of H1299 cells cultured in media +lipids or –lipids without or with phenformin (10 µM) and/or acetate (200 µM) as indicated (n = 3 per condition from a representative experiment). b, Proliferation rates of H1299 cells cultured in media +lipids or –lipids without or with antimycin A (15 nM) and/or acetate (200 µM) as indicated (n = 3 per condition from a representative experiment). c, Relative NAD+ /NADH ratio in H1299 cells cultured in media +lipids or –lipids without or with phenformin (10 µM) and/or acetate (200 µM) as indicated (n = 3 per condition from a representative experiment). d, Proliferation rates of H1299 cells cultured in media +lipids or –lipids in normoxia (21% oxygen), hypoxia (1% oxygen), and/or acetate (200 µM) as indicated. Data from the first four conditions are the same as those presented in Extended Data Fig. 2a. (n = 3 per condition from a representative experiment). All bar charts show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more.

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Extended Data Fig. 8 Effect of exogenous acetate on levels of TCA cycle intermediates.

a, Relative intracellular alpha-ketoglutarate (αKG) levels in HeLa cells cultured for 24 hours in medium –lipids without or with phenformin (100 µM) and/or acetate (200 µM) as indicated (n = 6 per condition from a representative experiment). b, Relative intracellular succinate levels in HeLa cells cultured for 24 hours in medium –lipids without or with phenformin (100 µM) and/or acetate (200 µM) as indicated (n = 6 per condition from a representative experiment). c, Relative intracellular fumarate levels in HeLa cells for 24 hours in medium –lipids without or with phenformin (100 µM) and/or acetate (200 µM) (n = 6 per condition from a representative experiment). d, Relative intracellular malate levels in HeLa cells cultured for 24 hours in medium –lipids without or with phenformin (100 µM) and/or acetate (200 µM) (n = 6 per condition from a representative experiment). All bar charts show means with error bars representing ± s.d. Unpaired Student’s t-test was performed where statistics are shown. All experiments were repeated three times or more.

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Extended Data Fig. 9 Gene expression correlations between lipid metabolism genes and hypoxia signature genes.

a, Pearson correlation coefficients and corresponding p-values, for each of 34 different tumor types, between expression of hypoxia signature genes and expression of fatty acid synthesis genes (third column), lipid uptake genes (fourth column), and beta-oxidation genes (fifth column). Insignificant correlations, based on the 1% FDR cutoff, are marked in red. Depicted p-values on Pearson correlation coefficients are calculated using two-sided Student’s t-test, and significance threshold is adjusted for multiple comparisons at 1% FDR using the Benjamini-Hochberg method. b, Scatter plots showing, for each considered tumor type, the correlation between the tumor hypoxia score and expression of genes participating in fatty acid synthesis, with dots representing individual TCGA samples. c, Scatter plots showing, for each considered tumor type, the correlation between the tumor hypoxia score and expression of genes participating in lipid uptake, with dots representing individual TCGA samples.

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Extended Data Fig. 10 Gene expression correlations between lipid metabolism genes and hypoxia signature genes.

a, Correlation of mRNA expression of SREBF1/2 and of gene markers of fatty acid synthesis within individual hypoxia score quintiles. Pearson’s correlation coefficients were calculated for each of five equally-sized bins of TCGA samples, corresponding to five hypoxia score quintiles, for SREBF1 (left) and SREBF2 (right). TCGA samples were sorted into quintiles based on their hypoxia scores from the lowest hypoxia score (quintile 1) to the highest score (quintile 5). b, Density plot of the correlation between the average mRNA expression of gene markers of tumor hypoxia and mRNA expression of Stearoyl-CoA desaturase 1 (SCD1) gene. Density counts represent the number of TCGA samples with the corresponding expression values, with red color representing high-density regions and blue color representing low-density regions, and the Pearson’s correlation coefficient (R) and the p-value are shown in the figure. Depicted p-value on Pearson correlation coefficients is calculated using two-sided Student’s t-test.

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Li, Z., Ji, B.W., Dixit, P.D. et al. Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited. Nat Metab 4, 711–723 (2022). https://doi.org/10.1038/s42255-022-00588-8

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