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Reductive carboxylation supports redox homeostasis during anchorage-independent growth

Abstract

Cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM)1. Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells2. Detachment from ECM is associated with enhanced production of reactive oxygen species (ROS) owing to altered glucose metabolism2. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumour spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.

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Figure 1: Reductive glutamine metabolism in spheroids.
Figure 2: Spheroid metabolism is distinct from the metabolic alterations induced by hypoxia.
Figure 3: Reductive carboxylation in spheroids is primarily dependent on cytosolic IDH1.
Figure 4: Reductive glutamine metabolism mitigates mitochondrial ROS and promotes spheroid growth.

References

  1. Bhowmick, N. A., Neilson, E. G. & Moses, H. L. Stromal fibroblasts in cancer initiation and progression. Nature 432, 332–337 (2004)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  2. Schafer, Z. T. et al. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 461, 109–113 (2009)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  3. Grassian, A. R., Metallo, C. M., Coloff, J. L., Stephanopoulos, G. & Brugge, J. S. Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation. Genes Dev. 25, 1716–1733 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rajagopalan, K. N. et al. Metabolic plasticity maintains proliferation in pyruvate dehydrogenase deficient cells. Cancer Metab. 3, 7 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kim, H. S. et al. Systematic identification of molecular subtype-selective vulnerabilities in non-small-cell lung cancer. Cell 155, 552–566 (2013)

    Article  CAS  PubMed  Google Scholar 

  6. Metallo, C. M. et al. Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature 481, 380–384 (2012)

    Article  CAS  ADS  Google Scholar 

  7. Friedrich, J., Seidel, C., Ebner, R. & Kunz-Schughart, L. A. Spheroid-based drug screen: considerations and practical approach. Nature Protocols 4, 309–324 (2009)

    Article  CAS  PubMed  Google Scholar 

  8. Hunnewell, M. G. & Forbes, N. S. Active and inactive metabolic pathways in tumor spheroids: determination by GC-MS. Biotechnol. Prog. 26, 789–796 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Schug, Z. T. et al. Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress. Cancer Cell 27, 57–71 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mullen, A. R. et al. Reductive carboxylation supports growth in tumour cells with defective mitochondria. Nature 481, 385–388 (2012)

    Article  CAS  ADS  Google Scholar 

  11. Garrett, R. H. & Grisham, C. M. Biochemistry 618 (Brooks Cole, 2004)

  12. Okoye-Okafor, U. C. et al. New IDH1 mutant inhibitors for treatment of acute myeloid leukemia. Nature Chem. Biol. 11, 878–886 (2015)

    Article  CAS  Google Scholar 

  13. Gnoni, G. V., Priore, P., Geelen, M. J. & Siculella, L. The mitochondrial citrate carrier: metabolic role and regulation of its activity and expression. IUBMB Life 61, 987–994 (2009)

    Article  CAS  PubMed  Google Scholar 

  14. Kaplan, R. S., Morris, H. P. & Coleman, P. S. Kinetic characteristics of citrate influx and efflux with mitochondria from Morris hepatomas 3924A and 16. Cancer Res. 42, 4399–4407 (1982)

    CAS  PubMed  Google Scholar 

  15. Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nature Methods 3, 281–286 (2006)

    Article  CAS  PubMed  Google Scholar 

  16. Lewis, C. A. et al. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol. Cell 55, 253–263 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Anastasiou, D. et al. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 334, 1278–1283 (2011)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  18. Piskounova, E. et al. Oxidative stress inhibits distant metastasis by human melanoma cells. Nature 527, 186–191 (2015)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  19. Jeon, S.-M., Chandel, N. S. & Hay, N. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature 485, 661–665 (2012)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  20. Sazanov, L. A. & Jackson, J. B. Proton-translocating transhydrogenase and NAD- and NADP-linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria. FEBS Lett. 344, 109–116 (1994)

    Article  CAS  PubMed  Google Scholar 

  21. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281–2308 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang, C. et al. Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport. Mol. Cell 56, 414–424 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cheng, T. et al. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. Proc. Natl Acad. Sci. USA 108, 8674–8679 (2011)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  24. Young, J. D. INCA: a computational platform for isotopically non-stationary metabolic flux analysis. Bioinformatics 30, 1333–1335 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Antoniewicz, M. R., Kelleher, J. K. & Stephanopoulos, G. Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions. Metab. Eng. 9, 68–86 (2007)

    Article  CAS  PubMed  Google Scholar 

  26. Young, J. D., Walther, J. L., Antoniewicz, M. R., Yoo, H. & Stephanopoulos, G. An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis. Biotechnol. Bioeng. 99, 686–699 (2008)

    Article  CAS  PubMed  Google Scholar 

  27. Waleh, N. S. et al. Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker. Cancer Res. 55, 6222–6226 (1995)

    CAS  PubMed  Google Scholar 

  28. Wu, R. F., Ma, Z., Liu, Z. & Terada, L. S. Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local Ras activation. Mol. Cell. Biol. 30, 3553–3568 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pietrak, B. et al. A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG. Biochemistry 50, 4804–4812 (2011)

    Article  CAS  PubMed  Google Scholar 

  30. Rendina, A. R. et al. Mutant IDH1 enhances the production of 2-hydroxyglutarate due to its kinetic mechanism. Biochemistry 52, 4563–4577 (2013)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Mitsche for help with analysis of lipogenic acetyl-CoA enrichment, A. Grassian for advice about IDH1, J. Garcia for hypoxia/hyperoxia experiments and C. Frezza for discussion of mitochondrial isolation. J. Kozlitina assisted with statistical analysis and R. Egnatchik provided advice about metabolic flux analysis. R.J.D. is supported by grants from the N.I.H (R01CA157996), Cancer Prevention and Research Institute of Texas (RP130272) and Robert A. Welch Foundation (I1733). C.M.M. is supported by N.I.H. grant R01CA188652.

Author information

Authors and Affiliations

Authors

Contributions

L.J. and R.J.D. designed the study. L.J., Q.A.W. and C.Y. performed molecular and cell biology experiments. P.S. and B.P.D. performed Seahorse experiments. L.S.T., S.J.P. and C.M.M. provided reagents and expertise for ROS and 2H tracing experiments. L.J. and A.A.S. performed metabolic flux analysis. N.D.A., M.T.M., B.P., S.S. and B.S. provided the IDH1 inhibitor. L.J. and R.J.D. wrote the paper.

Corresponding author

Correspondence to Ralph J. DeBerardinis.

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Competing interests

R.J.D. is on the advisory boards of Peloton Therapeutics and Agios Pharmaceuticals.

Extended data figures and tables

Extended Data Figure 1 Alternative pathways of isocitrate/citrate metabolism.

a, Predominant path of citrate formation in monolayer culture. b, Proposed pathway in anchorage-independent culture, emphasizing an alternative route of isocitrate/citrate metabolism and reducing equivalent flow.

Extended Data Figure 2 Nutrient metabolism in H460 spheroid culture.

a, Cell proliferation and doubling times of H460 cells cultured under monolayer and spheroid conditions (n = 4 cultures days 1–4; n = 3 cultures days 5–6 from a representative experiment). b, Effect of glucose or glutamine deprivation on cell counts in monolayer and spheroid culture (n = 4 cultures from a representative experiment). c, Rates of glucose consumption and lactate excretion in monolayer and spheroid culture (n = 4 cultures from two experiments). d, Rates of glutamine consumption; glutamate and ammonia excretion; ratio of ammonia excretion to glutamine consumption; and rate of excretion of 15NH4+ originating from [γ-15N]glutamine in monolayer and spheroid culture (n = 3 cultures from a representative experiment). e, Citrate mass isotopologue analysis in H460 cells in monolayer culture, aggregated into spheroids (P1), or disaggregated from spheroids then permitted to re-aggregate (P2) (n = 2 cultures from a representative experiment). f, Right, protein levels of phosphorylated PDHα (pPDH, Ser293), total PDHα (tPDH) and PDK1 in monolayer and spheroid culture with or without 2 mM dichloroacetate (DCA). Left, citrate mass isotopologue analysis in H460 spheroids cultured with [U-13C]glucose or [U-13C]glutamine, and treated with 2 mM DCA (n = 3 cultures from a representative experiment). All data represent mean ± s.d. *P < 0.05, Welch’s unequal variances t-test (a, c, d and f), or Welch’s unequal variances t-test, followed by multiple-comparison correction (b). All experiments were repeated 3 times or more.

Source data

Extended Data Figure 3 Reductive citrate metabolism in anchorage-independent spheroid culture.

a, Citrate m+5 may be generated in several ways from [U-13C]glutamine, including through reductive (left) or oxidative (middle) pathways. To test whether citrate m+5 arises from oxidative or reductive metabolism, spheroids were cultured with [1-13C]glutamine. Glutamine-C1 (green circle) is released as CO2 by α-ketoglutarate dehydrogenase in the oxidative TCA cycle, but is transferred to citrate via reductive metabolism. Citrate mass isotopologues in H460 cells cultured with [1-13C]glutamine (right). The m+1 fraction in this experiment is comparable to the m+5 fraction from [U-13C]glutamine (~20%), indicating that reductive labelling was enhanced in spheroids (n = 3 cultures from a representative experiment). b, Time-dependent evolution of succinate, fumarate and malate mass isotopologues in spheroids cultured with [U-13C]glutamine (n = 2 cultures for each time point). c, Citrate labelling from [U-13C]glutamine in immortalized, non-transformed bronchial epithelial cells (HBEC30) and lung cancer cells (HCC4017) from the same patient (n = 3 cultures from two experiments). All data represent mean ± s.d. *P < 0.05, Welch’s unequal variances t-test. All experiments were repeated 3 times or more.

Source data

Extended Data Figure 4 Effect of oxygen availability, nutrient availability and anchorage on reductive carboxylation.

a, Mass isotopologues of citrate in spheroids cultured with [U-13C]glutamine under 20% and 60% oxygen (n = 4 cultures from two experiments). b, Effects of reducing extracellular glucose and glutamine concentrations on citrate mass isotopologues in monolayer cells cultured with [U-13C]glutamine (n = 2 cultures from a representative experiment). c, Day 7 spheroids were allowed to attach to a conventional tissue culture dish for 24 h, and mass isotopologues of citrate were analysed with [U-13C]glutamine tracing (n = 4 cultures from two experiments). Insets are photomicrographs of cells in each of the culture conditions. Scale bars, 200 μm. All data represent mean ± s.d. *P < 0.05 comparing to monolayer, #P < 0.05 comparing to spheroid, Welch’s unequal variances t-test followed by multiple-comparison correction. All experiments were repeated 3 times or more.

Source data

Extended Data Figure 5 Hypoxia and anchorage independence elicit different effects on citrate metabolism.

a, Mass isotopologues of citrate and malate in H460 cells cultured with [U-13C]glutamine, in monolayer or spheroid conditions, under 21% or 1% oxygen (n = 4 cultures from two experiments). bd, Contribution of glutamine (b), glucose (c) and acetate (d) to the lipogenic acetyl-CoA pool used for palmitate synthesis, during 24 h of culture with each tracer (n = 3 cultures in panels b and c; n = 2 cultures in panel d from a representative experiment). All data represent mean ± s.d. *P < 0.05, Welch’s unequal variances t-test followed by multiple-comparison correction (a), or ANOVA (b), or Welch’s unequal variances t-test (c). The experiment in d was repeated twice, and all other experiments were repeated 3 times or more.

Source data

Extended Data Figure 6 Graphical view of metabolic flux analysis (MFA).

a, A conventional set of metabolic reactions and compartmentation produced an inadequate fit with the spheroid experimental data, with an unacceptable sum-of-squared residuals (SSR = 336). Poorly fit palmitate isotopologues from [U-13C]glucose and [U-13C]glutamine are shown on the right. v-CTP, bidirectional isocitrate/citrate trafficking flux. b, In the modified metabolic network, isocitrate/citrate produced from cytosolic reductive carboxylation enters the mitochondria and mixes with the isocitrate/citrate pool there. Adding this new reaction to the model (indicated in bold as v23) substantially improved the overall fit (SSR = 179) and the fit with palmitate isotopologues (right). Colour coding in b reflects flux changes in spheroids, expressed as the ratio of spheroid flux/monolayer flux. The dashed line indicates that the overall direction of malate transport was predicted to reverse from mitochondrial efflux in monolayer cells to mitochondrial import in spheroids. Flux terms are defined in Supplementary Table 2, and abbreviations and quantitative flux rates are in Extended Data Table 1.

Source data

Extended Data Figure 7 IDH1 inhibition suppresses reductive carboxylation in spheroids.

a, Abundance of citrate in vector control, wild-type, and IDH1, IDH2 or IDH3-deficient spheroids (n = 2 cultures from a representative experiment). b, Structure of IDH1 inhibitor compound GSK321 and structurally similar control compound GSK990. Compound GSK321 was initially identified as a potent inhibitor against an oncogenic allele of IDH1 containing the R132H mutation. Subsequent analysis revealed that at higher doses, the compound also inhibits wild-type IDH1. c, In vitro activity assay revealing effects of compound 321 on enzymatic activity of recombinant wild-type IDH1 and IDH2 (n = 4 repeats from a representative experiment). d, Time-dependent evolution of citrate mass isotopologues in 990- or 321-treated spheroids cultured with [U-13C]glutamine (n = 1 culture for each time point). e, Mass isotopologues of citrate in vector control and IDH1KO spheroids cultured with [U-13C]glutamine and treated with 5 μM IDH1 inhibitor (321) or control compound (990) (n = 4 cultures from two experiments). All data represent mean ± s.d. *P < 0.05, ANOVA. Experiments in d were repeated twice, and experiments in c and e were repeated 3 times or more.

Source data

Extended Data Figure 8 Mitochondria take up and metabolize citrate.

a, Protein expression of mitochondrial and cytosolic markers in subcellular fractions of monolayer and spheroid culture. b, Protein expression of CTP in control and CTP-deficient H460 cells. c, Mass isotopologues of malate and succinate in isolated mitochondria cultured with [U-13C]citrate (n = 4 cultures from two experiments). d, Mass isotopologues of malate and succinate in isolated mitochondria cultured with [U-13C]citrate, unlabelled pyruvate and glutamine (n = 4 cultures from two experiments). e, Mass isotopologues of fumarate, malate and succinate in isolated mitochondria cultured with [U-13C]glutamine and unlabelled pyruvate (n = 2 cultures from a representative experiment). All data represent mean ± s.d. *P < 0.05, Welch’s unequal variances t-test. Experiments in e were repeated twice, and all other experiments were repeated 3 times or more.

Source data

Extended Data Figure 9 Reductive glutamine metabolism mitigates mitochondrial ROS and promotes spheroid growth.

Mitochondrial (a) and cytosolic (b) ROS detected by a genetic hydrogen peroxide sensor in H460 spheroids containing or lacking IDH1 or IDH2. (n = 29 Vector spheroids; n = 26 IDH1-KO and IDH2-KO spheroids in panel a; n = 23 Vector spheroids; n = 22 IDH1-KO spheroids in panel b). c, Deuterium labelling of citrate in H460 spheroids without and with the pentose phosphate pathway inhibitor DHEA (n = 3 cultures from a representative experiment). d, Growth of H460 cells containing or lacking CTP in monolayer conditions (left) and as spheroids (right) (n = 6 monolayer cultures; n = 31 CTP-WT spheroids; n = 42 CTP-KO spheroids). e, Size of H460 spheroids containing or lacking IDH1 or IDH2, and treated with or without the mitochondrial ROS scavenger MitoTEMPO. (n = 40 Vector “-” spheroids; n = 52 Vector “+” spheroids; n = 46 IDH1KO “-“ and “+” spheroids; n = 48 IDH2KO “-” spheroids; n = 52 IDH2KO “+” spheroids). All data represent mean ± s.d. *P < 0.05, ANOVA (a), or Welch’s unequal variances t-test (be). All experiments were repeated 3 times or more.

Source data

Extended Data Table 1 Simulated metabolic fluxes in monolayer and spheroid culture

Supplementary information

Supplementary Information

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Supplementary Data

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Supplementary Data

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Supplementary Data

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Jiang, L., Shestov, A., Swain, P. et al. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 532, 255–258 (2016). https://doi.org/10.1038/nature17393

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