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Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours

A Publisher Correction to this article was published on 08 August 2018

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Abstract

As oxygen is essential for many metabolic pathways, tumour hypoxia may impair cancer cell proliferation1,2,3,4. However, the limiting metabolites for proliferation under hypoxia and in tumours are unknown. Here, we assessed proliferation of a collection of cancer cells following inhibition of the mitochondrial electron transport chain (ETC), a major metabolic pathway requiring molecular oxygen5. Sensitivity to ETC inhibition varied across cell lines, and subsequent metabolomic analysis uncovered aspartate availability as a major determinant of sensitivity. Cell lines least sensitive to ETC inhibition maintain aspartate levels by importing it through an aspartate/glutamate transporter, SLC1A3. Genetic or pharmacologic modulation of SLC1A3 activity markedly altered cancer cell sensitivity to ETC inhibitors. Interestingly, aspartate levels also decrease under low oxygen, and increasing aspartate import by SLC1A3 provides a competitive advantage to cancer cells at low oxygen levels and in tumour xenografts. Finally, aspartate levels in primary human tumours negatively correlate with the expression of hypoxia markers, suggesting that tumour hypoxia is sufficient to inhibit ETC and, consequently, aspartate synthesis in vivo. Therefore, aspartate may be a limiting metabolite for tumour growth, and aspartate availability could be targeted for cancer therapy.

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Fig. 1: Diversity of cancer metabolic responses to ETC inhibition.
Fig. 2: Aspartate import underlies the resistance of cancer cells to ETC inhibition.
Fig. 3: Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours.
Fig. 4: Aspartate levels inversely correlate with the expression of hypoxia-induced genes in primary human tumours.

Change history

  • 08 August 2018

    In the version of this Letter originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Letter.

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Acknowledgements

We thank all members of the Birsoy lab for helpful suggestions, and C. Moraes, and I. F. M. de Coo for providing the WT 143B and CYTB 143B cell lines. The KP cell lines are gifts from N. Bardeesy and T. Papagiannakopoulos. We also thank A. M. Hosios for natural abundance correction. This research is supported by an EMBO long-term fellowship to J.G.-B. (EMBO ALTF 887-2016). Profiling of human glioblastoma samples was supported by the Friedberg Charitable Foundation and Sohn Foundation grants to M.S. and Rachel Molly Markoff Foundation grant to M.S. and R.L.P. R.L.P. was supported by the NIH (R21CA198543), K.B. was supported by K22 (1K22CA193660), DP2 (DP2 OD024174-01), Irma-Hirschl Trust, AACR NextGen Grant and the Breast Cancer Research Foundation, and is a Searle Scholar, Sidney Kimmel Scholar and Basil O’Connor Scholar of March of Dimes.

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Authors and Affiliations

Authors

Contributions

K.B. and J.G.-B. conceived the project and designed the experiments. J.G.-B. and L.B. performed most of the experiments. K.L. performed computational analysis. X.G.Z assisted with the immunofluorescence experiments. C.A.L., J.F. and H.M. performed metabolite profiling experiments. T.P. assisted with the mouse xenograft experiments. R.L.P., M.S. and V.O.S. provided human tumour data and analysis. K.B. and J.G.-B wrote and edited the manuscript.

Corresponding author

Correspondence to Kıvanç Birsoy.

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Integrated Supplementary Information

Supplementary Figure 1 Metabolite changes in cancer cells upon ETC inhibition.

A) Sensitivity correlation of complex I inhibitor piericidin with ETC inhibitors of complex III (antimycin A) and V (oligomycin), as well as with the anti-diabetic drug phenformin. B) Metabolites detected in ETC inhibition resistant and sensitive cancer cell lines upon 10 nM piericidin treatment for 8 hrs. Heat map shows the changes of each metabolite relative to its levels in untreated cells, ranked by p-value (-log2 transformed) after comparing changes in the two groups. (n = 3 biologically independent samples per cell line). Statistics: two-tailed unpaired t-test. For individual P values, see Supplementary Table 1. Statistics source data are provided in Supplementary Table 1. C) Single Tandem Repeat (STR) profiling for the commonly misidentified cell lines U-937, RPMI 8402 and HPB-ALL.

Supplementary Figure 2 Aspartate uptake by aspartate/glutamate transporters is a major resistance mechanism to ETC inhibition.

A) Radioactive cpm measurements for the corresponding amounts of 14C-Aspartate (top). Time dependent uptake of 14C-Aspartate in COLO 320DM cell line after addition of 14C-Aspartate (2 nM) (bottom). B) Glutamate depletion does not sensitize resistant cancer cell lines to ETC inhibitors. L-Glutamate (150 μM), piericidin (10 nM), phenformin (100 μM) and antimycin (30 nM) were added where indicated. The boxes represent the median, and the first and third quartiles, and the whiskers represent the minimum and maximum of all data points (mean ± S.E.M., 5 cell lines in each group, n = 3 biologically independent samples). C) Canonical aspartate/glutamate transporters in mammalian cells. D) Ranked SLC1A2 mRNA expression data from the Cancer Cell Line Encyclopedia (CCLE) (log transformed) showing SLC1A2 expressing cancer cell lines (red dots). E) 14C-Aspartate uptake in A549 cancer cells transduced with control vector or expressing SLC1A3, SLC1A2 and SLC1A6 transporters. (mean ± S.E.M., n = 3 biologically independent samples). F) Immunofluorescence staining of SLC1A3 (green) in A549 cells transduced with control vector or expressing SLC1A3. Blue indicates nuclear staining by DAPI. Scale bar, 10 μm. G) SLC1A3 (red) is located in significantly amplified regions in epithelial cancers. H) Pharmacologic inhibition of SLC1A3 by its allosteric inhibitor UCPH 101 sensitizes ETC inhibition resistant cells to antimycin (30 nM) and piericidin (10 nM). Cells were grown for 5 days in the absence or presence of UCPH 101 (5 μM). The boxes represent the median, and the first and third quartiles, and the whiskers represent the minimum and maximum of all data points (mean ± S.E.M., 4 cell lines in each group, n = 3 biologically independent samples). I) Immunoblot analysis of SLC1A3, 14C-Aspartate uptake and proliferation upon antimycin (30 nM) treatment in wild-type and SLC1A3-null cell lines in which the gene is focally amplified (NCI-H596, SNU-182 and Detroit-562) (mean ± S.E.M., n = 3 biologically independent samples, ****p < 0.0001). Actin was used as a loading control. TFB-TBOA (20 μM) and pyruvate (1 mM) were added where indicated. J) Immunoblot analysis of SLC1A3 and 14C-Aspartate uptake of A549 cells transduced with control vector or expressing SLC1A3 (mean ± S.E.M., n = 3 biologically independent samples). Actin is included as a loading control. Statistics: two-tailed unpaired t-test. For individual P values, see Supplementary Table 1. Statistics source data are provided in Supplementary Table 1. All western blotting and immunofluorescence experiments were independently repeated a minimum of two times with similar results. Unprocessed original scans of blots are included in Supplementary Fig. 5.

Supplementary Figure 3 SLC1A3 enables cell proliferation under ETC inhibition or hypoxia at physiological aspartate levels.

A) NAD+/NADH ratio of corresponding cell lines under 21% and 0.5% oxygen. (mean ± S.E.M., n = 3 biologically independent samples). B) Relative intracellular amino acid abundances of A549 and PANC-1 cells upon piericidin (10 nM) treatment or under 0.5% oxygen, relative to untreated control cells under 21% oxygen. (mean ± S.E.M., n = 3 biologically independent samples). C) Serum aspartate levels of three independent NOD/SCID-gamma mice. (mean ± S.D, n = 3 biologically independent samples). D) Cancer cell competition experiment of A549 cancer cells under varying aspartate concentrations. Relative abundance of A549 cancer cells transduced with a control vector or SLC1A3 cDNA cultured under physiological (20 μM) and media (150 μM) aspartate concentrations in the presence or absence of piericidin treatment (10 nM) and 0.5% hypoxia. TFB-TBOA (20 μM) was added where indicated. Results are plotted relative to untreated cells under 21% oxygen. (mean ± S.E.M., n = 3 biologically independent samples, *p < 0.01, **p < 0.001). E) Relative proliferation of indicated cell lines transduced with control vector (Vector, black) or expressing SLC1A3 (SLC1A3, grey) in aspartate-depleted media supplemented with indicated concentrations of aspartate in the presence of piericidin (10 nM). (mean ± S.E.M., n = 3 biologically independent samples, *p < 0.01, **p < 0.001). F) Relative proliferation of indicated cell lines transduced with control vector (Vector, black) or expressing SLC1A3 (SLC1A3, grey) in aspartate-depleted media supplemented with indicated concentrations of aspartate under 21% or 0.5% oxygen. Results are plotted relative to cells under 21% oxygen and not supplemented with aspartate (mean ± S.E.M., n = 3 biologically independent samples, **p < 0.001). G) Aspartate abundance of established xenografts derived from A549 cell lines transduced with a control vector or with SLC1A3 cDNA. Results were normalized by lysine levels. (mean ± S.D., n = 8 biologically independent samples). H) Relative abundance of KP pancreas cancer cells grown in vivo as xenografts or in vitro under antimycin (30 nM) treatment or 0.5% oxygen. Results are plotted relative to untreated cells under 21% oxygen. (mean ± SD, n = 6 independent biological samples for in vitro conditions, n = 12 biologically independent samples for tumours, **p < 0.001, ***p < 0.0001). Statistics: two-tailed unpaired t-test. For individual exact P values, see Supplementary Table 1. Statistics source data are provided in Supplementary Table 1.

Supplementary Figure 4 Aspartate is shunted to nucleotide synthesis under hypoxia and ETC inhibition.

A) Schematic depicting the metabolic routes of aspartate in pyrimidine and purine synthesis. Filled circles represent 13C atoms derived from [U-13C]-L-aspartate (top). Relative levels of aspartate, citrate, malate, UMP and dTMP in control or SLC1A3-overexpressing A549 and PANC-1 cells cultured under piericidin (10 nM) treatment or 0.5% oxygen for 24 hr with [U-13C]-L-aspartate (150 μM), relative to untreated control cells under 21% oxygen. Colours indicate mass isotopomers (mean ± S.D., n = 3 biologically independent samples). B) Fold change in oxygen consumption rate (OCR) in A549, KP lung and KP pancreas overexpressing SLC1A3 (SLC1A3, grey), relative to cell lines transduced with control vector (Vector, black). (mean ± S.D., n = 10 biologically independent replicates). C) Relative levels of AMP and its mass isotopomer analysis in control or SLC1A3-overexpressing A549 cells cultured under 0.5% oxygen for 24 hr with [15N]-L-Aspartate (150 μM), relative to control cells under 0.5% oxygen. (mean ± S.D., n = 3 biologically independent samples). D) Fraction of labelled nucleotide precursors derived from labelled aspartate in control and SLC1A3-overexpressing A549 cells cultured for 24 hr with [U-13C]-L-aspartate (20 μM) or [15N]-L-Aspartate (20 μM) under 0.5% oxygen. Colours indicate mass isotopomers (mean ± S.D., n = 3 biologically independent samples). E) Correlation between aspartate levels and acyl-carnitine abundance (Top). Correlation of VEGF mRNA levels and aspartate levels (Bottom). F) Relative aspartate levels in CA9 and HK2 high (n = 12 biologically independent samples) and low (n = 12 biologically independent samples) tumours to population. The boxes represent the median, the first and third quartiles, and the whiskers represent the minimum and maximum of all data points. Statistics: two-tailed unpaired t-test. For individual exact P values, see Supplementary Table 1. Oxygen consumption assays and metabolite profiling experiments were done at least 2 independent times with similar results. Statistics source data are provided in Supplementary Table 1.

Supplementary Figure 5

Unprocessed images of western blots presented in Fig. 2d, e, Supp. Fig. 2I, 2J.

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Supplementary Information

Supplementary Figures 1–5 and Supplementary Table legends

Reporting Summary

Supplementary Table 1

Statistics source data

Supplementary Table 2

Correlation data for Figure 4

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Garcia-Bermudez, J., Baudrier, L., La, K. et al. Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours. Nat Cell Biol 20, 775–781 (2018). https://doi.org/10.1038/s41556-018-0118-z

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