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Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma - Nature Cell Biology

  • ️Gottlieb, Eyal
  • ️Mon Nov 23 2015

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Acknowledgements

This study has been supported by Cancer Research UK. S.T. is a recipient of an AIRC/Marie Curie International Fellowship for Cancer Research. The human and animal metabolomic studies were supported by The Norwegian Cancer Society, The Norwegian Research Council, Helse Vest, Haukeland University Hospital and the K.G-Jebsen Foundation. We acknowledge A. Golebiewska, V. Baus-Talko, N. Van Den Broek, G. MacKay, C. Nixon and E. MacKenzie for excellent technical assistance and A. King for excellent editorial work.

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Authors and Affiliations

  1. Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK

    Saverio Tardito, Liang Zheng, Andreas K. Hock & Eyal Gottlieb

  2. Department of Oncology, NorLux Neuro-Oncology Laboratory, Luxembourg Institute of Health, L-1526 Luxembourg, Luxembourg

    Anaïs Oudin, Fred Fack, Olivier Keunen, Rolf Bjerkvig & Simone P. Niclou

  3. Institute of Cancer Sciences, University of Glasgow, Glasgow G12 8QQ, UK

    Shafiq U. Ahmed & Anthony J. Chalmers

  4. Department of Biomedicine, Kristian Gerhard Jebsen Brain Tumour Research Center, University of Bergen, Bergen N-5009, Norway

    Hrvoje Miletic, Per Øystein Sakariassen, Rolf Bjerkvig & Simone P. Niclou

  5. The Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv 69978, Israel

    Adam Weinstock, Allon Wagner & Eytan Ruppin

  6. Institute of Infection, Immunity and inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK

    Susan L. Lindsay & Susan C. Barnett

  7. The Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

    Eytan Ruppin

  8. Department of Neurosurgery, Haukeland University Hospital, Bergen N-5021, Norway

    Svein Harald Mørkve & Morten Lund-Johansen

  9. Department of Clinical Medicine, University of Bergen, Bergen N-5020, Norway

    Morten Lund-Johansen

Authors

  1. Saverio Tardito

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  2. Anaïs Oudin

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  3. Shafiq U. Ahmed

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  4. Fred Fack

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  5. Olivier Keunen

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  6. Liang Zheng

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  7. Hrvoje Miletic

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  8. Per Øystein Sakariassen

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  9. Adam Weinstock

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  10. Allon Wagner

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  11. Susan L. Lindsay

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  12. Andreas K. Hock

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  13. Susan C. Barnett

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  14. Eytan Ruppin

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  15. Svein Harald Mørkve

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  16. Morten Lund-Johansen

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  17. Anthony J. Chalmers

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  18. Rolf Bjerkvig

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  19. Simone P. Niclou

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  20. Eyal Gottlieb

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Contributions

S.T. conceived the study, designed and performed most experiments, interpreted the data, and wrote the manuscript, A.O. performed the experiments in orthotopic xenograft models, S.U.A. and A.J.C. provided the differentiated and stem-like primary glioblastoma cells, L.Z. supervised the analysis of LC-MS samples, O.K. performed the MRI analysis, F.F. processed the orthotopic and clinical GBM samples, H.M. provided the tissue microarray, A.K.H. designed and provided the iRFP and iRFP–GS constructs, A.Weinstock, A.Wagner and E.R. generated and employed the metabolic modelling, S.C.B. and S.L.L. provided the primary astrocytes, M.L.-J., S.H.M. and P.Ø.S. provided the surgical specimens from the patients, S.P.N. and R.B. conceived and supervised the experiments in orthotopic models and human patients, and E.G. conceived and supervised the study, interpreted the data, and revised the manuscript.

Corresponding author

Correspondence to Eyal Gottlieb.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 5 Glutamine starvation reduces GBM cell proliferation.

(a) Representative microscopic fields of cells incubated for 6 days with or without Gln. (b) Cell cycle distribution of cells incubated for 3 days with or without Gln. Mean ± S.E.M. n = 3 independent experiments. p value refers to a two-tailed t test for unpaired samples. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Supplementary Figure 6 Exchange rates of metabolites in GBM cell lines.

Cells were incubated for 24 h +/− U-13C5 Gln, secretion rates (positive bars) and consumption rates (negative bars) of the indicated metabolites are shown. The sum of all isotopologues is reported for clarity. Alanine (Ala) exchange rates are magnified in the inset. Mean ± S.E.M. n = 3 independent experiments.

Supplementary Figure 7 Effects of Gln availability and Glutaminase inhibition on GBM cell lines growth and redox state.

(a) Correlation between Gln consumption and growth inhibition caused by Gln starvation is shown in six cell lines. Mean ± S.E.M. n = 3 independent experiments. (b) Citrate isotopologues distribution of 13C5-Gln derived atoms. 13C6-Citrate was not detected. Mean ± S.E.M. n = 3 independent experiments. (c) Oxidized (GSSG) to reduced (GSH) glutathione ratio. LN18 cells were incubated for 24 h +/− Gln and supplemented with Glu, α-ketoglutarate dimethylester (dm-αKG), sulfasalazine (SSZ), or cystine, as indicated. GSSG/GSH ratio was assessed by HPLC-MS, and shown as % of untreated control. Data derive from one experiment performed once. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (d–i) Cells were incubated with 0, 2.5, 5, 10, 15, 30 μM BPTES for 72 h, in the absence (solid line) or presence (dotted line) of 4 mM dm-αKG and counted. In all conditions cells were exposed to 0.3%DMSO. Mean ± S.E.M. n = 3 independent experiments.

Supplementary Figure 8 GS expression and its effects on colony formation capacity, and glucosamine biosynthetic pathway in GBM cells.

(a) Cells were incubated for 24h +/− Gln and GLUL (GS), GLS, and MYC mRNA relative expression was assessed by qPCR. Actin expression was used for normalization. Data derive from one experiment performed once. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (b) SF188 and U251 cells stably expressing a non-targeting control shRNA (shNTC) and two sequences targeting GS (shGS-1 and shGS-2) were cultured for 12–17 days +/− Gln in medium supplemented with glutamate (4 mM), and ammonia (0.8 mM) as indicated. The quantification of colonies surface area was obtained as described in the Methods section. Mean ± S.E.M., n = 4 independent experiments. (c) iRFP4 and iRFP-GS5 expressing LN18 cells were incubated +/− Gln for 24 h in medium supplemented with 0.8 mM 15NH4+. The intracellular isotopologues distribution of UDP N-acetylglucosamine is shown as % of control (value obtained for the metabolites in iRFP4 cells + Gln). Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Supplementary Figure 9 Heterogeneous GS expression in human GBM biopsies.

The whole bioptic tissues of three patients included in the tissue microarray were stained for GS, and Hematoxilin and Eosin [H&E], as indicated. Low magnifications of whole biopsies are reported in panels (a,b,h,i,n,o). For each biopsy, tissue regions framed in blue and green are magnified in d–g,j–m,q–t. Biopsy 1 shows necrotic tissue [nec] surrounded by a solid tumour core [TC] with uniformly low GS staining (d,f). (d,e) Invasive tumour areas [TI] show infiltrative cells with heterogeneous immunoreactivity for GS. (c) Normal astrocytes [black arrows] in the adjacent normal brain [Br] were positive for GS, while neurons [white arrows] were GS-negative. Biopsy 2 shows distinct areas with low/absent (k) and high (j) immunoreactivity for GS. GS-negative inflammatory tissue [i] surrounding a necrotic region [nec] is evident in panels h,i,k–m. Biopsy 3 shows distinct areas with low/absent (q) and high (r) immunoreactivity for GS. (p) GS-positive reactive astrocytes [arrows] with extensive cellular processes forming a network of GS positive fibers surrounding GS-negative tumour cells.

Supplementary Figure 10 Glucose and glutamine metabolic fates in GBM patients, and orthotopic mouse models.

(a) Serum levels of 13C6 and 13C0 glucose in three GBM patients injected with 13C6 glucose before surgical intervention for tumour removal. The dashed line represents the time at which glucose infusion starts. Black arrow points at the time of surgical resection. Values are reported as % of the basal values obtained before infusion. (b) 13C Lactate and (c) 13C Glutamate enrichment in serum at time of tumour resection, in tumour tissue, and in adjacent edematous tissue, of seven (1-7) GBM patients injected with 13C6-glucose. The % of lactate or glutamate incorporating one or more 13C carbons over the total amount of lactate or glutamate detected is reported; na: not available, nd: not detectable. Values were corrected for the natural abundance of 13C. (d,e) Isotopologue distribution of metabolites obtained in the liver of mice orthotopically xenografted with human P3 GBM and injected with 13C6 Glucose (d) or 13C5-Gln (e). The values are mean ± S.E.M. n = 3 mice. (f,g) Isotopologues of Gln (f) and glucose (g) found in the serum of mice orthotopically xenografted with the human GS-positive T16 GBM, and infused for 4h into the carotid artery with 13C5-Gln. Mean ± S.E.M. n = 3 mice. (h–j) Isotopologue distributions of Gln, Glu and α-kg in T16 tumours, contralateral brain, and liver tissues of mice infused as in (f,g). Mean ± S.E.M. n = 3 mice. (k) Astrocytes were incubated for 24 hours +/− Gln, and asparagine consumption is reported. Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Supplementary Figure 12 Unprocessed scans of western blots accompanied by size markers.

Red dotted lines delineate the region presented in the respective Figures as indicated. Images were obtained using a Licor Odyssey scanner and acquired using Image Studio 2.0.

Supplementary Table 1 Formulation of DMEM and Serum-like Modified Eagles Medium (SMEM).

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Supplementary Table 2 Exchange rates of metabolites in GBM cell lines.

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Supplementary Table 3 Representation of SMEM growth medium in the metabolic model Recon1.

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Supplementary Table 4 Weighted costs of growth limiting metabolites.

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Tardito, S., Oudin, A., Ahmed, S. et al. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma. Nat Cell Biol 17, 1556–1568 (2015). https://doi.org/10.1038/ncb3272

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  • Received: 30 October 2014

  • Accepted: 19 October 2015

  • Published: 23 November 2015

  • Issue Date: December 2015

  • DOI: https://doi.org/10.1038/ncb3272