Skip to main content

Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation

Tumor Biology volume 35, pages 5911–5920 (2014)Cite this article

Abstract

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP+ to NADPH. IDH1 mutations are frequently detected in grades 2–4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to d-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-l-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321(5897):1807–12. doi:10.1126/science.1164382.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  2. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol. 2008;116(6):597–602. doi:10.1007/s00401-008-0455-2.

    CAS  Article  PubMed  Google Scholar 

  3. Watanabe T, Nobusawa S, Kleihues P, Ohgaki H. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. Am J Pathol. 2009;174(4):1149–53. doi:10.2353/ajpath.2009.080958.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  4. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360(8):765–73. doi:10.1056/NEJMoa0808710.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  5. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature. 2009;462(7274):739–44. doi:10.1038/nature08617.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  6. Hirayama A, Kami K, Sugimoto M, Sugawara M, Toki N, Onozuka H, et al. Quantitative metabolome profiling of colon and stomach cancer microenvironment by capillary electrophoresis time-of-flight mass spectrometry. Cancer Res. 2009;69(11):4918–25. doi:10.1158/0008-5472.CAN-08-4806.

    CAS  Article  PubMed  Google Scholar 

  7. Fults D, Brockmeyer D, Tullous MW, Pedone CA, Cawthon RM. p53 mutation and loss of heterozygosity on chromosomes 17 and 10 during human astrocytoma progression. Cancer Res. 1992;52(3):674–9.

    CAS  PubMed  Google Scholar 

  8. Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol. 2009;118(4):469–74. doi:10.1007/s00401-009-0561-9.

    Article  PubMed  Google Scholar 

  9. Kato Y, Jin G, Kuan CT, McLendon RE, Yan H, Bigner DD. A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, the most common glioma-derived mutation. Biochem Biophys Res Commun. 2009;390(3):547–51. doi:10.1016/j.bbrc.2009.10.001.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  10. Takeuchi H, Jin S, Wang J, Zhang G, Kawanokuchi J, Kuno R, et al. Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner. J Bio Chem. 2006;281(30):21362–8. doi:10.1074/jbc.M600504200.

    CAS  Article  Google Scholar 

  11. Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, et al. Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption. J Bio Chem. 2006;281(24):16768–76. doi:10.1074/jbc.M601876200.

    CAS  Article  Google Scholar 

  12. Soga T, Igarashi K, Ito C, Mizobuchi K, Zimmermann HP, Tomita M. Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry. Anal Chem. 2009;81(15):6165–74. doi:10.1021/ac900675k.

    CAS  Article  PubMed  Google Scholar 

  13. Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010;17(1):98–110. doi:10.1016/j.ccr.2009.12.020.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  14. Noushmehr H, Weisenberger DJ, Diefes K, Phillips HS, Pujara K, Berman BP, et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell. 2011;17(5):510–22. doi:10.1016/j.ccr.2010.03.017.

    Article  Google Scholar 

  15. Ohka F, Natsume A, Motomura K, Kishida Y, Kondo Y, Abe T, et al. The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma. PLoS One. 2011;6(8):e23332. doi:10.1371/journal.pone.0023332.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  16. Songtao Q, Lei Y, Si G, Yanqing D, Huixia H, Xuelin Z, et al. IDH mutations predict longer survival and response to temozolomide in secondary glioblastoma. Cancer Sci. 2012. doi:10.1111/j.1349-7006.2011.02134.x.

    PubMed  Google Scholar 

  17. Seltzer MJ, Bennett BD, Joshi AD, Gao P, Thomas AG, Ferraris DV, et al. Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res. 2010;70(22):8981–7. doi:10.1158/0008-5472.CAN-10-1666.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  18. Kim JW, Dang CV. Cancer's molecular sweet tooth and the Warburg effect. Cancer Res. 2006;66(18):8927–30. doi:10.1158/0008-5472.CAN-06-1501.

    CAS  Article  PubMed  Google Scholar 

  19. Lopez-Lazaro M. The Warburg effect: why and how do cancer cells activate glycolysis in the presence of oxygen? Anticancer Agents Med Chem. 2008;8(3):305–12.

    CAS  Article  PubMed  Google Scholar 

  20. Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 2008;452(7184):230–3. doi:10.1038/nature06734.

    CAS  Article  PubMed  Google Scholar 

  21. Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, et al. Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature. 2011;480(7375):118–22. doi:10.1038/nature10598.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  22. Kefas B, Comeau L, Erdle N, Montgomery E, Amos S, Purow B. Pyruvate kinase M2 is a target of the tumor-suppressive microRNA-326 and regulates the survival of glioma cells. Neuro Oncol. 2010;12(11):1102–12. doi:10.1093/neuonc/noq080.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  23. Clower CV, Chatterjee D, Wang Z, Cantley LC, Vander Heiden MG, Krainer AR. The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci U S A. 2010;107(5):1894–9. doi:10.1073/pnas.0914845107.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  24. Reitman ZJ, Jin G, Karoly ED, Spasojevic I, Yang J, Kinzler KW, et al. Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome. Proc Natl Acad Sci U S A. 2011;108(8):3270–5. doi:10.1073/pnas.1019393108.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  25. Zhao S, Lin Y, Xu W, Jiang W, Zha Z, Wang P, et al. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. Science. 2009;324(5924):261–5. doi:10.1126/science.1170944.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  26. Chesnelong C, Chaumeil MM, Blough MD, Al-Najjar M, Stechishin OD, Chan JA, et al. Lactate dehydrogenase A silencing in IDH mutant gliomas. Neuro-oncology. 2013. doi:10.1093/neuonc/not243.

    Google Scholar 

  27. Koivunen P, Lee S, Duncan CG, Lopez G, Lu G, Ramkissoon S, et al. Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation. Nature. 2012;483(7390):484–8. doi:10.1038/nature10898.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  28. Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell. 2010;18(6):553–67. doi:10.1016/j.ccr.2010.11.015.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  29. Guo JU, Su Y, Zhong C, Ming GL, Song H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell. 2011;145(3):423–34. doi:10.1016/j.cell.2011.03.022.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  30. Lu C, Ward PS, Kapoor GS, Rohle D, Turcan S, Abdel-Wahab O, et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature. 2012;483(7390):474–8. doi:10.1038/nature10860.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  31. Turcan S, Rohle D, Goenka A, Walsh LA, Fang F, Yilmaz E, et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature. 2012;483(7390):479–83. doi:10.1038/nature10866.

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  32. Chatham JC, Blackband SJ. Nuclear magnetic resonance spectroscopy and imaging in animal research. ILAR J. 2001;42(3):189–208.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author information

Affiliations

  1. Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Fumiharu Ohka, Maki Ito, Melissa Ranjit, Ayako Motomura, Kazuya Motomura, Toshihiko Wakabayashi & Atsushi Natsume

  2. Division of Cancer Biology, Nagoya University School of Medicine, Nagoya, Japan

    Takeshi Senga

  3. Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan

    Yukinari Kato

  4. Institute for Advanced Bioscience, Keio University, Yamagata, Japan

    Kaori Saito, Keiko Kato & Tomoyoshi Soga

Authors
  1. Fumiharu Ohka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maki Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Melissa Ranjit
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takeshi Senga
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ayako Motomura
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kazuya Motomura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kaori Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Keiko Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yukinari Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Toshihiko Wakabayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Tomoyoshi Soga
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Atsushi Natsume
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fumiharu Ohka.

Additional information

Fumiharu Ohka and Maki Ito equally contributed to this work

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Figure 1

Microscopic imaging of cells expressing IDH1-WT or IDH1-R132H We generate HEK293 and U87 cells, expressing IDH1-WT or IDH1-R132H fused with GFP protein. In both cell lines, exogenous IDH1-WT and IDH1-R132H with GFP are located in the cytoplasm. (PPTX 2432 kb)

Supplemental Figure 2

Expression of endogenous IDH2 in cells expressing IDH1-WT or IDH1-R132H We detected expression of endogenous IDH2 expressing IDH1-WT or IDH1-R132H cells, compared with those expressions of β-actin. (PPTX 42260 kb)

Supplemental Figure 3

Cell growth rate of cells expressing IDH1-WT or IDH1-R132H in normal condition MTT assay revealed that cell growth rate of control cells, IDH1-WT cells and IDH1-R132H cells are almost equal in HEK293 and U87. (PPTX 80 kb)

Supplemental Figure 4

Restoration of cell viability by adding α-KG in glutamine deprivation condition In glutamine free medium, we cultured cells for 72 h with or without addition of 1 mM α-KG. Addition of α-KG, in glutamine deprivation condition, significantly helped to recover the cell viability of IDH1-R132H cells compared to that of IDH1-WT cells in U87 cell lines. (*: p < 0.05) (PPTX 119 kb)

Supplemental Figure 5

Analysis of α-KG level in U87 cells expressing IDH1-WT or IDH1-R132H Using CE-TOFMS, we analyzed α-KG level in U87 cells expressing IDH1-WT or IDH1-R132H. α-KG level in IDH1-R132H cells are significantly decreased, compared with those of IDH1-WT cells. (*: p < 0.05) (PPTX 55 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ohka, F., Ito, M., Ranjit, M. et al. Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation. Tumor Biol. 35, 5911–5920 (2014). https://doi.org/10.1007/s13277-014-1784-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-014-1784-5

Keywords

  • Glioma
  • IDH1 mutation
  • Metabolome
  • Glutaminolysis