Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells |
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| Institution: | 1. Department of Membrane Transport Biophysics, No.75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic;2. Institute of Medical Biochemistry and Laboratory Diagnostics, Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic;1. Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey;2. Department of Biology, Vytautas Magnus University, 44404 Kaunas, Lithuania;1. Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;2. Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;3. Annette G. Strauss Center for Neuro-Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;4. Center for Magnetic Resonance Research, Korea Basic Science Institute, Chungbuk 363-883, Republic of Korea;5. Department of Neurological Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;6. Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;7. McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;8. Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;9. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;10. Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;11. Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA;12. Veterans Affairs North Texas HealthCare System, Lancaster, TX 75216, USA;1. Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland;2. Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland;3. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan;4. Departments of Internal Medicine, Pathology and Human Genetics, University of Michigan, Ann Arbor, Michigan |
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| Abstract: | Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring 13C incorporation from 1-13C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, 13C-citrate, 13C-malate, and 13C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5 mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30–50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated 13C-accumulation into citrate and malate increased 1.5–2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. 13C–2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal 12C–2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and 13C–2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients. |
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| Keywords: | Reductive carboxylation NADPH-dependent isocitrate dehydrogenase IDH2 Hypoxia Breast carcinoma HTB-126 cells Breast adenocarcinoma MDA-MB-231 cells |
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