title: Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells.
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 (13)C incorporation from 1-(13)C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, (13)C-citrate, (13)C-malate, and (13)C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5mM 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 (13)C-accumulation into citrate and malate increased 1.5-2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. (13)C-2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal (12)C-2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and (13)C-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.
authors.affiliation: Department of Membrane Transport Biophysics, No.75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Electronic address: smolkova@biomed.cas.cz.
authors.forename: Katarína
authors.initials: K
authors.lastname: Smolková
authors.affiliation: Department of Membrane Transport Biophysics, No.75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; Institute of Medical Biochemistry and Laboratory Diagnostics, Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic. Electronic address: ales.dvorak@biomed.cas.cz.
authors.forename: Aleš
authors.initials: A
authors.lastname: Dvořák
authors.affiliation: Department of Membrane Transport Biophysics, No.75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Electronic address: zelenka@biomed.cas.cz.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Zelenka
authors.affiliation: Institute of Medical Biochemistry and Laboratory Diagnostics, Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic. Electronic address: vitek@cesnet.cz.
authors.forename: Libor
authors.initials: L
authors.lastname: Vítek
authors.affiliation: Department of Membrane Transport Biophysics, No.75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Electronic address: jezek@biomed.cas.cz.
authors.forename: Petr
authors.initials: P
authors.lastname: Ježek
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D005260:Female; D005977:Glutarates / Q000378:metabolism*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010313:Partial Pressure
chemical_list: D014408:Biomarkers, Tumor; D005977:Glutarates; C019417:alpha-hydroxyglutarate; C538784:IDH2 protein, human; D007521:Isocitrate Dehydrogenase; D010100:Oxygen
keywords: Breast adenocarcinoma MDA-MB-231 cells; Breast carcinoma HTB-126 cells; Hypoxia; NADPH-dependent isocitrate dehydrogenase IDH2; Oncometabolite r-2-hydroxyglutarate; Reductive carboxylation