Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, we provide a background for vital mitochondrial production of 2HG in breast cancer cells with outcomes towards cancer biology and possible future diagnosis of breast carcinoma.

4 th Department of Internal Medicine the General University Hospital and the 1st Faculty of Medicine Charles University 128 08 Prague Czech Republic

Department of Oncology the General University Hospital and The 1st Faculty of Medicine 128 08 Prague Czech Republic

Institute of Biophysics and Informatics the 1st Faculty of Medicine Charles University 120 00 Prague Czech Republic

Institute of Medical Biochemistry and Laboratory Diagnostics of the General University Hospital and the 1st Faculty of Medicine Charles University 121 08 Prague Czech Republic

Institute of Physiology of the Czech Academy of Sciences Laboratory of Mitochondrial Physiology 142 20 Prague Czech Republic

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