PCK2 balances TCA cycle activity, limits mitochondrial respiration and improves the redox balance in starved lung cancer cells Gabriele Grasmann1 , Mélanie Planque2,3, Corina T. Madreiter-Sokolowski4,5, Andelko Hrzenjak1,6, Wolfgang F. Graier4,7, Sarah-Maria Fendt2,3, Horst Olschewski1,6, Katharina Leithner1,7* 1 Division of Pulmonology, Department of Internal Medicine, Medical University of Graz, Graz, Austria 2 Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer Biology, VIB, Leuven, Belgium 3 Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), Leuven, Belgium 4 Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria 5 Energy Metabolism Laboratory, Institute of Translational Medicine, Department of Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland 6 Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria 7 BioTechMed-Graz, Graz, Austria Cancer cells frequently lack nutrients like glucose, due to insufficient vascular networks, requiring metabolic adaptations. Gluconeogenesis, in big parts the reverse pathway of glycolysis, has been recently described as survival strategy. Phosphoenolpyruvate carboxykinase (PEPCK) is the key gluconeogenic enzyme, catalyzing the conversion of oxaloacetate, a TCA cycle intermediate, to phosphoenolpyruvate. The mitochondrial isoform of PEPCK, PCK2 is expressed in different tumor types, including lung cancer. It has pro-survival and pro-proliferative effects in vitro and in vivo and mediates the biosynthesis of gluconeogenic/glycolytic intermediates in glucose deprived cancer cells. However, it remains unknown, whether PCK2 regulates TCA cycle activity and mitochondrial respiration in cancer cells under nutrient starvation. Here we show that PCK2 silencing increased the abundance and interconversion of TCA cycle intermediates in two different lung cancer cell lines in a PCK2 re-expression reversible manner. The TCA cycle is connected to mitochondrial respiration via its production of reducing equivalents. Glucose and serum starvation led to enhanced mitochondrial respiration compared to non-starvation conditions. Under starvation conditions mitochondrial respiration was additionally augmented by PCK2 silencing and glutathione oxidation was increased. Moreover, enhancing the TCA cycle activity by PCK2 inhibition severely reduced colony formation, an effect that was antagonized by external addition of antioxidants. As a conclusion, the cataplerotic activity of PCK2 limits oxygen consumption and contributes to maintaining a reduced glutathione pool upon starvation. The study sheds light on adaptive responses in cancer cells to nutrient deprivation and identifies gluconeogenesis as starvation-induced pathway that limits respiration-induced oxidative stress.