PYCR1 activity is essential for the maintenance of viable hypoxic regions in cancers Rebecca L Westbrook1, Esther Bridges2, Cristina Escribano Gonzalez1, Abeer Shaban3, Nathalie Escande-Beillard4, Katherine L Eales1, Lisa Vettore1, Paul Walker1, Federica Cuozzo1, Colin Nixon5, David J Hodson1, Bruno Reversade4,6, Adrian Harris2 and Daniel A Tennant1. 1Institute of Metabolism and Systems Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT. UK 2Hypoxia and Angiogenesis Group, Cancer Research UK Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, Department of Oncology, University of Oxford, Oxford OX3 9DS, UK 3University Hospital Birmingham NHS Foundation Trust and Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT. UK 4Institute of Medical Biology, Human Genetics and Embryology Laboratory; A*STAR, Singapore 138648, Singapore 5Beatson Institute for Cancer Research, University of Glasgow, Switchback Road, Glasgow. G61 1BD. UK 6Institute of Molecular and Cellular Biology, A*STAR, Singapore 138673, Singapore The demands of highly proliferative cancer cell populations alongside an inadequate blood supply lead to low oxygen and nutrient conditions in the tumour microenvironment. Hypoxic cells adapt their metabolic network, often increasing reliance on pathways that are independent of oxygen tension to maintain viability. However, to continue the efficient synthesis of anabolic precursors in these conditions, it is advantageous to continue use of oxidative TCA cycle metabolism, which is closely coupled to electron transport chain activity, and the reduction of molecular oxygen. We have previously shown that when redox homeostasis is perturbed by oncogenic mutations in isocitrate dehydrogenase 1, pyrroline 5-carboxylate reductase 1 (PYCR1) activity was increased to oxidise mitochondrial NADH, effectively uncoupling oxidative TCA cycle activity from respiration. We therefore hypothesised that this may also be true in hypoxic conditions. We show here that hypoxia elicits a PYCR1-dependent increase in proline synthesis and excretion, which is required for efficient growth of cells in 3D culture and in xenograft tumours. PYCR1 deficiency in hypoxia results in deficient oxidative TCA cycle and reduced growth, which cannot be recovered by exogenous proline supplementation. Finally, loss of PYCR1 in 3D spheroids and xenograft tumours increases intratumoural hypoxia through enforced respiration, which leads to loss of proliferative drive, increased cell death and necrosis. Our data therefore suggest that PYCR1 is an essential component of the cellular response to hypoxia, functionally uncoupling the mitochondrial electron transport chain from the TCA cycle to permit the efficient use of the limited supply of oxygen throughout the cell.