MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU Elena Favaro1,2., Anassuya Ramachandran2., Robert McCormick2, Harriet Gee2, Christine Blancher1, Meredith Crosby3, Cecilia Devlin4, Christopher Blick2, Francesca Buffa2, Ji-Liang Li2, Borivoj Vojnovic5, Ricardo Pires das Neves6, Peter Glazer3, Francisco Iborra6, Mircea Ivan4*, Jiannis Ragoussis1", Adrian L. Harris2*" 1 Genomics Group, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 2 Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom, 3 Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 4 Indiana University, Indianapolis, Indiana, United States of America, 5 Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford, United Kingdom, 6 Molecular Haematology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom Abstract Background: Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR- 210 (miR-210) which is associated with a poor prognosis. Methods and Findings: In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Conclusions: Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation. Citation: Favaro E, Ramachandran A, McCormick R, Gee H, Blancher C, et al. (2010) MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU. PLoS ONE 5(4): e10345. doi:10.1371/journal.pone.0010345 Editor: Andrei L. Gartel, University of Illinois at Chicago (UIC), United States of America Received December 16, 2009; Accepted March 29, 2010; Published April 26, 2010 Copyright: 2010 Favaro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Cancer Research UK and Friends of Kennington Cancer Research Fund (EF, ALH, JL), The Wellcome Trust (CB1, CC, IR), Royal College of Surgeons & British Urology Foundation (CB2, RMcC), European Union, ACGT (FB), Rhodes Scholarship (HG), Nuffield Medical Fellowship (AR), FIRC Studentship (EF). Biobanking supported by NIHR Biomedical Research Centre Oxford; Elsa Pardee Foundation (MI), American Cancer Society (MC, MI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mivan@iupui.edu (MI); aharris.lab@imm.ox.ac.uk (ALH) . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction Hypoxia is a major physiological difference between tumours and normal tissue, mainly generated by tumour growth with inadequate blood supply and consumption of oxygen by tumour cells [reviewed in [1]]. Hypoxia induces a complex transcriptional response mainly via induction of hypoxia inducible factor 1a (HIF1a), affecting many biological processes such as the glycolytic pathway, angiogenesis, pH regulation, invasion and immortalisa- tion [2]. An emerging paradigm in hypoxia is that mitochondria produce reactive oxygen species, mediated by electron transport continuing in hypoxia [3]. This free radical pathway contributes to upregulation of HIF [4] and enhanced growth in vivo [5], yet may also be toxic. A variety of pathways induced by HIF have already been reported to protect from the latter effect, for example induction of pyruvate dehydrogenase kinase inhibits the enzyme complex of pyruvate dehydrogenase and blocking the conversion of pyruvate to acetyl coenzyme A, the first step in the Krebs cycle [6] and enhances lactate production [7]. Mitophagy can be induced by the BH3 domain protein BNIP3, [8], and cytochrome C oxidase subunits may switch to more efficient ones [9]. Recently microRNAs (miRs), which are small (,22 nt) non- coding RNAs that regulate post-transcriptional gene expression by blocking translation of target mRNAs or by accelerating their degradation [10,11], have been reported to be induced by hypoxia. However, few of their targets or mechanisms of action are known [reviewed in [12]]. We and others [13] have shown PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10345