aconitase, a key enzyme of intermediary metabolism that requires Fe-S clusters, had decreased activity following ISCU reduction. Of specific relevance to cancer is the dual role of aconitase as IRP1 when it lacks its Fe-S cluster. IRP1 regulates the stability and translation of mRNA for transferrin and ferritin [24]. The effects of loss of aconitase would be to modify iron metabolism and increase its uptake, which is critical for tumour growth. Indeed, we observed an accumulation of intracellular iron upon transfection of cells with mimic-210 or siISCU. Mutations in ISCU give rise to lactic acidosis in individuals after moderate exercise, providing strong evidence for the importance of this target in maintaining efficient Krebs cycle activity [30,31]. An associated effect from decreased ISCU levels and subsequent decrease in the Krebs cycle activity was a switch to glycolysis and increased lactate production. This was induced in normoxia by miR-210 and thus is similar to the Warburg effect. This may be relevant in situations where miR-210 upregulation occurs in normoxia. For example, this has been shown in renal cancer cell lines with mutations in the von Hippel Lindau protein [14]. Conversely, in hypoxia, anti-210 reduced lactate production, reversing the Pasteur effect (switch to glycolysis in hypoxia). Iron-sulfur clusters are integral for efficient activity of the mitochondrial electron transport chain. We have shown that Complex I activity is decreased under hypoxia and upon transfection of mimic-210 in normoxia. In addition to Complex I, Complexes II and III also contain Fe-S clusters and we speculate that the activities of all three complexes could be decreased in a miR-210 dependent manner. An impaired electron transport chain would lead to an increase in free radical production as a consequence of electron leakage. In particular, complex III is a major contributor to ROS in hypoxia [32] and complex I and II contribute about 30% of the ROS [33]. In normoxic conditions, transfection of mimic-210 led to increased free radical production. Furthermore, the normoxic ROS production observed with mimic-210 was almost completely prevented with a miR-210 resistant ISCU construct. Conversely, in hypoxia, we observed that the induction of miR-210 was associated with an increase in ROS, and this could be almost completely reversed by anti-210. This provides a major new link to explain the mechanism of ROS induction in hypoxia, which has been reported by several groups [33] and may account for the majority of hypoxic ROS induction. The ROS released in hypoxia have been shown to function as O2 sensors, acting as signalling agents that activate HIF [32], mediate tumour growth in vivo via a HIF dependent mechanism [5] and extend lifespan of cells [34]. In contrast to the hypoxic induction of ROS and alleviation with anti-210 observed in our study, Chan et al. could not measure any significant change in ROS production after exposure to hypoxia, and showed an inverted trend when miR-210 was inhibited [20]. The reason for this discrepancy is unclear but may potentially reflect an underlying difference in cancer compared to normal endothelial cells. Finally, we analysed whether there was any association of ISCU downregulation in primary human cancers with hypoxia and outcome, and found an aggressive phenotype was associated with this change in studies of over 1000 patients, and downregulation compared to normal tissues in many tumour types. In conclusion, we have found a major new mechanism for the response of cancer cells to hypoxia, co-ordinated by miR-210 suppression of ISCU and the subsequent decrease in activity of iron-sulfur cluster proteins (Supporting Figure S5). In addition to aconitase, many metabolic enzymes require Fe-S clusters for their activity suggesting that downregulation of ISCU would have far reaching consequences on cell metabolism. Mutations in many of miR-210 Regulates ISCU these Fe-S cluster enzymes including succinate dehydrogenase subunits SDHD, SDHB and SDHC [35] and fumarate hydratase [36] are implicated in hyperproliferative disorders and cancer, demonstrating an important link between cellular metabolism and subsequent transformation and highlights a role for HIF, via a miR-210-ISCU axis in these processes. In addition to metabolic enzymes, several DNA repair enzymes [37] with Fe-S clusters are potentially targets. We have previously shown miR-210 can be detected at an elevated level in serum of cancer patients [38] so it will be of interest if they reflect the metabolic state of their primary cancer and hence could be used in future therapy selection. The regulation of distinct biological pathways by ISCU downregulation suggests a potential therapeutic approach of synthetic lethality whereby drugs that mediate DNA damage repaired by the iron- sulfur cluster containing helicases Rad3 [39] or Fanconi anaemia [37] could be use in synergy with PARP inhibitors, or inhibitors of glycolysis [36] and glutaminolysis in the subgroup of patients with highest miR-210 levels. While this work was in preparation, three new miR-210 targets were validated, i.e. HOXA1, HOXA9 and FGFRL1, and the analysis of tumour xenografts derived from cancer cell lines overexpressing miR-210 suggested a potential involvement of miR-210 in tumour growth initiation [40]. Materials and Methods Cell culture Cell exposure to hypoxia (1%, or 0.1%, or 0.01% oxygen) was undertaken in a hypoxia incubator (MiniGalaxy A, RS Biotech, Scotland, UK), using a continuous flow of a humidified a mixture of 95% N2 and 5% CO2. miRNA mimic or inhibitor transfection Transfection was performed with Dharmacon anti-210, miR- 210 mimic, or control oligos (Thermo Scientific, CO, USA), at a final concentration of 20 nM, using Optimem serum-free medium and Oligofectamine reagent (both from Invitrogen, Paisley, UK) as per the manufacturer’s protocol. Transfection and luciferase assays Luciferase reporter plasmids containing 39 untranslated regions (UTR) of ISCU or random genomic sequence (control) were obtained from SwitchGear Genomics (Menlo Park, CA. A Renilla luciferase expression plasmid (pRL-TK vector, Promega, South- ampton, UK) was used as transfection control. Enzyme activity assays Aconitase activity was using the Bioxytech Aconitase-340 assay (Oxis International Inc, Beverly Hills, CA). Data are presented as % of activity compared to control. Cells were assayed for Complex I activity using the Mitoprofile Complex I Rapid Elisa Kit (MitoSciences, Eugene, OR, USA) according to manufacturer’s instructions. Data are presented as % of activity compared to control. Lactate and pyruvate measurements Lactate and pyruvate were assayed using kits from Instruchemie (Delfzijl, The Netherlands). MitoSOX staining Mitochondrial superoxide was assayed using MitoSOX Red (Molecular Probes, Eugene, OR, USA). Images were acquired with a confocal microscope (Radiance 2000, Bio-Rad Laborato- PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10345