Hypoxia is a hallmark of the tumor microenvironment and contributes to the failure of conventional cancer treatments. My previous work identified the first hypoxia-induced miRNA, miR-210, which plays important roles in the adaptive process to low oxygen tension. miR-210 is a direct target of the hypoxia inducible factor (HIF) and has high clinical relevance, being associated with negative prognosis in several tumor types, including breast cancer. Our recent data suggest that miR-210 is a central regulator of tumor metabolism, by repressing genes involved in mitochondrial respiration. Following up on this early insight, we are proposing a detailed study of miR-210's role in shifting the balance between mitochondrial respiration and glycolysis. The central hypothesis of our proposal is that miR-210 inhibits mitochondrial respiration and stimulates glycolysis. The hypothesis will be tested in breast cancer context, by pursuing the following specific aims:
AIM 1 : To investigate the role of miR-210 in the glycolytic metabolism. Our recent data indicate that miR- 210 downregulates electron transport at the level of mitochondrial complex I and II and suppresses Krebs cycle activity. We also preliminarily demonstrate that miR-210 increases production of reactive oxygen species and succinate, thus inducing HIF and its glycolytic targets. To directly investigate the role miR-210 in stimulating the glycolytic flux, we will take a combined approach, involving in vivo metabolic imaging, NMR-based metabolomic analyses and enzymatic assays. The studies will be performed in vitro and on breast cancer xenografts.
AIM 2 : To examine the role of miR-210 targets in the glycolytic shift and tumor growth. Screens for genes that respond to miR-210 in normoxia and hypoxia identified targets with functions in the mitochondrial electron transport: ISCU (Iron Sulfur Cluster Scaffold Homolog), SDHD (succinate dehydrogenase subunit D) and NDUFA4 (subunit of the NADH-ubiquinone oxidoreductase complex). These genes will be rescued from miR-210's control, using shRNA and overexpression approaches, and the resulting effects on glycolysis and on tumor growth will be determined.
AIM 3 : To examine the impact of miR-210 on the effect of drugs acting at the level of glycolysis. Based on the preliminary data and on the rationale described in Aims 1 and 2, we hypothesize that by increasing the dependence of cancer cells on the glycolytic pathway, miR-210 increases the effect of experimental antitumor agents that block glycolytic enzymes and reactivate mitochondrial respiration. In particular, we will concentrate on the response of breast cancer cells with altered levels of miR-210 to 2-Deoxyglucose and Dichloroacetate, (inhibitors of hexokinase and pyruvate dehydrogenase kinase, respectively). The effects will be studied in normoxic and hypoxic cultures and in orthotopic breast cancer xenografts.
We propose the first systematic studies of miR-210 as a link between hypoxia and energy metabolism in tumors. We investigate how hypoxia-induced miR-210 contributes to glucose utilization by the tumors and how it affects the response to specific antineoplastic agents. We propose a highly integrated approach, combining metabolic assays, in vivo imaging, and novel animal models to answer these important questions.
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