Abstract 5505: A genome-wide siRNA screen for novel regulators of HIF-1α activity in normoxia and hypoxia

The ability to survive hypoxic stress is an essential component of tumor development. Many rapidly proliferating solid tumors outgrow their vasculature while the abnormal tumor vasculature leads to small, transient pockets of hypoxic cells. In addition to being deprived of oxygen, these cells are also deprived of nutrients and unable to export harmful byproducts of cellular metabolism out of their immediate environment. One mechanism cancer cells employ to survive hypoxic stress is the stabilization and activation of the transcription factor hypoxia inducible factor 1α (HIF-1α), which translocates to the nucleus and induces pro-survival genes such as VEGF, Glut-1, and glycolytic enzymes. MiaPaCa-2 pancreatic carcinoma cells stably transfected with a 5X hypoxia response element (HRE) promoter regulating the firefly luciferase gene were generated, and shown to express luciferase only when HIF-1α was stabilized in hypoxia(1% O 2 ). Using these cells, a genome wide siRNA screen was performed using the Dharmacon 22,000 gene siRNA library under the conditions of normoxia, 16hr and 72 hr hypoxia, to discover novel effectors of the HIF-1α pathway. Luciferase expression was corrected for cell number using a WST-1 cell proliferation assay prior to luciferase detection. Several hits were identified including genes which, when silenced, both increased and decreased HIF-1α transcriptional activity. Genes whose inhibition increased HIF-1α transcriptional activity in normoxia, in some cases up to 19 fold, include members of the E3 ubiquitin ligase complex which bind to and degrade HIF-1α under normal oxygen tensions, and also a number of other genes with uncharacterized function. There were also genes whose inhibition decreased hypoxia induced HIF-1α activity. Many were not well characterized, but there were a number of genes belonging to the MAP kinase pathway; the DNA repair pathway; the NF-κB pathway; and glycolysis genes, that were identified as necessary for HIF-1α transcriptional activity. Hits in these pathways were validated by secondary siRNAs and Western blots. It was discovered that silencing key genes involved in glycolysis inhibited HIF-1α transcriptional activity due to a decrease in HIF-1α protein levels. They include hexokinase-1, pyruvate dependent kinases-1,2 and 3, and 6-phosphofructo-1-kinase(M). These results suggest that increased glycolysis found in many tumors under aerobic conditions (the Warburg effect), which has been suggested to be caused by increased Myc expression, mutant Ras, mutant p53 and PI-3-kinase signaling, has an important role in increasing HIF-1α expression and activity. This stabilization and increase in activity then further induces the expression of target glycolytic genes and glycolytic activity in a feed-forward loop. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5505.