Regulation of hypoxic response by HIF isomerization

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Project Summary When a tumor becomes large, hypovascularized and hypoxic regions are formed inside the tumor. The cancer cells in the regions become resistant to therapy due to poor delivery of chemotherapy drugs and a lack of reactive oxygen species after irradiation. In addition, the cancer cells upregulate transcription factors of the hypoxia- inducible factor (HIF) family as master regulators of their response to hypoxia. The HIF-1 protein, one of the best characterized HIF subunits, is stabilized by hypoxia and forms a dimer with HIF-1 to activate more than 2,500 genes. The target genes are involved in angiogenesis, glycolysis, and growth factor signaling, which collectively facilitate cancer progression and metastasis. Indeed, increased levels of HIF indicate a poor prognosis for cancer patients. Thus, it is important to understand the functions and regulation of HIFs to improve cancer therapy. The principal investigator’s group recently found that some peptide bonds preceding prolines are cis-trans isomerized by one of the FKBP family of peptidyl prolyl isomerases. This isomerization stabilizes HIF-1 by preventing ubiquitin-mediated degradation. Based on these findings, the group hypothesized that the isomerization inhibits HIF-1 phosphorylation of serine or threonine near the prolines that is mediated by GSK3, which is a major step for degradation. To test this hypothesis, they proposed the following three aims. In Aim 1, they will use in vitro peptide assays and cell transfection assays to study how isomerization regulates phosphorylation. In Aim 2, cell biological consequences of FKBP depletion and HIF-1 depletion will be compared. This includes cell proliferation assays, PCR of HIF-target genes, cell migration assays, and angiogenesis assays. In Aim 3, transcription activation of the FKBP by HIF-1 will be studied with ChIP-seq, luciferase reporter assays and electrophoretic mobility shift assays. This aim will determine a positive feedback loop between FKBP and HIF-1. These studies are expected to unravel a novel molecular mechanism regulating HIF-1 stability in hypoxia and potential drug targets for cancer therapy.