Identification of a Novel Small Molecule HIF-1α Translation Inhibitor

Abstract

Purpose: Hypoxia inducible factor-1 (HIF-1), the central mediator of the cellular response to low oxygen, functions as a transcription factor for a broad range of genes that provide adaptive responses to oxygen deprivation. HIF-1 is overexpressed in cancer and has become an important therapeutic target in solid tumors. In this study, a novel HIF-1α inhibitor was identified and its molecular mechanism was investigated. Experimental Design: Using a HIF-responsive reporter cell-based assay, a 10,000-member natural product-like chemical compound library was screened to identify novel HIF-1 inhibitors. This led us to discover KC7F2, a lead compound with a central structure of cystamine. The effects of KC7F2 on HIF-1 transcription, translation, and protein degradation processes were analyzed. Results: KC7F2 markedly inhibited HIF-mediated transcription in cells derived from different tumor types, including glioma, breast, and prostate cancers, and exhibited enhanced cytotoxicity under hypoxia. KC7F2 prevented the activation of HIF-target genes such as carbonic anhydrase IX, matrix metalloproteinase 2 (MMP2), endothelin 1, and enolase 1. An investigation into the mechanism of action of KC7F2 showed that it worked through the down-regulation of HIF-1α protein synthesis, an effect accompanied by the suppression of the phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 and p70 S6 kinase, key regulators of HIF-1α protein synthesis. Conclusion: These results show that KC7F2 is a potent HIF-1 pathway inhibitor and its potential as a cancer therapy agent warrants further study. (Clin Cancer Res 2009;15 (19):6128-36) Hypoxia, a reduction in partial oxygen pressure, is a major hindrance to effective solid tumor therapy. The microenvironment of rapidly growing solid tumors shows increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia (1). The hypoxic fraction of tumors is resistant to traditional therapies. Radiotherapy is compromised because of the reduced reaction of oxygen with radiationinduced DNA free radicals (2). Chemotherapy is hampered by the diffusion-limited drug delivery to hypoxic regions from distant vasculature. Moreover, many anticancer drugs are most effective against rapidly proliferating cells, and hypoxia (and deficiencies in other nutrients such as glucose) can cause a reduction in cell proliferation rate (3). This is compounded by the induction of the multidrug resistance (MDR1) gene product P-glycoprotein in hypoxic tissue (4), further reducing drug efficacy. Hypoxic tumor regions also impede immune responses, and may promote the growth of cancer stem cells (5,6).Hypoxia drives malignant tumor progression. Tumor hypoxia increases malignant progression and metastasis by promoting angiogenesis through the induction of proangiogenic proteins such as vascular endothelial growth factor (VEGF) and metabolic adaptation through elevation of glycolytic enzymes (7,8). Hypoxia also generates selective pressure for cells to acquir...

MDM2 Regulates Hypoxic Hypoxia-inducible Factor 1α Stability in an E3 Ligase, Proteasome, and PTEN-Phosphatidylinositol 3-Kinase-AKT-dependent Manner

“…The LN229-HRE-AP reporter cell line for HIF transcriptional activity was created by stably transfecting LN229 cells with the pACN188 plasmid, which contains an alkaline phosphatase gene driven by six hypoxia response elements (HREs) derived from the VEGF promoter (36). PRK5-HA-MDM2 plasmid is a kind gift from Dr. David Schlaepfer.…”

Hypoxia-inducible TAp73 supports tumorigenesis by regulating the angiogenic transcriptome

“…2d). Furthermore, addition of KC7F2, which selectively suppresses cellular HIF-1α synthesis 25 , or chetomin (CHT), which blocks the interaction between HIF-1α and p300/CREB, and hence affects HIF-1α-mediated transcriptional activity 26 -though having nonspecific effects on baseline TAp73 levels-inhibited the upregulation of TAp73β following hypoxia or DFX treatment respectively, and the concomitant induction of glut-1 (Fig. 2e,f), together confirming the requirement of HIF-1α and its transactivation potential in regulating TAp73 abundance.…”

4′,6-Dihydroxy-4-methoxyisoaurone Inhibits the HIF-1α Pathway Through Inhibition of Akt/mTOR/p70S6K/4E-BP1 Phosphorylation

“…Because PI3K-Akt-mTOR-p70S6K-4E-BP1 pathways have been implicated in the regulation of HIF-1a protein synthesis at the translational level (10,17,26), we tested whether ISOA affects the phosphorylation of these translation initiation factors. As shown in Fig.…”

“…HIF-1 is a heterodimeric transcriptional factor composed of a and b subunits (8,9). The HIF-1a subunit is highly regulated by oxygen concentration and serves as a marker of hypoxia (10). In normoxic conditions, specific HIF-1a prolyl hydroxylase hydroxylates two proline residues (P402 and P564) within the oxygen-dependent degradation domain of HIF-1a, resulting in HIF-1a recognition by Von HippelLindau (VHL) ubiquitin ligase complex that targets HIF-1a for degradation through the 26S proteasome pathway (11,12).…”