Identification of Novel Small Molecule Inhibitors of Hypoxia-Inducible Factor-1 That Differentially Block Hypoxia-Inducible Factor-1 Activity and Hypoxia-Inducible Factor-1α Induction in Response to Hypoxic Stress and Growth Factors

Abstract

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1A subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HREluc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1-dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1A protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1A protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1A protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factormediated induction of HIF-1A. (Cancer Res 2005; 65(11): 4918-28)

Selective inhibition of MEK1/2 reveals a differential requirement for ERK1/2 signalling in the regulation of HIF-1 in response to hypoxia and IGF-1

“…U2OS cells stably expressing either the luciferase reporter construct pGL-HREluc (U2OS-HRE-luc) or the pGL3 luciferase control construct (U2OS-luc control) were used. These cell lines have been described previously (Chau et al, 2005). We found that transient overexpression of WT ERK1 clearly enhanced luciferase activity in U2OS-HREluc cells (Figure 1a) without affecting HIF-1a protein levels induced in response to both hypoxia and the hypoxia mimetic agent, deferoxamine mesylate (DFX) (Figure 1b and data not shown).…”

“…repeat fused to the luciferase gene (Ba´rdos et al, 2004;Chau et al, 2005). Twenty-four hours after transfection, the cells were treated with either PD98059 or PD184352 30 min before hypoxia or DFX treatment.…”

“…Twenty-four hours after transfection, the cells were treated with either PD98059 or PD184352 30 min before hypoxia or DFX treatment. Alternatively, U2OS-HRE-luc or U2OS-luc control cells were used (Chau et al, 2005). Hypoxia induced a two to three fold increase in luciferase activity in MCF-7 cells when compared with normoxia ( Figure 4a) whereas DFX induced a 20-to 35-fold increase in luciferase activity ( Figure 4a).…”

“…The RCC4 cells were a kind gift from Professor Patrick Maxwell (Imperial College, London, UK) and have been described previously (Maxwell et al, 1999). The U2OS-HRE-luc and USOS-luc control cells have been described previously (Chau et al, 2005). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL), containing 10% foetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 mg), and L-glutamine (2 mM).…”

Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer

“…This might complicate translating our knowledge into an effective treatment. Although HIF, a downstream target of some PHDs, is a very attractive target for treating cancer in general (Hewitson and Schofield, 2004) and several inhibitors have already been tested in preclinical trials (Kung et al, 2004;Welsh et al, 2004;Chau et al, 2005), HIF may only play a supportive rather than a definitive role in the formation or maintenance of these tumours. The role of PHD3 in apoptosis has not been fully elucidated and so downstream candidates for therapeutic targeting have not been identified.…”