[Show abstract][Hide abstract] 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-1alpha 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-HRE-luc 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-1alpha protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1alpha protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1alpha 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 factor-mediated induction of HIF-1alpha.
[Show abstract][Hide abstract] ABSTRACT: The hypoxia-inducible factor 1 (HIF-1) transcriptional complex is regulated by cellular oxygen levels and growth factors. The phosphoinosotide 3-kinase (PI-3K)-Akt/protein kinase B (PKB) pathway has been shown to regulate HIF-1 activity in response to oncogenic signals and growth factors. We assessed whether the HDM2 oncoprotein, a direct target of Akt/PKB, could regulate HIF-1alpha expression and HIF-1 activity under normoxic conditions. We found that growth factor stimulation, overexpression of Akt/PKB, or loss of PTEN resulted in enhanced expression of both HIF-1alpha and HDM2. Growth factor-mediated induction of HIF-1alpha was ablated by transient expression of a dominant negative form of Akt/PKB or by treatment with LY294002. Transient expression of HDM2 led to increased expression of HIF-1alpha. Pulse-chase and cycloheximide experiments revealed that HDM2 did not significantly affect the half-life of HIF-1alpha. Growth factor-induced HIF-1alpha and HDM2 proteins were localized to the nucleus, and induction of both proteins was observed in both p53(+/+) and p53(-/-) HCT116 cells to comparable levels. Importantly, insulin-like growth factor 1-induced HIF-1alpha expression was observed in p53-null mouse embryo fibroblasts (MEFs) but was significantly impaired in p53 Mdm2 double-null MEFs, indicating a requirement for Mdm2 in this process. Finally, we showed that phosphorylation at Ser166 in HDM2 contributed in part to growth factor-mediated induction of HIF-1alpha. Our study has important implications for the role of the PI-3K-Akt/PKB-HDM2 pathway in tumor progression and angiogenesis.
Full-text · Article · May 2004 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Metastasis in breast cancer significantly increases morbidity and mortality. The 5-year survival rate reduces from 90% for localised disease to about 20% once metastasis has taken place. The phosphoinositide 3-kinase/Akt signalling pathway has an important role in cell motility, invasion and metastasis. However, the precise contribution of the Akt kinase family members, Akt1, Akt2 and Akt3, in mediating these processes is unclear. The possibility that they have distinct functions in tumour progression is particularly interesting.
Full-text · Article · Feb 2004 · Breast cancer research: BCR