Targeting PI3K/Akt represses Hypoxia inducible factor-1α activation and sensitizes Rhabdomyosarcoma and Ewing's sarcoma cells for apoptosis

Background: Hypoxia inducible factor-1 alpha (HIF-1 alpha) has been identified as an important novel target in apoptosis resistance of pediatric tumors such as Rhabdomyosarcoma (RMS) and Ewing's sarcoma (ES). Evidence suggests that PI3K/Akt signaling plays a role in regulation of HIF-1 alpha activation as well as apoptosis resistance in various adult tumors. However the relevance of PI3K/Akt signaling in HIF-1b alpha activation and apoptosis resistance in childhood tumors has not been addressed yet. Thus, this study was to investigate whether PI3K/Akt signaling is involved in hypoxia induced activation of HIF-1 alpha as well as in resistance to hypoxia-induced apoptosis in childhood tumors such as RMS and ES. Methods: Constitutive activation of PI3K/Akt signaling was analyzed by Western blotting. Hypoxic activation of HIF-1 alpha was determined by Western Blot analysis and electrophoretic mobility shift assay. Apoptosis was determined by flow cytometric analysis of the propidium iodine stained nuclei of cells treated with PI3K inhibitor LY294002 in combination with either TNF-related apoptosis-inducing ligand (TRAIL) or doxorubicin. Results: This study demonstrated that PI3K/Akt signaling was constitutively activated in RMS and ES cell lines, A204 and A673, respectively. Targeting PI3K/Akt signaling by the inhibitor LY294002 (30 mu M) significantly decreased the protein expression as well as DNA binding activity of HIF-1 alpha and restored the apoptosis-inducing ability of cells in hypoxia Additionally, pretreatment with LY294002 sensitized A204 and A673 cells to TRAIL or doxorubicin induced apoptosis under hypoxia. Conclusion: These results suggest that the constitutively active PI3K/Akt signaling contributes to hypoxic activation of HIF-1 alpha as well as HIF1 alpha-mediated apoptosis resistance in RMS and ES cells under hypoxia.