Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

Background: Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. Methods: HIF-1 alpha and HIF-2 alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. Results: 17/56 Ewing's tumours were HIF-1 alpha-positive, 15 HIF-2 alpha-positive and 10 positive for HIF-1 alpha and HIF-2 alpha. Expression of HIF-1 alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1 alpha and HIF-2 alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2 alpha in Ewing's. Downstream transcription was HIF-1 alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Conclusions: Co-localisation of HIF-1 alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.