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-1alpha and HIF-2alpha 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-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.


CONCLUSIONS
Co-localisation of HIF-1alpha 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.
Knowles
Helen J
HJ
Botnar Research Centre, University of Oxford, Nuffield Orthopaedic Centre, Oxford, OX3 7LD, UK. helen.knowles@ndorms.ox.ac.uk
Schaefer
Karl-Ludwig
KL
Dirksen
Uta
U
Athanasou
Nicholas A
NA
D017209:Apoptosis; D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human