Expression and stability of hypoxia inducible factor 1α in osteosarcoma

Abstract

Hypoxia contributes to both physiological and pathological processes and its effects are mainly mediated through the transcription factors hypoxia-inducible factor 1α and 2α (HIF1α and HIF2α). The purpose of this study was to examine the role of these proteins in osteosarcoma progression. We developed a method to isolate primary human osteoblast cell lines. HIF1α and HIF2α expression were then compared in osteoblast and osteosarcoma cell lines under 21% oxygen (normoxia) and 1% oxygen (hypoxia). We also used hypoxia-responsive element (HRE)-driven reporter constructs in conjunction with siRNAs specific to HIF1α or HIF2α to determine the contribution of each protein to HRE-mediated transcription. Finally, we measured HIF1α expression in primary osteosarcoma tumors by immunohistochemistry. We found that mainly HIF1α transcript was significantly higher in osteosarcoma cell lines compared to normal osteoblasts under both normoxia and hypoxia. At the protein level, HIF1α was preferentially stabilized in osteosarcoma cell lines under both conditions. HIF1α expression was required for the observed increases in HRE activity. Finally, nuclear or nucleocytoplasmic HIF1α staining in osteosarcoma cases was associated with high-grade tumors. These findings point to a role for HIF1α in osteosarcoma progression and suggest that the observed differences in HIF1α oxygen dependent degradation may play an important pathophysiological role in this disease. Pediatr Blood Cancer 2012; 59: 1215-1222. © 2012 Wiley Periodicals, Inc.