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

Abstract

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.

Figure 1

Expression of HIF in Ewing's sarcoma. (a) HIF-1α and (b) HIF-2α expression in Ewing's tumour cells; scale bar = 50 μm. Expression of HIF-1α (c) co-localises with that for cleaved caspase 3 (d); scale bar = 100 μm.

Figure 2

HIF induction in ES and OS cell lines. Western blots showing induction of HIF-1α (120 kDa), HIF-2α (120 kDa) and Glut-1 (50 kDa) in a panel of ES (A673, RD-ES, SK-ES-1, SK-N-MC, TC-71) and OS (MG-63, Saos2, G292, 143B) cell lines in response to 24 h (a) hypoxia (H; 0.1% O₂) or (b) low glucose (G; over-exposed in comparison to hypoxia blots) compared with the untreated/normoxic (N) control. (c) Activation of the HRE-luciferase reporter construct and (d) secretion of VEGF in response to hypoxia (light grey bars) or low glucose (dark grey bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 3

HIF siRNA in ES and OS cell lines. (a) Western blots showing expression of HIF-1α (120 kDa), HIF-2α (120 kDa) and Glut-1 (50 kDa) in response to siRNA targeting HIF-1α (H1), HIF-2α (H2), HIF-1α and HIF-2α (H12) or control siRNA (scr) in ES (SK-N-MC) and OS (143B) cells. Effects of HIF siRNA on (b) secretion of VEGF and (c) activation of the HRE-luciferase reporter construct and in response to hypoxia or low glucose in ES and OS cell lines. Bar graph data is normalised to the siRNA mock control (-) for each condition and represents pooled data from the entire panel of ES or OS cell lines. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus scr siRNA control. (d) Secretion of TGFα from ES cell lines in response to hypoxia (light grey bars) or low glucose (dark grey bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4

Phenotypic effects of hypoxia and low glucose. Effect of hypoxia (light grey bars) or low glucose (dark grey bars) on (a) cell number and (b) apoptosis in ES and OS cell lines and (c) mitotic index in OS cell lines. Bar graph data is normalised to the normoxic control (white bars) for each condition: *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. (d) Proliferation assessed in ES cell lines by Western blot for Ki-67. N, normoxia; H, hypoxia; G, low glucose. Numerical values represent mean fold change in Ki-67 as assessed by densitometry:*, p < 0.05; **, p < 0.01 versus normoxia.

Figure 5

Phenotypic effects of HIF siRNA. Effect of HIF siRNA on (a) cell number, (b) apoptosis and (c, d) proliferation under hypoxia in ES and OS cell lines. Bar graph data is normalised to the relevant scrambled siRNA control (white bars): *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. (c) Proliferation assessed in ES cell lines by Western blot for Ki-67. N, normoxia; H, hypoxia. Numerical values represent mean fold change in Ki-67 as assessed by densitometry:*, p < 0.05; **, p < 0.01 versus hypoxic scrambled (scr) control.

Figure 6

Effect of hypoxia on migration of ES and OS cell lines. (a) Fraction closure of a scratch wound due to cell migration during 18 h normoxia (white bars) or hypoxia (grey bars). (b) Effect of siRNA targeting HIF-1α (H1) or HIF-2α (H2) on migration of ES (combined data from A673, SK-ES-1 and SK-N-MC) or OS (combined data from MG63, Saos2 and 143B) cell lines under hypoxia. N, normoxia; H, hypoxia. Bar graph data is normalised to the (a) normoxic or (b) scrambled hypoxic siRNA control: *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.