Vascular endothelial growth factor expression is up-regulated by EWS-ETS oncoproteins and Sp1 and may represent an independent predictor of survival in Ewing's sarcoma

Purpose: Tumor markers ideally allow monitoring and prediction of disease progression. In Ewing's sarcoma, a devastating childhood cancer, only a few reliable prognostic markers have been identified. To this end, we analyzed the expression of four tumor-promoting proteins, cyclin D1, HER2/Neu, Mdm2, and vascular endothelial growth factor (VEGF), in Ewing's sarcoma. Experimental Design and Results: Thirty-one tissue samples from patients with Ewing's sarcoma were stained with antibodies against cyclin D1, HER2/Neu, Mdm2, or VEGF. Whereas no significant expression of HER2/Neu and Mdm2 was detected, positive cyclin D1 and VEGF staining was observed in 42% and 55% of all tumors, respectively. Importantly, VEGF expression was found to be an independent negative predictor of survival in Ewing's sarcoma patients, whereas cyclin D1 expression did not correlate with survival in these patients. Consistently, the Ewing's sarcoma-specific EWS-ETS oncoproteins were capable of activating both the cyclin D1 and VEGF promoters in transient transfections of tissue culture cells. Furthermore, this activation was enhanced by coexpression of the Sp1 transcription factor. Using a mammalian two-hybrid system, some evidence was obtained that this may involve a physical interaction between EWS-ETS and Sp1 proteins. Conclusions: Our data reveal that VEGF may serve as a prognostic marker in Ewing's sarcoma patients and provide a molecular mechanism by which VEGF and cyclin D1 expression is up-regulated in approximately half of all Ewing's sarcomas.