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Ewing’s sarcoma is the second most common malignant bone tumor occurring in children and adolescents. It is a small round-cell tumor of neuroectodermal origin belonging to the Ewing’s family tumors (EFTs), which also include related peripheral Primitive Neuroectodermal Tumor (pNET). Eighty three percent of Ewing’s sarcoma carry a t(11;22)(q24;q12) chromosome translocation leading to the fusion of a portion of the EWS gene to a gene of the ETS family of transcription factors, FLI1. Downstream targets of EWS/FLI1 were investigated and manic fringe (MNFG), a member of the Fringe gene family, was identified as an upregulated target gene. Furthermore, since MNFG action is mediated through the Notch signaling pathway, this suggests that MNFG may influence normal Notch receptor function and contribute to the pathogenesis of Ewing’s sarcoma. Interestingly, Notch signaling plays an important role in neuroectodermal development. In order to investigate the involvement of Notch signaling in Ewing’s sarcoma, we generated tetracycline regulated GFP-tagged expression constructs with a full length Notch-1 receptor (FL-N1), a dominant negative form of the Notch-1 receptor (DN-N1) and a constitutively active Notch-1 receptor (A-N1). They were transfected along with the tet-off expression vector into two Ewing’s cell lines, HTB-86 and HTB-166. These cells were expanded in culture and injected subcutaneously into 4 week-old nude BALB/C mice. Mice were then observed for tumor growth for 45 days or until tumor size had reached a maximum of 20 mm, after which they were sacrificed. Tumors were excised and a portion of each was used for protein and RNA extraction, as well as for histology. Cells were also grown for apoptosis and proliferation studies. Tumors expressing the constitutively active Notch (A-N1) grew larger in size compared to controls. A larger size tumor was seen in mice expressing the dominant negative Notch (DN-N1). The change in tumor size correlated with a change in the apoptosis rate. It appears that a delicate balance of Notch-1 levels is maintained in Ewing’s sarcoma, as a deregulation of Notch signaling alters the apoptosis rate and tumor cell growth.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]