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Background: Ewing's sarcoma (ES) is the second most common bone or soft-tissue sarcoma in childhood and adolescence and features a high propensity to metastasize. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is a membrane-bound mesenchymal stem cell marker highly expressed in ES. Here, we investigated the role of STEAP1 as an immunohistological marker for outcome prediction in patients with ES., Patients and Methods: Membranous STEAP1 immunoreactivity was analyzed using immunohistochemistry in 114 primary pre-chemotherapy ES of patients diagnosed from 1983 to 2010 and compared with clinical parameters and patient outcome. Median follow-up was 3.85 years (range 0.43-17.51)., Results: A total of 62.3% of the ES samples displayed detectable STEAP1 expression with predominant localization of the protein at the plasma membrane. High membranous STEAP1 immunoreactivity was found in 53.5%, which correlated with better overall survival (P=0.021). Accordingly, no or low membranous STEAP1 expression was identified as an independent risk factor in multivariate analysis (hazard ratio 2.65, P=0.036)., Conclusion: High membranous STEAP1 expression predicts improved outcome and may help to define a specific subgroup of ES patients, who might benefit from adapted therapy regimens.

Ewing sarcoma is a malignant bone and soft tissue tumor of children and young adults, which is known to be highly aggressive and invasive. It expresses specific chimeric genes (EWS-FLI-1, EWS-ERG, EWS-ETV1, and EWS-E1AF), the 3' portions of which are all members of the ETS family. ETS-related proteins, such as FLI-1, ERG, and E1AF, transactivate the promoters of matrix metalloproteinase (MMP) genes, which play important roles in the processes of invasion and metastasis. Therefore, we hypothesize that the Ewing sarcoma-specific chimeric genes also transactivate the MMP genes, contributing to the tumor's invasiveness and propensity for metastasis. To verify this hypothesis, we investigated the expression of MMPs in eight Ewing sarcoma cell lines. Surprisingly, MMP-1 and MMP-3 were not expressed at all in any of the cell lines. MMP-9 was expressed in four out of the eight cell lines, and MMP-2 and MT1-MMP in all of the cell lines. Ewing sarcoma-specific chimeric genes have been shown to transactivate the promoter of the MMP-1 gene by the reporter assay, and bind to the putative recognition sites in the MMP regulatory elements by the gel shift assay. However, an in vivo formaldehyde cross-linking study revealed that the chimeric protein did not bind to the predicted ETS recognition sites in the regulatory elements of the MMPs. These results indicate that the absence of the MMP expression in the tumor cells is at least in part due to the loss of accessibility of the ETS recognition sites in the regulatory elements of the MMP genes. Therefore, we should be careful before theorizing simply that a putative binding site is essential for the transcription of critical genes, since the binding of this fusion protein was found to be modulated in tumor cells in this study.

Fas ligand (FasL) exists in transmembrane and soluble forms and induces apoptosis on cross-linking with the Fas receptor. We evaluated the biological significance of FasL and Fas in 61 tumor tissues and 9 cell lines of the Ewing's sarcoma family of tumors (ESFT). FasL was present in 62.5% and Fas in 79.4% of primary ESFT. Metastatic tumors had higher expression of FasL (95%), suggesting association with a metastatic phenotype. FasL was detected in the cytoplasm and membrane of ESFT cells by immunofluorescence. Western blotting revealed transmembrane and soluble FasL in cytosolic extracts and soluble FasL in conditioned media. Both transmembrane and soluble FasL induced apoptosis of Fas-sensitive Jurkat cells in co-culture experiments with ESFT cells or their media. Treatment with phenanthroline and the synthetic metalloproteinase inhibitor BB-3103 reduced the levels of soluble FasL in the media, suggesting that in ESFT, FasL is processed by a metalloproteinase and released in the extracellular milieu. The released soluble FasL may serve to attack cells of the immune system and/or interfere with the binding of transmembrane FasL with Fas, and results in down-regulation of transmembrane FasL. Synthetic metalloproteinase inhibitors may modify the ratio of transmembrane to soluble FasL.