In this study we also conducted an analysis comparing the
In this study, we also conducted an analysis comparing the expression profiling of Hh genes among the sarcomas investigated. In these tumors, the IHH gene showed a higher expression level than PTCH1 gene in RMS and SS samples (p<0.0001 and p=0.0133, respectively) and GLI1 in ES samples (p<0.0001). We observed an altered expression of the Hh genes investigated in RMS, SS and ES. This altered expression suggests an activation of the Hedgehog pathway and its participation in the biogenesis of sarcomas investigated. Several studies have demonstrated the use of Hedgehog pathway inhibitors as an alternative treatment of sarcomas , .
Eichenmüller et al.  used the apoptosis-inducing betulinic ARCA Cy5 EGFP in human RMS cell lines as well as mouse NIH-3T3 fibroblasts. The efficacy of the betulinic acid was determined in RMS cell cultures by measuring cell viability, survival, apoptosis and hedgehog signaling activity. The exposure of hedgehog-activated RMS cells to betulinic acid resulted in a strong decrease in GLI1 and PTCH1 gene expressions. Tostar et al. (2010) investigated eight ERMS samples and five ERMS cell lines and observed a certain HH signaling target genes, including HHIP, PTCH1, SFRP1, and GLI1 genes. Moreover, treatment of the cell lines with cyclopamine and GANT61, an inhibitor of GLI1, reduced cell proliferation in all ERMS cell lines analyzed. Interestingly, GANT61 was more effective, and this was accompanied by increased apoptosis, while cyclopamine promoted necrotic events. Specific knockdown of SMO had no effect on the proliferation of ERMS cells, indicating the presence of an SMO-independent HH signaling pathway in the ERMS cell lines.
Declaration of interest
Acknowledgments This work was supported by awards from the FAPESP (The State of São Paulo Research Foundation: 04/12150-8, 07/53869-3) and GRAACC (Grupo de Apoio ao Adolescente e Criança com Câncer).
Introduction Ewing sarcoma (ES) is a member of a family of tumors that includes ES, peripheral primitive neuroectodermal tumor, and Askin tumor . ES is composed of undifferentiated primitive mesenchymal cells that exhibit features of primitive neuroectodermal differentiation . ES arises most frequently in bones, mainly in flat bones and diaphyses of long bones. Soft tissue ES is less frequent and often arises in deep tissues. ES occurs more frequently in adolescents and young adults and more commonly in whites , . Approximately 15% to 25% of patients present with overt metastasis at the time of diagnosis . ES is categorized as one of the small round blue cell tumors (SRBCTs) and may be challenging to differentiate from other SRBCTs , , . Although immunohistochemical analysis is useful in the workup of ES because most cases are CD99 positive with a membranous pattern, no immunohistochemical markers alone are sufficient to distinguish ES from other SRBCTs. The t(11;22) or t(21;22) translocations result in fusion of the 5′ end of ES breakpoint region 1 (EWSR1) gene with the 3′ end of the erythroblast transformation-specific (ETS) family genes that act as a transcription factor. EWSR1-FLI1 and EWSR1-ERG are 2 of the most common fusions reported in approximately 90% and 5% of ES, respectively. Other rare fusion partners such as ETV1 (7q22) , ETV4 (17q12), E1AF (17q12), and FEV (2q33) are reported in approximately 5% of the patients with ES . However, EWSR1 is rearranged in other soft tissue sarcomas such as EWSR1-WT1 in desmoplastic small round cell tumor, EWSR1-ATF1 in soft tissue–clear cell sarcoma, and EWSR1-CHN in extraskeletal myxoid chondrosarcoma . Therefore, a positive EWSR1 rearrangement detected by fluorescence in situ hybridization (FISH) is insufficient to make a definitive diagnosis. Molecular-based testing such as reverse transcriptase polymerase chain reaction (RT-PCR) ,  plays an additional important role for diagnostic purpose, by identifying the specific fusion gene partner .