GLI1 in Ewing Tumors Figure 5. EWS/FLI1 and Gli1 targets overlap in ESFT. Panel A. EWS/FLI1 antisense (see Figure 1C) demonstrates that NKX2.2 and GAS1 are transcriptionally upregulated by EWS/FLI1 in TC71 and TC32. Panel B. GLI1 overexpression in TC32 shows that EWS/FLI1 targets PTCH, NKX2.2 and GAS1 are transcriptionally modulated by GLI1 expression in a Ewing cell background. Panel C. GLI1 shRNA reduces GLI1 expression and also reduces expression of these EWS/FLI1 targets. Panel D. GANT58 (10 mcM), an inhibitor of GLI transcriptional activation, results in transcriptional downregulation of our three putative downstream targets of GLI1. doi:10.1371/journal.pone.0007608.g005 hierarchy can be of tremendous benefit in the design of new therapies. Certainly, targeting an EWS/FLI1 mediator which is more upstream, such as GLI1, could be expected to have more extensive effects than targeting a gene further downstream in the process. Our work with the GLI1 inhibitor GANT58 suggests that this pathway may be a valid target for translational research in Ewing Sarcoma. A secondary benefit of employing GLI1 inhibition in Ewing tumors is that GLI1, unlike EWS/FLI1, is PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7608