is a Hedgehog independent phenomenon in ESFT, suggesting non-canonical mechanism of pathway activation. Finally, in multiple ESFT cell lines, we demonstrate that several loci known to be transcriptionally modulated by EWS/FLI1 are dependent upon GLI1 expression. This establishes GLI1 as a higher order target in the EWS/FLI1 signaling network and begins to define a hierarchy in the EWS/FLI1 signaling network. Results Primary tumors demonstrate significant GLI1 expression Our earlier findings focused on EWS/FLI1 activation of GLI1 in an NIH3T3 model transformation system[4] with added data from ESFT cell lines. However, HH-GLI pathway activity has been found to be diminished in in vitro cultured medulloblastoma lines[12], so the cell lines we evaluated may not reflect the condition in primary ESFT. To see how well these findings apply to clinical disease, we evaluated the status of a panel of 12 ESFT primary tumor specimens. As is illustrated in Figure 1, the expression of mediators of the HH-GLI pathway closely resembles that found in EWS/FLI1 expressing NIH3T3 cells. The most characteristic indicators of oncogenic signaling via this pathway are the expression levels of GLI1, GLI2 and the direct GLI1 target Patched1. These are important components of what has been termed the GLI code[13]. In these twelve ESFT specimens, we found expression levels of these pathway mediators to be similar or higher than those in specimens from cell lines known to be in the upper quartile for expression in microarray data obtained for the NCI-60 panel of tumor cell lines (Novartis, http://wombat.gnf. org). Our earlier data from NIH3T3 indicated little or no expression of Sonic Hedgehog (SHH)[4], which mediates pathway activity in development and in some tumor systems. Once again, our primary tumor specimens demonstrate very little to no expression of SHH transcript when compared to a positive control expressing SHH cDNA. Similar low levels of Indian Hedgehog GLI1 in Ewing Tumors (IHH) were also found (data not shown). Some of the variability of expression of GLI pathway members from sample to sample could reflect variable tumor cell content in the primary specimens. All specimens showed either an EWS/FLI1 or an EWS/ERG transcript by RT-PCR, indicating at least some ESFT tumor cell content. However the percentage of the specimen which was tumor cells could not be assessed. Another source of variability could relate to some of the other signaling pathways such as PI3- AKT, RAS-MEK1, and PKC-delta which have been shown to affect signaling in the HH-GLI1 pathway[14]. Nevertheless, overall these findings indicate that primary ESFT demonstrate significant expression of HH-GLI1 mediators and appear to demonstrate little expression of Hedgehog species. Multiple means of inhibition demonstrate GLI1 to be EWS/FLI1 dependent in ESFT Our earlier findings demonstrated that GLI1 expression is dependent on EWS/FLI1 expression in one set of experimental conditions using in vivo EWS/FLI1 siRNA. If GLI1 upregulation by EWS/FLI1 is of biologic importance to ESFT, we would expect to find such a relationship applies to multiple means of targeting EWS/FLI1 and in multiple ESFT cell lines. Since off target effects can produce spurious results in RNAi experiments, we assessed the relationship between EWS/FLI1 and GLI1 activity in ESFT cell lines using several shRNA targeting sequences. In all cases, polyclonal cell lines were derived using lentivirally delivered shRNA followed by rapid selection in puromycin. As is seen in Figure 2A, the degree of EWS/FLI1 knockdown of expression mirrors the reduction in GLI1 and of Patched1 transcript. This close relationship is highly unlikely to have been produced by an off target effect. It also establishes Patched1 as a target of transcrip- tional modulation by EWS/FLI1 in ESFT cell lines. Furthermore, similar polyclonal shRNA experiments have been shown to yield reductions of GLI1 and of Patched1 in both A673 Figure 1. Expression of HH-GLI pathway species in primary ESFT specimens. Twelve CHTN banked tumor specimens are assessed for HH- GLI1 related gene expression by real time quantitative PCR. Also included is TTC475 in both primary tumor specimen and cell line form. For comparison, two Rhabdomyosarcoma (RMS-R and -T) and an Osteosarcoma (Saos) cell line are included. SaOS2 is in the upper quartile for GLI1 expression in NCI60 microarray data. For reference, TC71 cells transduced with SHH or empty retroviral vector are included in the SHH panel. doi:10.1371/journal.pone.0007608.g001 PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7608