block the pathway at this level [22,23,24,25]. As is shown in Figure 4C, treatment of ESFT cells with FSK results in diminished expression of GLI1 transcript. As a consequence, anchorage independent growth is inhibited. This further suggests that GLI1 is important to ESFT biology and that it is activated downstream of Smoothened. Finally, GANT58 (NSC75503) has been shown to inhibit transcriptional activation by GLI1 (as well as by the other GLI species) [9]. We exposed A673 cells to increasing concentrations of GANT58 and found a dramatic reduction in anchorage independent growth with concentrations as low as 5 mcM. This further supports a mechanism of activation which is downstream of Smoothened and is consistent with the recent observation of direct transcriptional activation of GLI1 by EWS/FLI1[5]. Some GLI1 and EWS/FLI1 transcriptional targets overlap GLI1 is an oncogenic transcription factor. If GLI1 is more than a peripheral event in ESFT biology, one might expect it to play a significant role in the transcriptional profile of EWS/FLI1 in ESFT. To evaluate this possibility, we assessed a published set of EWS/FLI1 targets[26]. Generated in A673 cells, this data set identified 31 genes upregulated by EWS/FLI1 as assessed by two separate shRNA hairpin sequences combined with data on restored expression of EWS/FLI1. In this target list, one finds GLI1. Also on this list are NKX2.2 and GAS1, two loci known to be affected by HH-GLI signaling. NKX2.2 is a Class II HH target in the developing neural tube[27]. Its expression is induced in neural ectoderm subsequent to SHH release by the notochord. More recently, it has been shown to be directly targeted by GLI1[28], much like Patched1. GAS1 is more indirectly involved as part of a feedback network of SHH binding factors. An enhancer of HH-GLI signaling, its expression is modulated based on the signaling conditions of the target tissue. Under conditions of low HH stimulation, GAS1 expression is enhanced; while in conditions of strong SHH signaling, its expression is downregu- lated[29]. Our own data indicate that the GLI1 target Patched1 is also transcriptionally upregulated by EWS/FLI1 in multiple ESFT lines (see Figure 2). The finding that several HH-GLI1 targets have also been identified EWS/FLI1 targets suggests a model in which EWS/FLI1 reaches these targets through GLI1. To test this hypothesis, we first sought to demonstrate that NKX2.2 and GAS1 were also EWS/FLI1 modulated targets in TC32 and TC71, as has been shown in A673[26]. Figure 5A demonstrates that both are indeed transcriptionally upregulated by EWS/FLI1, based on data from cells transduced with EWS/FLI1 antisense. Next, we tested whether these EWS/FLI1-modulated targets are also GLI1 responsive in an ESFT background. Since TC32 cells have the lowest level of GLI1 expression of common ESFT cell lines[4], we overexpressed GLI1 in TC32 (Figure 5B). As direct GLI1 targets, Patched1 and NKX2.2 are upregulated by GLI1. This supraphysiologic GLI1 expression results in downreg- ulation of GAS1, as has been observed with prolonged SHH stimulation in developmental models[29]. Nevertheless, the transcript levels of all three loci are clearly GLI1 modulated in a Ewing cell background. As a further test that EWS/FLI1 targets these three loci through GLI1, we measured the effect of GLI1 inhibition via GLI1 shRNA. Figure 5C demonstrates that all three loci are transcriptionally downregulated by GLI1 hairpin, just as they diminish with EWS/FLI1 hairpin. The fact that GAS1 is downregulated by both GLI1 expression and by GLI1 knockdown reflects the different states of pathway activation between the more physiologic levels of GLI1 expression in the shRNA experiment GLI1 in Ewing Tumors and the dramatic overstimulation of the pathway with GLI1 overexpression. As an additional proof of this model, we employed a different method of GLI1 transcriptional inhibition. The compound GANT58 has been shown to inhibit GLI transactivation [5,9]. We have already shown that it has effects similar to GLI1 shRNA on anchorage independent growth in an ESFT line (see Figure 4D). If our model is correct, GANT58 should produce effects on downstream transcriptional targets similar to those seen with GLI1 shRNA. To test this, we exposed the ESFT cell line A673 to GANT58 at doses shown to inhibit GLI1 transactivation. Figure 5D demonstrates the anticipated transcriptional downreg- ulation of Patched, NKX2.2 and GAS1 by treatment with GANT58. Discussion Our findings demonstrate the widespread nature of EWS/FLI1 dependent GLI1 deregulation in ESFT cell lines. Deregulated expression of GLI1 is a characteristic of primary ESFT and many ESFT cell lines. GLI1 expression is important to support the malignant phenotype of ESFT cell lines whether inhibited by shRNA, endogenous regulators, or by pharmacologic agents. This deregulation appears to be Hedgehog independent. Finally, we have identified the first members of a subset of EWS/FLI1 targets which require GLI1 for their deregulation. The significant biologic effects of GLI1 inhibition in ESFT cell lines is not surprising, since inhibition of NKX2.2 alone is sufficient to produce similar significant effects[26]. The biologic effects of altered expression of our other two overlapping targets, Patched1 and GAS1 remain to be elucidated in ESFT. As we have observed in a model systems [4] and has been seen in other ESFT cell lines [5], EWS/FLI1 deregulation of GLI1 appears to the a Hedgehog independent phenomenon. The mechanism by which GLI1 is upregulated has been recently described as being direct transcriptional upregulation by EWS/ FLI1[5]. It is gratifying that data from this publication agrees with our hypothesis of activation of GLI1 by a Hedgehog independent mechanism downstream of Smoothened. These observations will enable a broader understanding of the range of activity of EWS/ FLI1 and will add to the growing literature on non-canonical mechanisms of activating HH-GLI signaling[30]. With the recent opportunities afforded by microarray analysis, a more consistent set of EWS/FLI1 targets is emerging[3]. Such target sets are biased by the means by which they are identified. As such, these methods are likely to favor targets whose transcript levels are inherently high, such as those which may be strong direct EWS/FLI1 transcriptional targets [31]. However, there is no reason to assume that the list of biologically significant EWS/ FLI1 targets must necessarily be restricted to the ‘‘high amplitude’’ set favored by microarray analysis. Given the literature suggesting that Patched1, NKX2.2, and GAS1 are components of the HH-GLI signaling network, the most likely model is for EWS/FLI1 induced elevation of GLI1 expression leading to altered expression of a number of target loci. Further characterization of this system has the potential to provide some order to the complex picture of transcriptional deregulation by EWS/FLI1. While it is likely that only a subset of EWS/FLI1 targets are deregulated through GLI1, the data presented here and the known biology of HH-GLI signaling suggest that many may be of biologic import. Since GLI1 acts as a transcription factor, analysis of shRNA-manipulated ESFT cell lines by expression microarray would allow the identification of common targets of EWS/FLI1 and GLI1. Knowledge of such a PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7608