GLI1 in Ewing Tumors Figure 2. RNAi mediated reduction in EWS/FLI1 expression results in a consistent reduction of GLI1 signaling. Panel A: Four different shRNA sequences targeting EWS/FLI1 were introduced into the Ewing cell line TC71. The sequences are EF-2[26], EF-bp (from the Type 1 EWS/FLI1 breakpoint)[34], EF-4 [26] and EF-818 [33]. These are compared to a non-targeting control shRNA directed to a sequence in the luciferase gene by qPCR. Reduced expression of EWS/FLI1 is always accompanied by a reduction of GLI1 and its direct target Patched1. Panel B: Similar qPCR results are obtained from other ESFT cell lines, A673 and CHLA9. Panel C: An unrelated antisense construct inhibits EWS/FLI1 expression by Western blot (left of panel). This reduction in EWS/FLI results similar alterations in the expression of GLI1 and Patched1 by real time qPCR (right of Panel C). doi:10.1371/journal.pone.0007608.g002 and CHLA9 (see Figure 2B), indicating that the EWS/FLI1 dependence of GLI1 expression and signaling is shared among several well studied ESFT cell lines. Finally, to further evaluate the soundness of this relationship, we employed an antisense form of EWS/FLI1 and selected stable knockdown clones from TC71 and TC32[15]. Figure 2C demonstrates once again that an experimentally induced reduction in EWS/FLI1 expression results in a reduction of GLI1 and Patched1 expression. Multiple means of GLI1 inhibition establish the importance of GLI1 expression to ESFT phenotype We have also demonstrated that GLI1 hairpin is capable of diminishing anchorage independent growth in ESFT cell lines. To rule out a possible off-target effect, we evaluated a panel of GLI1 shRNA targeting sequences in the ESFT cell line TC71. Figure 3A illustrates the targeting sequences used and depicts the degree of GLI1 expression decrease and the degree of decrease in anchorage independent growth. The consistent observation is that the degree of GLI1 knockdown is associated with a reduction in anchorage independent growth. Figure 3B demonstrates the efficacy of these constructs at reducing GLI1 protein levels. These findings suggest that GLI1 is clearly important to this aspect of tumor cell line phenotype and that this observation is unlikely to be due to an off target effect. To further confirm the effect of HH-GLI inhibition in ESFT, we employed alternate means of inhibition. Suppressor of Fused (SUFU) is an endogenous inhibitor of the HH-GLI1 pathway acting by either cytoplasmic sequestration of GLI1 or by inhibition of GLI1 transcription at select loci[16,17]. We drew upon our prior observation that overexpression of Suppressor of Fused (SUFU) is capable of diminishing anchorage independent growth of EWS/FLI1 transformed NIH3T3 cells[4]. To see if ESFT lines behave in a similar fashion, we transduced a total of three ESFT lines with SUFU under the control of a high activity retroviral promoter. High levels of SUFU transcript were exhibited by these polyclonal lines (data not shown). Figure 3C shows that all three lines exhibited diminished anchorage independent growth. These experiments were verified in 2–4 independent assays per line. They further strengthen the case that GLI1 dependence of ESFT lines is a widespread phenomenon. GLI1 signaling in ESFT is Hedgehog independent The most common mechanism for activation of the HH-GLI pathway involves activation of Smoothened, either by overexpression PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7608