GLI1 in Ewing Tumors Figure 4. GLI1 upregulation is Hedgehog independent. Panel A: Exogenous SHH in culture media effectively upregulates GLI1 and Patched in NIH3T3, an SHH responsive cell line. Cyclopamine (5 mcM) effectively blocks this stimulation. However, the same human SHH protein is relatively ineffective in altering GLI1 expression in ESFT cell lines, with 2/4 lines showing no response. Patched1 expression is mildly altered (,50% change) in TC32, with 3/4 lines showing no change. Statistical comparisons are made with diluent. NS means ‘‘not significant.’’ Panel B: Consistent with the minimal expression changes produced by Cyclopamine, 10 mcM Cyclopamine is ineffective at altering anchorage independent growth in ESFT cell lines as was observed with GLI1 shRNA. Panel C: Forskolin, which blocks the pathway at the level of GLI1 at 100 mcM concentration, has similar inhibitory effects to those observed with shRNA and endogenous inhibitors. Panel D: Unlike Cyclopamine increasing concentrations of the specific GLI transcriptional inhibitor GANT58 results in abrogation of anchorage independent growth in the Ewing cell line A673. doi:10.1371/journal.pone.0007608.g004 (Figure 4A, right), the picture is different. GLI1 transcript is upregulated by SHH to generally lesser degree, with 2/4 lines showing no response. While this increase in GLI1 transcript can be blocked by Cyclopamine, none of the cell lines demonstrate even a 50% enhancement of Patched1 with exogenous human SHH. So this lesser degree of GLI1 activation in ESFT cell lines is not accompanied by any major enhanced signaling downstream of GLI1. We conclude that, in addition to having little HH expression, ESFT cell lines are relatively HH insensitive. The consequence of this insensitivity is shown in Figure 4B as incubation of these lines with Cyclopamine produces little measurable effect on anchorage independent growth. These results contrast with the results presented above for GLI1 hairpin or overexpression of SUFU. Cyclopamine acts on the HH-GLI pathway at the cell surface by inhibiting the activation of Smoothened[18]. The failure of Cyclopamine to recapitulate our shRNA and SUFU inhibitory studies suggest that GLI1 activation is a more central phenomenon is ESFT. To further confirm this impression we used another means of blockade. Forskolin (FSK) is a compound which effects cytoplasmic PKA stimulation and is known to inhibit the HH-GLI pathway at the level of GLI1[19,20,21]. While it is not a pure GLI1 inhibitor, it has been extensively used experimentally to PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7608