Ewing family tumors (EFT), classically Ewing's sarcoma and peripheral primitive neuroectodermal tumor, share a common class of tumor-specific fusion genes thought to be key mediators of tumor biology. Here we demonstrate that the most common Ewing's fusion, EWS/FLI1, produces transcriptional upregulation of GLI1 and its direct transcriptional target PATCHED1 in a model transformation system. This deregulation of GLI1 is common to other EWS/ets chimera and depends on the functional transcriptional regulatory domains. Inhibition of GLI1 via RNAi or via overexpression of endogenous inhibitors results in a reduction of EWS/FLI1 transformation activity. Activation of GLI1 appears to occur in a Hedgehog-independent fashion as blockade of Hedgehog signaling has only a modest effect on EFT cells. We present evidence that EWS/FLI1 upregulation of cMYC may play a role in the upregulation of GLI1 in EWS/ FLI1-transformed NIH3T3 cells. Finally, we demonstrate that observations made in a model transformation system translate to an Ewing cellular background. EFT cell lines express GLI1 and PATCHED and this expression is EWS/FLI1 dependent. Inhibition of GLI1 expression via RNAi results in reduced anchorage-independent growth in an EFT cell line. GLI1 appears to be a transcriptionally deregulated target of EWS/FLI1 that mediates a portion of its tumorigenic phenotype.

The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.

Ewing Family Tumors (Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor) are common bone and soft tissue malignancies of childhood, adolescence and young adulthood. Chromosomal translocation in these tumors produces fusion oncogenes of the EWS/ETS class, with EWS/FLI1 being by far the most common. EWS/ETS chimera are the only well established driver mutations in these tumors and they function as aberrant transcription factors. Understanding the downstream genes whose expression is modified has been a central approach to the study of these tumors. FOXM1 is a proliferation associated transcription factor which has increasingly been found to play a role in the pathogenesis of a wide range of human cancers. Here we demonstrate that FOXM1 is expressed in Ewing primary tumors and cell lines. Reduction in FOXM1 expression in Ewing cell lines results in diminished potential for anchorage independent growth. FOXM1 expression is enhanced by EWS/FLI1, though, unlike other tumor systems, it is not driven by expression of the EWS/FLI1 target GLI1. Thiostrepton is a compound known to inhibit FOXM1 by direct binding. We show that Thiostrepton diminishes FOXM1 expression in Ewing cell lines and this reduction reduces cell viability through an apoptotic mechanism. FOXM1 is involved in Ewing tumor pathogenesis and may prove to be a useful therapeutic target in Ewing tumors.