GLI1 Is a Direct Transcriptional Target of EWS-FLI1 Oncoprotein

Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis. Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis. Ewing Sarcoma Family of Tumors (ESFT) 2The abbreviations used are: ESFT, Ewing sarcoma family of tumors; Hh, Hedgehog; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ChIP, chromatin immunoprecipitation; RT, reverse transcription; siRNA, small interfering RNA; shRNA, small hairpin RNA. affects patients between the ages of 3 and 40 with most cases occurring during the second decade of life. It is an undifferentiated small round cell tumor of the bone and soft tissue with an unknown cell of origin. Currently, the cure rate for patients with localized disease is only 70%, and is less than 30% for patients showing metastatic disease despite intensive multimodal treatment strategies (1Grier H.E. Krailo M.D. Tarbell N.J. Link M.P. Fryer C.J. Pritchard D.J. Gebhardt M.C. Dickman P.S. Perlman E.J. Meyers P.A. Donaldson S.S. Moore S. Rausen A.R. Vietti T.J. Miser J.S. N. Engl. J. Med. 2003; 348: 694-701Crossref PubMed Scopus (956) Google Scholar). There is a need for more effective therapies to treat ESFT, especially in patients with metastases. ESFT is characterized by chromosomal translocations occurring between the genes for the TET (TAF15, EWS, and TLS) family member protein EWS and members of the ETS family of DNA-binding transcription factors. In ∼90% of the cases, the translocation occurs between chromosome 22 and chromosome 11 (2Sandberg A.A. Bridge J.A. 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Eisenacher M. Braun Y. Brachwitz K. Wai D.H. Dirksen U. Lanvers-Kaminsky C. Juergens H. Herrero D. Stegmaier S. Koscielniak E. Eggert A. Nathrath M. Gosheger G. Schneider D.T. Bury C. Diallo-Danebrock R. Ottaviano L. Gabbert H.E. Poremba C. Eur. J. Cancer. 2008; 44: 699-709Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Because EWS-FLI1 functions as a transcription factor, that GLI1 is a direct transcriptional target of EWS-FLI1 and a role in ESFT tumorigenesis. We data that GLI1 is a direct transcriptional target of EWS-FLI1 and is important in ESFT tumorigenesis. GLI1 be a target in and Hedgehog cells in with fetal bovine of the ESFT cell in with and with the of and EWS-FLI1 cell been F. K. A. B. P. O. Cancer Cell. 2007; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). cells in with cells in with and cells in with and from The GLI1 from the and of Cancer and Cancer of the in chromatin immunoprecipitation to the and as (6Abaan O.D. Levenson A. Khan O. Furth P.A. Uren A. Toretsky J.A. Oncogene. 2005; 24: 2715-2722Crossref PubMed Scopus (65) Google Scholar). the cell with for with or the with for activity a to a promoter by P. A. and a by Stephen cells with an EWS-FLI1 expression or in to the and with the In studies the cells with and then activity GLI1 promoter activity the which the GLI1 promoter by that used as a cells with or EWS-FLI1 in to the with the activity The GLI1 promoter by all the DNA-binding and to and mutations have been shown to EWS-FLI1 ability A. O. Toretsky J.A. 2004; PubMed Scopus Google Scholar). of the a the cell from cells to to and then to a The then to in in for for at at and at or at to the in in for The then three in and or in for The than three in and then the detected a from cell by and reverse reverse to the as R. A. C. C. T. A. J. 2003; PubMed Scopus Google Scholar). from The is as GLI1 from that of the GLI1 and cells in a cells with at and in at in a cells of to the The of the cells in a and for cells with at and in at in a cells of to the 3 The of the cells in a and 3 for to cell by a cell for and and then a for and proliferation by at a of in a or at or in to cells the cells or cells of to the soft agar in as a and in agar at as to of a cell and to at of cells in of agar and as The at for the agar and in a cell for with for at of by and colony and by The at GLI1 a of of EWS-FLI1 expression associated with increased GLI1 mRNA levels in R. Owen L.A. Trem D.J. Wong J.S. Whangbo J.S. Golub T.R. Lessnick S.L. Cancer Cell. 2006; 9: 405-416Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). in GLI1 mRNA be a direct or of EWS-FLI1 the of GLI1 promoter a direct target of EWS-FLI1. The GLI1 promoter been and characterized Yang J.T. J.W. E. K. D. P. 1998; PubMed Scopus Google Scholar, J.W. D.J. 2002; PubMed Scopus Google Scholar). of the GLI1 promoter potential which are characterized by X. S. Yang H. J.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). a direct between EWS-FLI1 and the GLI1 promoter in ESFT We used the for immunoprecipitation of EWS-FLI1 because is in ESFT cells (6Abaan O.D. Levenson A. Khan O. Furth P.A. Uren A. Toretsky J.A. Oncogene. 2005; 24: 2715-2722Crossref PubMed Scopus (65) Google Scholar). We to the of GLI1 promoter between EWS-FLI1 protein and of the GLI1 and and in the and the a result because of PTPL1, a direct transcriptional target of EWS-FLI1 (6Abaan O.D. Levenson A. Khan O. Furth P.A. Uren A. Toretsky J.A. Oncogene. 2005; 24: 2715-2722Crossref PubMed Scopus (65) Google Scholar), used as a in the absence of and for the in the absence of results suggest that EWS-FLI1 is to of the GLI1 promoter in ESFT cells. The to study the of EWS-FLI1 protein and the GLI1 promoter GLI1 by EWS-FLI1 transcription from the GLI1 cells with EWS-FLI1 in a expression and the GLI1 gene promoter in a We that EWS-FLI1 protein is increased activity of the GLI1 The been a of with the of The activity of the GLI1 promoter is also increased in a to the with the expression of EWS-FLI1 a in promoter of EWS-FLI1 an promoter which used as a to the that EWS-FLI1 activate reporter We then all the DNA-binding as shown in to the ability of EWS-FLI1 to activity of the GLI1 When the and GLI1 promoter in cells with EWS-FLI1, a in activity that the activity to the EWS-FLI1 GLI1 Expression and in the of an in GLI1 promoter activity EWS-FLI1 then at the of EWS-FLI1 GLI1 protein expression and the activity of GLI1 as a transcription We cells with EWS-FLI1 and GLI1 protein expression by We a in GLI1 protein expression cells EWS-FLI1 protein cells also with EWS-FLI1 and a reporter that a promoter to the of EWS-FLI1 GLI1 transcriptional activity The GLI DNA-binding to the promoter by the The between the used in and used in is that is activity of the GLI1 gene promoter by EWS-FLI1 protein, and the is activity of a promoter by GLI1 Expression of EWS-FLI1 in cells increased reporter activity data suggest that EWS-FLI1 GLI1 protein expression to a which GLI1 transcriptional that the in the promoter be to direct transcriptional activity by EWS-FLI1, used of GLI1 protein We used which been characterized as a specific of GLI1 transcriptional activity M. A. T. Toftgard R. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). We also used which is a Smoothened J. Chen B. L. M.P. Beachy P.A. Nature. 2000; PubMed Scopus Google Scholar). to have to GLI1 activation by EWS-FLI1, because EWS-FLI1 acts downstream of Smoothened to activate When cells with EWS-FLI1 and with the GLI1 a in GLI1 transcriptional activity as by the reporter decreased GLI1 activity as to a results that the in GLI1 promoter activity is mediated by an in expression of activated GLI1 GLI1 in ESFT the GLI1 promoter is activated by EWS-FLI1, GLI1 protein is in ESFT cell lines. We GLI1 protein expression in a of ESFT cell that EWS-FLI1. a cell used as a because levels of GLI1 ESFT cell GLI1 expression with cells as by of ESFT cells a of GLI1 protein expression with cells We also for Hedgehog pathway ESFT and cells ESFT cells GLI1 as as the GLI target and also the GLI target cells GLI genes or used as a We that GLI1 is in a of ESFT cell which suggests that GLI1 expression is a of EWS-FLI1 GLI1 cells with for EWS-FLI1 with to cells used because are the only ESFT cell that EWS-FLI1 EWS-FLI1 expression decreased treatment in the cells When EWS-FLI1 expression decreased a in GLI1 levels as by data suggest that EWS-FLI1 GLI1 protein expression in ESFT cells. of GLI1 in ESFT and cells with or GLI1 GLI1 GLI1 protein in cells by and in cells by 3 cells GLI1 a in proliferation with the The result cells GLI1 a 30% in proliferation with the This result also and GLI1 protein expression with a of for cells and with a of for cells. with a of for cells and a in proliferation with a of for cells. GLI1 with a ESFT and GLI1 a role in EWS-FLI1 tumorigenesis. The ESFT cell and with the GLI1 at and in cells used as a to that the to cell the at inhibition of cellular proliferation in cells and inhibition in cells. There inhibition in the cells. a of the inhibition in inhibition in and inhibition in cells We also and cells with at and and with the to have a in proliferation of ESFT cells because the EWS-FLI1 acts downstream of Smoothened to activate We to that at and the proliferation to a in and cells with the inhibition in and inhibition in of which than the inhibition in and inhibition in of which than We then the of proliferation in ESFT cells to a cell This that are proliferation in a cell ESFT cells. cells and cells proliferation cells. cells and cells proliferation with cells that the in proliferation to the of the GLI1 transcription activity in ESFT cells with the reporter and of or activity reporter activity of the in the reporter activity at and The GLI1 also to soft agar colony formation in cells The colony formation at and results that GLI1 a role in cell proliferation and of ESFT tumor of ESFT cells with a small of GLI1 soft agar colony cells in soft agar in at a of in a tissue The cells with or at and which in of shown is the of A. The are the a of The ability of EWS-FLI1 to a is in its ability to act as a transcription factor and alter gene Many have been as or by EWS-FLI1. studies have that EWS-FLI1 increases GLI1 study by microarray that GLI1 gene expression decreased in an ESFT cell EWS-FLI1 by R. Owen L.A. Trem D.J. Wong J.S. Whangbo J.S. Golub T.R. Lessnick S.L. Cancer Cell. 2006; 9: 405-416Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). study that cells with EWS-FLI1 increased expression of GLI1 and its transcriptional (27Zwerner J.P. Joo J. Warner K.L. Christensen L. Hu-Lieskovan S. Triche T.J. May W.A. Oncogene. 2008; 27: 3282-3291Crossref PubMed Scopus (98) Google Scholar). However, study a for EWS-FLI1 increases GLI1 (27Zwerner J.P. Joo J. Warner K.L. Christensen L. Hu-Lieskovan S. Triche T.J. May W.A. Oncogene. 2008; 27: 3282-3291Crossref PubMed Scopus (98) Google Scholar) that increased expression of GLI1 protein be an of EWS-FLI1. evidence that expression of GLI1 In that EWS-FLI1 to the GLI1 promoter and increases expression of GLI1 Our results the potential role of in GLI1 gene However, suggest that direct activation of GLI1 promoter by EWS-FLI1 is the increased GLI1 protein GLI1 with a small ESFT proliferation and results by GLI1 with and showing a in (27Zwerner J.P. Joo J. Warner K.L. Christensen L. Hu-Lieskovan S. Triche T.J. May W.A. Oncogene. 2008; 27: 3282-3291Crossref PubMed Scopus (98) Google Scholar) also that an ESFT cell GLI1 expression by Our that GLI1 is important for ESFT tumor We that inhibition of GLI1 function of and cell in and in soft In of a small of GLI1 as to shRNA, siRNA, and are to protein expression in have been in target in molecules that to be for and study of GLI1 gene in ESFT cell and that of or of the GLI1 gene W.M. Douglass E.C. Peiper S.C. Houghton P.J. Look A.T. Cancer Res. 1989; 49: 5407-5413PubMed Google Scholar). data that increased GLI1 expression in ESFT cells is to EWS-FLI1 and by mechanisms such as gene Our GLI1 to the of genes that are by EWS-FLI1 and to ESFT been characterized as a specific of GLI1 transcriptional activity M. A. T. Toftgard R. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). However, its for GLI1 is It is that also GLI and cells GLI1 and in to cells only GLI1 the between cell in to be to expression levels of GLI the in to and suggests the activation of Hedgehog pathway at the GLI the that GLI1 expression is an important to ESFT GLI1 is in by Hh mechanisms such as overexpression of Hh ligand, inactivation of Patched1, or activation of Smoothened. However, recent evidence also shown that activation of GLI1 by or signaling is important for tumor formation of cancers such as pancreas, and B. C. V. M. R. V. F. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar, Z. Mei Xie J. X. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, di Magliano M. S. A. J. Dlugosz A.A. Hebrok M. 2006; PubMed Scopus Google Scholar, S. J. I. Li A. T. P. F. A. Cancer Res. 2007; PubMed Scopus Google Scholar). However, the of and signaling GLI1 activation been shown to be a direct mechanism. We a of GLI1 activation EWS-FLI1 GLI1 by to the GLI1 to an in expression of GLI1 of ESFT cell with cell expression levels of GLI1 and its target gene ESFT cell expression or very levels with with GLI1 transcriptional activity in ESFT cells the that the of EWS-FLI1 GLI1 is of the Hedgehog and a Smoothened are M. A. T. Toftgard R. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). However, the evidence that in cancers GLI1 activation occurs by a as in suggests that the of GLI be be used to treat cancers ESFT, which have activation of the pathway at the of GLI. Because cancers have is that inhibition of GLI1 a cure for EWS-FLI1 important genes to cellular the from specific GLI1 most from We for the GLI1 gene promoter for the EWS-FLI1 cell and for the modified with