GLI1 Is a Direct Transcriptional Target of EWS-FLI1 Oncoprotein

William Matsui,Gulay Bulut,Elspeth Beauchamp,Jeffrey S Rubin,Jeffrey Toretsky,Akil Merchant,Ogan Abaan,Aykut Üren,Yoshimi Endo,Kevin Chen

doi:10.1074/jbc.m806233200

Abstract

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.

Highlights

Ewing Sarcoma Family of Tumors (ESFT)2 affects patients between the ages of 3 and 40 with most cases occurring during the second decade of life
GLI1 Is Expressed in ESFT Cell Lines—Because the GLI1 was statistically significant. These experiments were promoter is activated by EWS-FLI1, we explored whether GLI1 repeated four times, and GLI1 protein expression was reduced protein is expressed in ESFT cell lines
The ability of EWS-FLI1 to cause a malignant phenotype is dependent in part on its ability to act as a transcription factor and alter gene expression

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Hedgehog Pathway Drugs—COS7 cells were grown in DMEM with 10% fetal bovine serum (FBS). COS7 cells were cotransfected with an EWS-FLI1 expression vector or empty vector control (CIneo) in addition to the pGL38xGLI luciferase and Renilla TK mutant constructs with FuGENE 6 (Roche Applied Science) according the manufacturer’s protocol. COS7 cells were transfected with either empty vector control or EWS-FLI1 construct in addition to the luciferase constructs with FuGENE 6 (Roche Applied Science) according the manufacturer’s protocol. Viable cells were quantified using 10 ␮l of WST-1 reagent (Roche Applied Science) according to the manufacturer’s protocol after 3 days. Cyclopamine or 75503 at varying concentrations or vehicle alone (1% DMSO) were added in media containing 5% FBS to cells 4 h after plating once the cells had attached.

RESULTS

Relative Luciferase Activity

DISCUSSION

Colony Number