The GLI transcription factors as a therapeutic target in childhood cancers
Beauchamp, Elspeth Morrison.
Thesis (Ph.D.)--Georgetown University, 2010.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. The function of EWS-FLI1 is essential for the maintenance of Ewing's Sarcoma Family of Tumors' (ESFT) cell survival and tumorigenesis. GLI transcription factor activity as the mediator of Hedgehog signaling is recognized as a critical pathway for tumor maintenance. 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 is 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 role for GLI1 in ESFT tumorigenesis. Most attempts at inhibiting this pathway focus on disruption of the ligand (Hedgehog) or the receptor Smoothened. In this study we present evidence for characterization of an inhibitor of GLI1. Arsenic trioxide (ATO) is an FDA approved drug used for the treatment of acute promyelocytic leukemia (APL) as a second line of therapy. Our data show that ATO directly binds to recombinant GLI1 protein, inhibits GLI1 transcriptional activity, and decreases the expression of endogenous GLI target genes. Unlike its mechanism of action in APL, ATO does not reduce GLI1 protein levels. ATO does not alter the subcelluar localization of GLI1 or inhibit binding of recombinant GLI1 protein to DNA in surface plasmon resonance (Biacore) experiments. We show that ATO inhibits the growth of ESFT cell lines in vitro in cell proliferation assays and in vivo in a mouse xenograft model. Furthermore, ATO significantly improved survival of the ND2-SmoA1 mouse model of medulloblastoma that is driven by an activating Smoothened mutation. Our results establish ATO as a novel Hedgehog pathway inhibitor acting at the level of GLI1 both in vitro and in vivo by utilizing two separate mouse models. These results warrant the clinical investigation of ATO for tumors with activated Hedgehog/GLI signaling and in patients who develop resistance to current Smoothened antagonists.
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