O-GlcNAcylation is involved in the transcriptional activity of EWS-FLI1 in Ewing's sarcoma

Abstract

The oncogene EWS-FLI1 encodes a chimeric transcription factor expressed in Ewing's sarcoma family tumors (ESFTs). EWS-FLI1 target gene expression is thought to drive ESFT pathogenesis and, therefore, inhibition of EWS-FLI1 activity holds high therapeutic promise. As the activity of many transcription factors is regulated by posttranslational modifications, we studied the presence of modifications on EWS-FLI1. The immuno-purified fusion-protein was recognized by an antibody specific for O-linked b-N-acetylglucosaminylation, and bound readily to a phosphoprotein-specific dye. Inhibition of Ser/Thrspecific phophatases increased EWS-FLI1 molecular weight and reduced its O-GlcNAc content, suggesting that phosphorylation and O-GlcNAcylation of EWS-FLI1 interact dynamically. By mutation analysis, O-GlcNAcylation was delineated to Ser/Thr residues of the amino-terminal EWS transcriptional-activation domain. Metabolic inhibition of the hexosamine biosynthetic pathway abrogated O-GlcNAcylation of EWS-FLI1 and interfered specifically with transcriptional activation of the EWS-FLI1 target Id2. These results suggest that drugs modulating glycosylation of EWS-FLI1 interfere functionally with its activity and might, therefore, constitute promising additions to the current ESFT chemotherapy.

Biochemical Properties and Biological Functions of FET Proteins

“…The LC domain of EWSR1 is modified by O-GlcNAcylation. Reduction of O-GlcNAcylation with small-molecule inhibitors reduces the expression level of the fusion protein EWSR1-FLI1 and represses the expression of genes regulated by EWSR1-FLI1 (95). Finally, methylation of EWSR1 or TAF15 by protein arginine N-methyltransferase 1 (PRMT1) causes these FET proteins to relocalize to the cytoplasm and subsequently reduces their ability to regulate transcription in the nucleus (96)(97)(98).…”

A transcription assay for EWS oncoproteins in Xenopus oocytes

“…Frog oocytes are thus likely to harbor crucial endogenous factors that have co-evolved with the EAD and this should be advantageous over alternative (non-mammalian) systems for EAD analysis, such as yeast (Zhou and Lee, 2001). For example Tyr phosphorylation can modulate EAD activity under particular circumstances (Kim et al, , 2000 and other phosphorylation events (Olsen and Hinrichs, 2001) and O-GlcNAcylation (Bachmaier et al, 2009) have been shown to influence EFP/EAD activity. Transcriptional activity of N3Z and N3ZA in mammalian cells was detected by Cat assay using the Z7Cat reporter.…”

More and better cure for an orphan: priorities for future paediatric cancer research in Europe – Meeting report of the EC-funded science-communication project DIRECT “Overcoming Cancer with Research”

“…1 and summarised as follows: I) Prognosis and therapeutic targets in Ewing tumours (PROTHETS) [18] PROTHETS was realised from 01/2005 to 12/2007 through collaborative studies in Germany, Austria, France, Russia, Finland and Spain with the goal to defi ne prognostic markers and new therapeutic targets for the Ewing's sarcoma family of tumours (ESFT), thereby providing rigorous scientifi c justifications for the development of clinical trials for this rare, but fatal childhood disease. Th e fact that about 10 associated research articles have been published in various international, medium and high impact scientifi c journals [19][20][21][22][23][24][25][26][27] whilst and shortly after PROTHETS was active, allows the conclusion that this FP6-project funded with approximately 2.5 million €, has already been successful. approach, which is expected to improve child health, provide the biotech industry with new options for drug developments, and serve as a model for targeting other cancers.…”