Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma.

S Niedan,M Kauer,R Kofler,D N T Aryee,U Kontny,U Pötschger,A Meier,H Kovar,R Schwentner

doi:10.1038/onc.2013.361

Abstract

The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.

Highlights

Ewing sarcoma (ES) is characterized by aberrant activation of ETS transcription factors due to rearrangement with the EWS gene
FOXO1 is expressed at lower levels in primary ES compared with a wide variety of tissues[14] (Figure 1d), and its promoter is directly bound by EWS-FLI1 in chromatin immunopreciptation-PCR experiments (Figure 1e).[15]
Very little is known about the mechanisms of EWS-FLI1-mediated gene repression, even though there is evidence that part of its repressive signature can be assigned to activation of transcriptional repressors NKX2.2 and NR0B1,25,26 to epigenetic mechanisms involving the NuRD (Nucleosome remodelling and deacetylation) complex[27] or to post-transcriptional mechanisms.[28]

Summary

Introduction

Ewing sarcoma (ES) is characterized by aberrant activation of ETS transcription factors due to rearrangement with the EWS gene. The most common gene fusion combines EWS with FLI1,1 which has a rate-limiting role in ES pathogenesis,[2] as interference with. Experimental evidence suggests that the EWS domain confers transcription activating properties to EWS-FLI1,4 knockdown of EWS-FLI1 uncovers similar numbers of repressed and activated genes.[5,6,7] We focused on the repressive EWS-FLI1 regulatory network as the mechanisms and mediators of transcriptional repression downstream of EWS-FLI1 are poorly understood. We identified the enrichment of forkhead box (FOX) recognition motifs in EWS-FLI1-repressed genes. FOX transcription factors are a superfamily of more than 100 structurally related proteins with a conserved 100-residue DNA-binding domain, the forkhead domain

Methods

Results

Conclusion