The EWS/FLI1 oncogenic protein inhibits expression of the Wnt inhibitor DICKKOPF-1 gene and antagonizes β-catenin/TCF-mediated transcription

Abstract

Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.

Activation of Wnt/β-Catenin in Ewing Sarcoma Cells Antagonizes EWS/ETS Function and Promotes Phenotypic Transition to More Metastatic Cell States

“…S2B). These data corroborate a prior study of gene expression in a different tumor dataset wherein a correlation was also discovered between levels of LEF1 and EWS/ETS repressed targets (30). Together these data provide compelling evidence that activation of Wnt/beta-catenin antagonizes the transcriptional function of EWS/ETS fusions in Ewing sarcoma.…”

“…S2A). Thus, transcriptional antagonism exists between EWS/ERG and Wnt/beta-catenin in CHLA25 cells, corroborating a prior study of A673 cells which reported an inverse relationship between EWS/FLI1 and TCF/LEF target genes (30). …”

“…These findings suggest that either Wnt/beta-catenin can inhibit EWS/FLI1 transcription or that heterogeneity in expression of EWS/FLI1 prior to ligand exposure determines Wnt-responsiveness. In support of this latter possibility, Navarro et al (30) previously showed that cells with reduced expression of EWS/FLI1 were more responsive to Wnt3a, an observation that we have also now independently validated (Supplemental Figure S3A-C). …”

“…Thus, the data from Navarro et al and from our current work show that antagonism between these transcriptional axes is bidirectional. The mechanistic basis of this antagonism remains to be fully elucidated but the studies of Navarro et al suggest that physical interactions between EWS/FLI1 and LEF1 inhibit transcription of TCF/LEF target genes in response to beta-catenin activation (30). Since LEF1 is strongly induced by Wnt/beta-catenin in Ewing sarcoma cells we speculate that this might lead to sequestration of EWS/ETS proteins by LEF1 and inhibition of EWS/ETS-dependent transcription.…”

Integrated microRNA and mRNA expression profiling in a rat colon carcinogenesis model: effect of a chemo-protective diet

“…Recently, it has been reported that miR-204 represses SIRT1 whose expression is regulated during embryonic stem cell differentiation (60). We also noted a downregulation of FLI1 and upregulation of its targeting miR-193a, which may participate in tumor progression by activating ␤-catenin transcription (47). Next, we applied LDA to classify treatment effects on colonic microRNAs.…”