Abstract
Simple SummaryEwing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 defines a specific transcriptomic profile in Ewing sarcoma cells, which determines the tumorigenesis process. Our study focused on the identification of transcription factors regulated by EWSR1-FLI1. FEZF1 (FEZ family zinc finger protein 1), a transcription factor involved in neural cell identity, was identified as one of the most strongly upregulated genes by EWSR1-FLI1. Functional studies were carried out to characterize the involvement of FEZF1 in Ewing sarcoma pathogenesis. As a result, the inhibition of FEZF1 diminished clonogenicity and cell proliferation in three Ewing sarcoma cell lines. Transcriptomic analysis revealed several neural-specific genes transcriptionally regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1, which could explain the neural-like phenotype observed in several Ewing sarcoma cell lines and tumors.Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.
Highlights
Ewing sarcoma is an aggressive cancer affecting children and young adults
We looked for transcription factors that were upregulated or downregulated upon EWSR1-FLI1 knockdown in the well-characterized Ewing sarcoma cell model A673/TR/shEF
We analyzed the expression levels (RNAseq data) of a total of 1674 transcription factors that belong to the Gene Ontology term “DNA binding transcription factor activity” (GO:0003700) [33] (Table S1), in A673/TR/shEF cells incubated in the absence (EWSR1-FLI1high) or presence (EWSR1-FLI1low) of doxycycline during 72 h
Summary
Ewing sarcoma is an aggressive cancer affecting children and young adults. Ewing sarcoma cells are highly undifferentiated, and tumors initially respond well to chemotherapy and radiotherapy, local and distant relapses are frequent [1]. From a molecular point of view, Ewing sarcoma is characterized by chromosomal translocations that give rise to chimeric proteins that act as aberrant transcription factors. The most frequent chromosomal translocation is t (11;22), which produces an aberrant transcription factor formed by the fusion of the EWSR1 gene with the ETS transcription factor FLI1. The resulting aberrant transcription factor EWSR1-FLI1 affects the expression, directly or indirectly, of hundreds of genes that globally induce cell proliferation and block cell differentiation [2]. Since these chimeric transcription factors are present in all Ewing sarcoma tumors, it is widely accepted that they are the main oncogenic driver in Ewing sarcoma [1]
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