Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicase DHX9.

Marco Fidaleo,Elisabetta Volpe,Daniela Caporossi,Francesca Svetoni,Belén Miñana,Maria Paola Paronetto

doi:10.18632/oncotarget.5033

Marco Fidaleo, Elisabetta Volpe + Show 4 more

Open Access

https://doi.org/10.18632/oncotarget.5033

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Abstract

Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability.

Highlights

Ewing Sarcomas (ES) are aggressive tumors of bone and soft tissues
We provide evidence that DNA damage regulates DHX9 expression by alternative splicing (AS) through the inclusion of a new PTCcontaining exon and consequent targeting to nonsense-mediated decay (NMD)
UV light regulates this event in a carboxy terminal domain (CTD)-dependent manner by slowing down the elongation rate of RNA polymerase II (RNAPII) and promoting the inclusion of the alternative exon 6A in DHX9 pre-mRNA

Summary

Introduction

Ewing Sarcomas (ES) are aggressive tumors of bone and soft tissues They are caused by different chromosomal translocations that yield in-frame fusion proteins comprising the amino terminus of the EWS protein fused to the carboxyl terminus of various ETS transcription factors [1]. These chimeric proteins activate a specific oncogenic program to direct neoplastic transformation [2, 3]. EWS-FLI1, generated by chromosomal translocation between chromosome 22 and 11 [3], is the most common product, representing a landmark of ES Both EWS and EWS-FLI1 modulate gene expression [4, 5]. EWSR1 haploinsufficiency might contribute, at least in part, to ES cells sensitivity to genotoxic stress

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