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EWS-WT1 Oncogene Activates a Neuronal Reprogramming Factor ASCL1 and Mediates Partial Neural Differentiation

ABSTRACT: A chromosomal translocation fusion gene product EWS-WT1 is the defining genetic event in Desmoplastic Small Round Cell Tumor (DSRCT), a rare but aggressive tumor with a high rate of mortality. EWS-WT1 oncogene acts as an aberrant transcription factor that drives tumorigenesis, but the mechanism by which EWS-WT1 causes tumorigenesis is not well understood. To delineate the oncogenic mechanisms, we generated the EWS-WT1 fusion in the mouse using a gene targeting (knock-in) approach, enabling physiologic expression of EWS-WT1 under the native Ews promoter. We derived mouse embryonic fibroblasts (MEFs) and performed genome-wide expression profiling to identify transcripts directly regulated by EWS-WT1. Remarkably, expression of EWS-WT1 led to a dramatic induction of many neuronal genes. Notably, a neural reprogramming factor, ASCL1 (achaete-scute complex-like 1), was highly induced by EWS-WT1 in MEFs and in primary DSRCT. Further analysis demonstrated that EWS-WT1 directly binds to the proximal promoter region of ASCL1 and activates its transcription through multiple WT1-responsive elements. Depletion of EWS-WT1 in a DSRCT cell line resulted in severe reduction in ASCL1 expression and cell viability. Remarkably, when stimulated with neuronal induction media, cells expressing EWS-WT1 expressed neural markers and generated neurite-like projections. These results demonstrate for the first time that EWS-WT1 activates neural gene expression and is capable of directing partial neuronal differentiation, likely via ASCL1. These findings suggest that stimulating DSRCT tumor cells with biological or chemical agents that promote neural differentiation might be a useful approach to develop novel therapeutics against this incurable disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE53301 | GEO | 2014/09/11

SECONDARY ACCESSION(S): PRJNA231625

REPOSITORIES: GEO

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Project description:The t(11;22)(p13;q12) translocation is pathognomonic for the highly aggressive desmoplastic small round cell tumour (DSRCT). The translocation fuses exon 7 of the EWS gene to exon 8 of the WT1 gene (EWS/WT1). Two splice variants of EWS/WT1 exist, arising from the presence (+KTS) or absence (-KTS) of three amino acids: lysine, threonine and serine between zinc fingers 3 and 4. To investigate the oncogenic properties of both isoforms of EWS/WT1, we over-expressed EWS/WT1 in untransformed murine embryonic fibroblasts (MEFs). We demonstrate that neither isoform of EWS/WT1 is sufficient to transform wild type MEFs, however the oncogenic potential of both isoforms is unmasked by the loss of p53. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 resulted in enhanced cell proliferation, clonogenic survival and anchorage-independent growth. In addition EWS/WT1 expression in wild type MEFs attenuated several p53-dependent responses, including cell cycle arrest after irradiation and daunorubicin induced apoptosis. We show that DSRCT commonly have copy number amplification of MDM2 and MDMX, suggesting loss of p53 function in the tumours. Expression of either isoform of EWS/WT1 in MEFs induced characteristic mRNA expression profiles, including up-regulation of canonical Wnt pathway signaling. This was validated in cell lines and in a series of DSCRT, which confirmed canonical Wnt pathway activation in the tumours. We show for the first time that both isoforms of EWS/WT1 have oncogenic potential and that in addition to co-operating with loss of p53 function can also further attenuate p53-mediated responses. In addition we provide the first link between EWS/WT1 and Wnt pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterizes DSCRT. Four independently generated pools of wild type MEFs were infected with tetracycline repressible EWS/WT1+KTS, EWS/WT1-KTS or GFP, selected with hygromycin and protein expression confirmed. Four GFP, four KTS+ and four KTS- samples were hybridized to the Ilumina BeadChip.

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EWS-WT1 Oncogene Activates a Neuronal Reprogramming Factor ASCL1 and Mediates Partial Neural Differentiation

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