The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

Bandopadhayay, Pratiti; Jabbour, Anissa; Riffkin, Christopher D.; Salmanidis, Marika; Gordon, Lavinia; Popovski, Dean; Rigby, Lin; Ashley, David M.; Watkins, D. Neil; Thomas, David M.; Algar, Elizabeth; Ekert, Paul G.

doi:10.1186/1471-2407-13-585

Cited by 11 publications

(11 citation statements)

References 37 publications

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“…However, EWSR1-WT1 was not sufficient to transform the cells to overcome growth arrest: Whereas both control and clone 2 cells proliferated slowly upon subsequent passages, clone 2 ceased proliferating after Cre expression, suggesting that EWSR1-WT1 expression in nonimmortalized cells is not sufficient for cellular transformation. These results are consistent with previous studies of EWSR1-WT1 expression in mouse fibroblasts and suggest that additional "hits" including p53 mutation may be required (35).…”

Section: Ewsr1-wt1supporting

confidence: 92%

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

Vanoli

Tomishima

Feng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.CRISPR-Cas9 | chromosomal translocation | homology-directed repair | EWSR1-WT1 | DSRCT

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Section: Ewsr1-wt1supporting

confidence: 92%

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

Vanoli

Tomishima

Feng

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Neither isoform of EWS-WT1 is sufficient to transform wild-type murine embryonic fibroblasts (MEFs). The oncogenic potential of both can be unmasked by p53 loss as seen by nuclear localization of p53, and copy-number amplification and gene-set enrichment analysis demonstrated augmentation of the WNT pathway [ 39 ]. In the absence of intact p53 protein, WT1 acts as a transcriptional activator [ 40 ].…”

Section: Molecular Findingsmentioning

confidence: 99%

Desmoplastic Small Round Blue Cell Tumor: A Review of Treatment and Potential Therapeutic Genomic Alterations

Bulbul

Fahy

Xiu

et al. 2017

Sarcoma

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Desmoplastic small round blue cell tumors (DSRCTs) originate from a cell with multilineage potential. A molecular hallmark of DSRCT is the EWS-WT1 reciprocal translocation. Ewing sarcoma and DSRCT are treated similarly due to similar oncogene activation pathways, and DSRCT has been represented in very limited numbers in sarcoma studies. Despite aggressive therapy, median survival ranges from 17 to 25 months, and 5-year survival rates remain around 15%, with higher survival reported among those undergoing removal of at least 90% of tumor in the absence of extraperitoneal metastasis. Almost 100% of these tumors contain t(11;22) (p13;q12) translocation, and it is likely that EWS-WT1 functions as a transcription factor possibly through WT1 targets. While there is no standard protocol for this aggressive disease, treatment usually includes the neoadjuvant HD P6 regimen (high-dose cyclophosphamide, doxorubicin, and vincristine (HD-CAV) alternating with ifosfamide and etoposide (IE) chemotherapy combined with aggressively attempted R0 resection). We aimed to review the molecular characteristics of DSRCTs to explore therapeutic opportunities for this extremely rare and aggressive cancer type.

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“…RT-PCR and sequencing analysis showed a chimeric transcriptional message of the Ewing’s sarcoma gene exon 10 fused to the Wilms’ tumor gene exon 8. Alternative splicing in exon 9 of WT1 and EWS-WT1 generates an insertion of three aminoacids -lysine, threonine and serine (KTS)- between zinc fingers 3 and 4, producing + KTS and –KTS isoforms [ 10 ]. Both EWS-WT1 -KTS and EWS-WT1 + KTS have been described in DSRCT, though is still not clear from which isoform the oncogenic properties of EWS-WT1 come [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of action of trabectedin in desmoplastic small round cell tumor cells

et al. 2017

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BackgroundDesmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive disease, that can be described as a member of the family of small round blue cell tumors. The molecular diagnostic marker is the t(11;22)(p13;q12) translocation, which creates an aberrant transcription factor, EWS-WT1, that underlies the oncogenesis of DSRCT. Current treatments are not very effective so new active drugs are needed. Trabectedin, now used as a single agent for the treatment of soft tissue sarcoma, was reported to be active in some pre-treated DSRCT patients. Using JN-DSRCT-1, a cell line derived from DSRCT expressing the EWS-WT1 fusion protein, we investigated the ability of trabectedin to modify the function of the chimeric protein, as in other sarcomas expressing fusion proteins. After detailed characterization of the EWS-WT1 transcripts structure, we investigated the mode of action of trabectedin, looking at the expression and function of the oncogenic chimera.MethodsWe characterized JN-DSRCT-1 cells using cellular approaches (FISH, Clonogenicity assay) and molecular approaches (Sanger sequencing, ChIP, GEP).ResultsJN-DSRCT-1 cells were sensitive to trabectedin at nanomolar concentrations. The cell line expresses different variants of EWS-WT1, some already identified in patients. EWS-WT1 mRNA expression was affected by trabectedin and chimeric protein binding on its target gene promoters was reduced. Expression profiling indicated that trabectedin affects the expression of genes involved in cell proliferation and apoptosis.ConclusionsThe JN-DSRCT-1 cell line, in vitro, is sensitive to trabectedin: after drug exposure, EWS-WT1 chimera expression decreases as well as binding on its target promoters. Probably the heterogeneity of chimera transcripts is an obstacle to precisely defining the molecular mode of action of drugs, calling for further cellular models of DSRCT, possibly growing in vivo too, to mimic the biological complexity of this disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3091-1) contains supplementary material, which is available to authorized users.

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The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

Cited by 11 publications

References 37 publications

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

Desmoplastic Small Round Blue Cell Tumor: A Review of Treatment and Potential Therapeutic Genomic Alterations

Mechanism of action of trabectedin in desmoplastic small round cell tumor cells

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