A zebrafish transgenic model of Ewing's sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis

Abstract

Ewing's sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing's sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing's sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing's sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing's sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

Fig. 1.

EWS-FLI1 induces SRBCTs in zebrafish. (A) Schematic of Tol2 transposons including the hsp70 or the β-actin promoter, FLAG-tag (black rectangle), human EWS-FLI1 coding sequence, IRES-GFP sequence (gray rectangle), flanked by Tol2 recombination sites (triangles). (B-H) H&E staining of sagittal sections of adult zebrafish. (B) Control adult zebrafish. Wild-type zebrafish injected with EWS-FLI1 developed an invasive leukemia like-tumor (C magnified in D) or a solid small round blue cell tumor of the eye (arrow in E) (E magnified in F). tp53 mutants injected with EWS-FLI1 transposons also develop leukemia (G) or solid tumors (H) with similar histology. Scale bars: 200 μm (B,C,E), 50 μm (D,F–H).

Fig. 2.

H&E staining of selected serially transplanted tumors originating from two different zebrafish SRBCTs. (A) Tumor D85; (B) tumor D86 (see supplementary material Table S1). Numbers indicate serial transplants (primary, secondary, tertiary etc.).

Fig. 3.

Gene expression in zebrafish SRBCTs is distinct from MPNSTs and similar to human EWS-FLI1 gene expression data. (A) Histology of representative zebrafish MPNST (left) and SRBCT (right) that were used for microarray analysis. Scale bars: 50 μm. (B) Heat map of 421 probes that exhibited differential expression between MPNSTs and SRBCTs. Yellow, high expression; blue; low expression. (C) Quantitative RT-PCR of selected SRBCT upregulated genes. (D,E) GSEA using human homologs of zebrafish genes shown in B compared with human gene expression data from (D) stable knockdown of EWS-FLI1 (red, higher expression in A673 Ewing’s cell line; blue, higher expression following EWS-FLI1 knockdown in A673 cells) or (E) inducible expression of EWS-FLI1 in a bone-marrow-derived stromal cell line (red, higher expression in EWS-FLI1 expressing cells; blue, higher expression in control cells).

Fig. 4.

EWS-FLI1 induces developmental defects in zebrafish embryos. (A) Non-transgenic (GFP–) embryos are wild type. (B) Transgenic embryos (GFP+) carrying a heat-shock-inducible EWS-FLI1 transgene exhibit pericardial edema (arrow) and reduced head and eye size. (C) A morpholino against the transgene decreases the penetrance of the pericardial edema phenotype. (D) Percentage of transgenic embryos exhibiting pericardial edema is decreased using a morpholino targeting the EWS-FLI1 transgene. (E) RT-PCR for EWS-FLI1 shows low level of expression without heat shock (–HS) and increased expression following heat shock (+HS). no RT, no RT negative control.

Fig. 5.

EWS-FLI1 affects convergence and extension in zebrafish embryos. Heat-shocked control (A) and EWS-FLI1 transgenic (B) embryos at 2 d.p.f. reveals that transgenic embryos are shorter with reduced head development. At the 18 somite stage, in situ hybridization for myoD in heat-shocked control (C) and EWS-FLI1 transgenic (D) reveals a wider somite width (E) and a shorter anterior-posterior axis as measured by the angle between the head and tail (F) in transgenic embryos (*P<0.02, **P<0.001; error bars indicate s.e.m.).