The identification of human EWS-FLI1-driven tumors in zebrafish shows that critical downstream transcriptional targets and protein interactions that allow EWS-FLI1 to generate tumors are conserved between zebrafish and humans. The developmental phenotypes caused by EWS-FLI1 in zebrafish embryos provides further support for this conservation and provides a second, independent context in which to dissect the downstream targets of EWS-FLI1. Creating regulated or inducible models of EWS-FLI1 expression will improve this system by allowing us to induce EWS-FLI1 in a temporal or tissue-specific fashion to increase the tumor incidence. A model in which we could reliably produce SRBCTs in the zebrafish would provide an ideal platform for chemical or genetic screening for EWS-FLI1 inhibitors.
The identification of both solid and infiltrating tumors suggests that EWS-FLI1 can induce the formation of both of these tumor types in the zebrafish. Both of these tumor types have also occurred in transgenic mice expressing EWS-FLI1
using tissue-specific promoters, suggesting that the cellular context in which EWS-FLI1
is expressed dictates the tumor type that subsequently develops (Torchia et al., 2007
; Lin et al., 2008
). The finding that EWS-FLI1 promotes leukemia in both mouse and zebrafish indicates that this property of EWS-FLI1 is conserved. Although EWS-FLI1-associated leukemia is not observed in humans, a closely related fusion of TLS-ERG is associated with acute myeloid leukemia (Ichikawa et al., 1994
). Rare cases of pre-B-cell lymphoblastic leukemia with the EWS-FLI1
translocation have also been reported (Ozdemirli et al., 1998
; Jakovljevic et al., 2010
). These cases suggest a connection between EWS translocation proteins and cancers of the hematopoietic lineage. Differences in cellular context, cellular niche, expression level, or temporal expression in animal models may account for the lack of EWS-FLI1-associated leukemia in humans.
Serial transplantation of two zebrafish tumors demonstrated the independent growth of the tumor cells. The histological variation observed following several rounds of transplantation could be caused by tumor evolution, stochastic differences in subpopulations of cells transplanted, or differences in signals or environment within the host tissue. Although some rounds of transplantation required a longer time for tumor development, the number of transplant recipients was not large enough to confirm that this difference is statistically significant. Presently, serial transplantation studies performed in zebrafish are becoming a powerful tool to study cancer in this model organism [reviewed by Taylor and Zon (Taylor and Zon, 2009
)]. Most zebrafish cancer models to date have been shown to be serially transplantable; however, some differences are becoming evident; a KRAS-induced MPD was unable to be serially transplanted (Le et al., 2007
) and a T-ALL line showed increasing malignancy with subsequent transplants (Frazer et al., 2009
). Understanding the mechanisms underlying engraftment and proliferation in zebrafish cancer models might shed light on tumor progression and metastasis in human cancer.
In the zebrafish model, we identified tumors in animals injected with a hsp70:EWS-FLI1 transgene, even in the absence of heat shock. In these tumors, the heat shock promoter is probably acting as a minimal promoter that allows EWS-FLI1 expression to be controlled by local enhancers at the genomic site of transgene integration. Under this model, the two different histologies of SRBCTs that we identified might arise because of trapping of different types of tissue-specific enhancers by the hsp70:EWS-FLI1 transgene. Identification of these enhancers, and the tissue type they define, could perhaps help clarify the Ewing’s sarcoma cell of origin.
Mutation of tp53
provided a sensitized background for the formation of SRBCTs in zebrafish. Although mutations in tp53
occur in only a subset of Ewing’s sarcomas (Huang et al., 2005
), the p53 pathway might also be inhibited by other mechanisms (Ban et al., 2008
; Li et al., 2010
). Interestingly, the solid SRBCTs that we identified most frequently arose in proximity to the eye and abdomen, the locations in which MPNSTs also develop in tp53
mutant zebrafish (Berghmans et al., 2005
). This cell type might be predisposed to tumor development in the zebrafish, or the MPNSTs and SRBCTS might both be able to arise from the same type of nerve sheath cell. An alternative possibility is that the tumors originate as a result of the loss of p53, but that the presence of EWS-FLI1 drives the tumor cells to a less differentiated, more primitive state, which results in the SRBCT histology. These possibilities cannot be distinguished from this study.
The microarray analysis provides firm support for the validity of this system as a model of human Ewing’s sarcoma. The identification of relevant target genes and the enrichment of the zebrafish gene set in both Ewing’s sarcoma cell lines and primary tumors reveal that EWS-FLI1 produces similar gene expression changes in the zebrafish tumors. By comparing the gene expression profiles to MPNSTs, we eliminated those genes that are generally affected in tumors and also those genes that might be changed because of the absence of p53. Therefore, additional gene expression changes caused by EWS-FLI1 might also exist if they share these properties. Our gene set is therefore a conservative estimate of the transcriptional changes caused by EWS-FLI1, but is also more likely to include specific targets and features of Ewing’s sarcoma.
We identified some genes that were upregulated in zebrafish SRBCTs but downregulated in human EWS-FLI1-dependent expression datasets, or vice versa. Differences in regulatory elements, protein-protein interactions, cellular niche and growth factors, and many other differences might exist between animal models and humans. Of course, animal models will almost never completely recapitulate the human conditions. In this context, the similarity of these tumors suggests that these differences are not crucial to tumorigenesis. Therefore, the gene expression profile we found is likely to include relevant biological targets downstream of EWS-FLI1. These gene sets also show enrichment in multiple, independent datasets generated from experiments in human cell lines. Even among the various microarray analyses performed from human cell lines, the differences between experiments are much greater than the similarities. A meta-analysis used to generate a EWS-FLI1 core-expression signature concluded that the core was more similar to primary Ewing’s sarcoma, than any of the individual experiments (Hancock and Lessnick, 2008
). Therefore, each additional model of Ewing’s sarcoma provides new information toward understanding the transcriptional consequences of EWS-FLI1 and, by extension, facilitates the development of improved, targeted therapies of Ewing’s sarcoma.
The stable EWS-FLI1
-expressing transgenic line that we established represents a valuable new tool for the study of EWS-FLI1 cellular function. Many signaling pathways that function in zebrafish embryonic development have been dissected. The phenotypes observed in the EWS-FLI1
embryos are consistent with defects in convergence and extension during gastrulation. The non-canonical Wnt-PCP and BMP signaling pathways as well as other genes, including those encoding Stat3, heterotrimeric G proteins and cell adhesion molecules contribute to proper convergence and extension in zebrafish embryos (Roszko et al., 2009
). The convergence and extension defect we observed in transgenic EWS-FLI1
embryos suggests that EWS-FLI1 directly interferes with genes that function during this process; a finding made possible by the expression of the transgene in the context of a developing embryo, in which the interplay of multiple signaling pathways determines embryo morphology. Future experiments will be required to fully dissect the relationship between EWS-FLI1 and these morphological defects.
The phenotype we observed in the hsp70:EWS-FLI1
line was not identical to that previously reported following mRNA injection (Embree et al., 2009
). Similarly to our analysis, embryos with severe trunk defects, including a shorter axis, were observed, as well as abnormal mitoses. We speculate that mRNA injection results in a higher level of EWS-FLI1
expression as well as an earlier onset of expression that might account for some of these differences.
This animal model of EWS-FLI1 function is amenable to future pathway and genetic analysis and provides a new approach to dissecting the role of EWS-FLI1. Genes that contribute to cancer frequently have crucial roles in embryonic development; therefore, elucidating the mechanism by which EWS-FLI1 perturbs development will also shed light on its contribution to tumorigenesis. Importantly, these zebrafish models represent the first vertebrate model for EWS-FLI1 function that is amenable to genetic and chemical screening. A system to perform suppressor screens for EWS-FLI1-induced phenotypes in a whole animal context has until now, been elusive. Future genetic and chemical screens for EWS-FLI1 suppressors in the zebrafish should reveal novel downstream effectors and therapeutics for the treatment of Ewing’s sarcoma.