Dysregulated heparan sulfate proteoglycan metabolism promotes Ewing sarcoma tumor growth
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Evaluation Summary:
This manuscript describes a model of Ewing sarcoma , which represents an improvement upon previous zebrafish sarcoma models and the data suggest that the methods employed yield tumours that resemble human disease. This new model may be used to better understand sarcoma progression so that new therapeutic targets may be realised.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The Ewing sarcoma family of tumors is a group of malignant small round blue cell tumors (SRBCTs) that affect children, adolescents, and young adults. The tumors are characterized by reciprocal chromosomal translocations that generate chimeric fusion oncogenes, the most common of which is EWSR1-FLI1. Survival is extremely poor for patients with metastatic or relapsed disease, and no molecularly targeted therapy for this disease currently exists. The absence of a reliable genetic animal model of Ewing sarcoma has impaired investigation of tumor cell/microenvironmental interactions in vivo. We have developed a new genetic model of Ewing sarcoma based on Cre-inducible expression of human EWSR1-FLI1 in wild-type zebrafish, which causes rapid onset of SRBCTs at high penetrance. The tumors express canonical EWSR1-FLI1 target genes and stain for known Ewing sarcoma markers including CD99. Growth of tumors is associated with activation of the MAPK/ERK pathway, which we link to dysregulated extracellular matrix metabolism in general and heparan sulfate proteoglycan catabolism in particular. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of Ewing sarcoma cells in vitro and in vivo. These results highlight the important role of the extracellular matrix in Ewing sarcoma tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease.
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Evaluation Summary:
This manuscript describes a model of Ewing sarcoma , which represents an improvement upon previous zebrafish sarcoma models and the data suggest that the methods employed yield tumours that resemble human disease. This new model may be used to better understand sarcoma progression so that new therapeutic targets may be realised.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The current manuscript describes a model of Ewing sarcoma wherein the cre-inducible expression of human EWSR1-FLI1 in wild type zebrafish causes the rapid onset of small round blue cell tumors (SRBCTs). The tumors appear to resemble human tumors, expressing CD99 as well as elevated ERK1/2 signaling. Proteomics indicated that progression was associated with dysregulated extracellular matrix metabolism in general and heparan sulfate catabolism in particular. Accordingly, targeting heparan sulfate proteoglycans with Surfen reduced ERK1/2 signaling and tumour cell growth in vitro and in the zebrafish model. Overall, this study reveals a model that may be used to better understand the evolution of sarcoma. However, greater comparisons may be needed in order for this model to be used as a model of human disease.
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Reviewer #2 (Public Review):
The authors of this paper have generated an inducible transgenic zebrafish that expresses human EWSR1-FLI1, the most common oncofusion in Ewing sarcoma, a common bone and soft tissue cancer in children and adolescents. These zebrafish represent the first in vivo genetic model of Ewing sarcoma that develops spontaneous tumors solely from expression of the oncofusion, with tumor formation in defined anatomic sites. Specifically, these tumors tested positive for CD99 expression and PAS staining, two diagnostic markers of Ewing sarcoma. Additionally, increased ERK1/2 signaling was observed in both the initial cellular outgrowths and mature adult tumors and transgenic embryos displayed an upregulation of proteins associated with extracellular matrix reorganization, proteoglycan metabolism, and protein synthesis. …
Reviewer #2 (Public Review):
The authors of this paper have generated an inducible transgenic zebrafish that expresses human EWSR1-FLI1, the most common oncofusion in Ewing sarcoma, a common bone and soft tissue cancer in children and adolescents. These zebrafish represent the first in vivo genetic model of Ewing sarcoma that develops spontaneous tumors solely from expression of the oncofusion, with tumor formation in defined anatomic sites. Specifically, these tumors tested positive for CD99 expression and PAS staining, two diagnostic markers of Ewing sarcoma. Additionally, increased ERK1/2 signaling was observed in both the initial cellular outgrowths and mature adult tumors and transgenic embryos displayed an upregulation of proteins associated with extracellular matrix reorganization, proteoglycan metabolism, and protein synthesis. This led the authors to examine the efficacy of surfen, a sulfated heparan sulfate antagonist that interferes with proteoglycan signaling, which resulted in the impairment of ERK1/2 signalling and decreased proliferation and survival in two Ewing sarcoma cell lines. In vivo, surfen treatment inhibited the formation of EWSR1-FLI1-associated cellular outgrowths in zebrafish embryos and rescued abnormal fin shape in transgenic larvae. Thus, this Ewing sarcoma transgenic zebrafish line may shed light on additional mechanisms contributing to tumorigenesis and can serve as a robust drug screening tool to further identify therapies to target Ewing sarcoma.
Strengths
Overall, this is a well-written manuscript. The authors did a thorough job showing their novel zebrafish transgenic line recapitulates Ewing sarcoma phenotypes and that these phenotypes were specific to the expression of the oncofusion. Their data nicely shows the comparable histology between the zebrafish and human tumors, especially as they are the first group to demonstrate the successful immunohistochemical staining with CD99, diagnostic (but not entirely specific) for Ewing sarcoma. Additionally, they have described a quantifiable phenotypic readout (Ccurv analysis) for drug discovery that can be used to determine the efficacy of potential therapeutic compounds for Ewing sarcoma. This study demonstrates the utility of using a representative zebrafish model to uncover key mechanisms of disease progression and opportunities for molecularly targeting these mechanisms to rescue disease phenotypes.
Weaknesses
The ability of surfen treatment to cause morphological changes and inhibit cell proliferation and survival in two Ewing sarcoma cell lines would need to be further compared to other cancer cell lines, especially those that do not have upregulated ERK1/2 signaling to determine specificity of proteoglycan metabolism in Ewing sarcoma progression. Surfen treatment decreased expression of active ERK1/2 in the Ewing sarcoma cell lines, and a similar approach should be used to investigate if decreased pERK1/2 expression is observed in vivo in correlation with decreased cellular outgrowths. Additionally, images showing the ability of surfen to rescue normal fin curvature would make these results more compelling.
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Reviewer #3 (Public Review):
In this manuscript, Vasileva et al developed an Ewing sarcoma in vivo model by introducing EWSR1-FLI1 fusion oncogene into zebrafish. They used the Cre/loxp system to delay the expression of the oncoprotein, thus avoided the developmental toxicity of EWSR1-FLI1 which they observed in previous studies. Injected zebrafish developed malignant small round blue cell tumors similar to human Ewing sarcoma at multiple levels, including histology, immunohistology of Ewing markers and elevated ERK1/2 signaling. Embryonic proteomics analysis showed that proteins involved in ECM and proteoglycan metabolism were alternated by EWSR1-FLI1 in zebrafish embryos, especially the enzymes involved in heparan sulfate proteoglycan catabolism. Similar signatures were also identified in a human Ewing sarcoma microarray dataset. …
Reviewer #3 (Public Review):
In this manuscript, Vasileva et al developed an Ewing sarcoma in vivo model by introducing EWSR1-FLI1 fusion oncogene into zebrafish. They used the Cre/loxp system to delay the expression of the oncoprotein, thus avoided the developmental toxicity of EWSR1-FLI1 which they observed in previous studies. Injected zebrafish developed malignant small round blue cell tumors similar to human Ewing sarcoma at multiple levels, including histology, immunohistology of Ewing markers and elevated ERK1/2 signaling. Embryonic proteomics analysis showed that proteins involved in ECM and proteoglycan metabolism were alternated by EWSR1-FLI1 in zebrafish embryos, especially the enzymes involved in heparan sulfate proteoglycan catabolism. Similar signatures were also identified in a human Ewing sarcoma microarray dataset. Based on these findings, the authors used a sulfated heparan sulfate antagonist, surfen, to treat human Ewing sarcoma cell lines and observed reduction of pERK1/2, cell proliferation and colony formation. Subsequently, they added surfen to the fish water containing the injected zebrafish to evaluate the in vivo tumor suppression effect of this compound. This is an interesting and important study defining the first robust animal model of Ewing sarcoma caused by EWSR1-FLI in vivo. And the authors use the model to document changes in protein expression that suggest treatment with a novel agent that is in use in humans for another condition.
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