Abstract
Simple SummaryEwing sarcoma is an aggressive tumor with still unacceptable survival rates, particularly in patients with metastatic disease and for which it is necessary to develop new and innovative therapies. These tumors are characterized by the presence of chromosomal translocations that give rise to chimeric transcription factors (i.e., EWSR1–FLI1) that govern the oncogenic process. In this article, we describe an efficient strategy to permanently inactivate the EWSR1–FLI1 oncogene characteristic of Ewing sarcoma using CRISPR/Cas9 gene editing technology. Although the application of gene therapy in cancer still has many limitations, for example, the strategy for delivery, studies like ours show that gene therapy can be a promising alternative, particularly for those tumors that are highly dependent on a particular oncogene as is the case in Ewing sarcoma.Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1–FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1–FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1–FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1–FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1–FLI1 at the cell level should be considered a therapeutic approach to develop in the future.
Highlights
Ewing sarcoma is a highly aggressive rare bone and soft tissue tumor arising in children and young adults [1]
We chose the cell line A673 as a model to analyze the effect of EWSR1–FLI1 gene inactivation in Ewing sarcoma cells, since this cell line is probably the Ewing sarcoma cell line more broadly used to study Ewing sarcoma pathogenesis, with regard to the mechanism of EWSR1–FLI1-mediated oncogenesis
EWSR-FLI1 silencing in A673/TR/shEF produced a significant increase in the percentage of cells in the G1 phase in comparison to control cells (42%) but to a lesser extent than that observed in A673/Cas9/FLI1-EX9. These results demonstrate that EWSR1–FLI1 gene inactivation by gene editing produces a practically complete blockage of cell proliferation
Summary
Ewing sarcoma is a highly aggressive rare bone and soft tissue tumor arising in children and young adults [1]. From a molecular perspective, Ewing sarcoma is characterized by reciprocal chromosomal translocations that give rise to fusion proteins that govern tumorigenesis [1]. The most frequent of these fusions proteins is EWSR1–FLI1 in which the N-terminal region of the EWSR1 gene is fused to the C-terminal region of the transcription factor FLI1 containing the DNA binding domain [6]. Other chromosomal aberrations are rare and point mutations affecting oncogenes or tumor suppressor genes are even less frequent [7,8]. In light of this data, it is thoroughly accepted that these chimeric transcription factors are the main oncogenic drivers in Ewing sarcoma [1]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
