EWS/Fli‐1 chimeric fusion gene upregulates vascular endothelial growth factor‐A

Abstract

Vascular endothelial growth factor (VEGF)‐A plays an important role in the pathological angiogenesis that occurs in soft‐tissue sarcoma and in about half of Ewing's sarcoma cases, where it is highly overexpressed. EWS/Fli‐1 is considered to be a transcriptional activator and to play a significant role in tumorigenesis of Ewing's sarcoma. However, the relationship between EWS/Fli‐1 and VEGF‐A is still unclear. The aim of this research is to investigate the relationship between EWS/Fli‐1 and VEGF‐A, and to determine whether small interfering RNA (siRNA)‐targeting of VEGF‐A can be developed as a novel treatment for Ewing's sarcoma. Knockdown of EWS/Fli‐1 using siRNA on a Ewing's sarcoma cell line (A673) suppressed VEGF‐A expression, and transfection of EWS/Fli‐1 into a human osteosarcoma cell line increased VEGF‐A expression. To inhibit VEGF‐A secretion from Ewing's sarcoma, we developed a chemically synthesized siRNA that targets VEGF‐A. Transfection of the VEGF siRNA into the Ewing's sarcoma cell line significantly suppressed VEGF‐A secretion by up to 98% in vitro, compared with a control. In vivo, we established Ewing's sarcoma xenograft models and performed intratumoral injection of the siRNA mixed with atelocollagen. We observed that the inhibition of tumor growth occurs in a dose‐dependent manner. Histological examination revealed decreased microvessel density and morphological change around microvessels in the Ewing's sarcoma xenografts treated with the siRNA. It is considered that a combination of chemically synthesized siRNA that targets VEGF‐A and atelocollagen might be a novel and effective option for treating Ewing's sarcoma that secretes VEGF‐A.

Expression of therapeutic targets in Ewing sarcoma family tumors

“…The angiogenic growth factor VEGF has been identified in ESFT cell lines where it was shown to contribute to tumor vasculogenesis [30], and inhibition or blockade of VEGF resulted in suppression of Ewing sarcoma tumor growth [31][32][33]. These findings have led some investigators to advocate for the use of the anti-VEGF inhibitor bevacizumab in treating ESFT [33].…”

Overexpression of Fli-1 in astrocytoma is associated with poor prognosis

“…Epithelial cells have been reported to express high levels of Fli-1 [30], a feature that has also been observed in the majority of benign and malignant vascular tumors [31–33]. The EWS-Fli-1 fusion protein directly activates VEGF-A, leading to increased angiogenesis and malignant progression [34]. High-levels of VEGF, which plays a critical role in tumor initiation, have also been detected in the tumor microenvironments of Fli-1-overexpressing erythroleukemias [35].…”

“…ETS family transcription factors regulate the expression of oncogenes, tumor suppressor genes, and other genes associated with vessel formation, invasion, and metastasis, and their expression often correlates with poor survival in some types of cancers [21, 37–39]. Fli-1 plays a critical role in normal development, hematopoiesis and oncogenesis by functioning as both transcriptional activator and repressor [34, 40, 41]. Knocking-down Fli-1 expression in erythroleukemic cells leads to a marked growth inhibition and cell death, demonstrating a possible therapeutic approach to induce tumor suppression [42, 43].…”

Englerin A Inhibits EWS-FLI1 DNA Binding in Ewing Sarcoma Cells

“…Reagents-Synthetic EWS-FLI1 siRNA were AGCAGAAC-CCUUCUUAUGACUU (sense) and GUCAUAAGAAGGGU-UCUGCUUU (antisense) (8). RNAi directed at TRPC4 (sc-42668) and TRPC5 (sc-42670) were commercially obtained from Santa Cruz Biotechnologies.…”