ABSTRACT
The EWS/Fli-1 fusion gene, a product of the translocation t(11;22, q24;q12), is detected in 85% of Ewing sarcomas and primitive neuroectodermal tumors. It is thought to be a transcriptional activator that plays a significant role in tumorigenesis. In this study, we developed a novel EWS/Fli-1 blockade system using RNA interference and tested its application for inhibiting the proliferation of Ewing sarcoma cells in vitro and the treatment of mouse tumor xenografts in vivo. We designed and synthesized a small interfering RNA (siRNA) possessing an aromatic compound at the 3'-end targeting the breakpoint of EWS/Fli-1. As this sequence is present only in tumor cells, it is a potentially relevant target. We found that the siRNA targeting EWS/Fli-1 significantly suppressed the expression of EWS/Fli-1 protein sequence specifically and also reduced the expression of c-Myc protein in Ewing sarcoma cells. We further demonstrated that inhibition of EWS/Fli-1 expression efficiently inhibited the proliferation of the transfected cells but did not induce apoptotic cell death. In addition, the siRNA possessing the aromatic compound at the 3'-end was more resistant to nucleolytic degradation than the unmodified siRNA. Administration of the siRNA with atelocollagen significantly inhibited the tumor growth of TC-135, a Ewing sarcoma cell line, which had been subcutaneously xenografted into mice. Moreover, modification of the 3'-end with an aromatic compound improved its efficiency in vivo. Our data suggest that specific downregulation of EWS/Fli-1 by RNA interference is a possible approach for the treatment of Ewing sarcoma.
Subject(s)
Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA, Small Interfering/genetics , Sarcoma, Ewing/therapy , Xenograft Model Antitumor Assays/methods , Animals , Apoptosis , Base Sequence , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Chromosome Breakpoints , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Structure , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/chemical synthesis , RNA-Binding Protein EWS , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathology , Tumor Burden/geneticsABSTRACT
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.
Subject(s)
Bone Neoplasms/metabolism , Gene Expression Regulation/physiology , Gene Fusion , Oncogene Proteins, Fusion/genetics , Osteosarcoma/metabolism , Sarcoma, Ewing/metabolism , Vascular Endothelial Growth Factor A/metabolism , Alternative Splicing , Animals , Antineoplastic Combined Chemotherapy Protocols , Blotting, Western , Bone Neoplasms/genetics , Bone Neoplasms/prevention & control , Collagen/pharmacology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoenzyme Techniques , Mice , Osteosarcoma/genetics , Osteosarcoma/prevention & control , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/genetics , Sarcoma, Ewing/prevention & control , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Ewing sarcoma cells, of which over 85% retain chimeric fusion gene EWS/Fli-1, are by and large more resistant to chemotherapeutics compared to nonneoplastic cells. The purpose of this study is to determine the role of EWS/Fli-1 fusion and its downstream targets regarding the cells' resistance against actinomycin D (ActD), which is one of the most commonly used antitumor agents in combination chemotherapy of Ewing sarcomas. METHODS: Cytotoxicity was measured by WST-8 assay. Caspase-dependent and -independent cell death was examined by fluorescence microscope. Protein expression was analyzed by western blotting. Caspase activity was determined by Caspase-Glo assay. RESULTS: ActD-induced caspase-dependent apoptotic cell death to Ewing sarcoma TC-135 cells in a dose- and time- dependent manner. Knockdown of EWS/Fli-1 fusion by siRNA resulted in enhancement of ActD-induced apoptosis. ActD treatment activated both mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways although in a distinctive manner. Combined administration of U0126 (MEK inhibitor) and LY294002 (PI3K inhibitor) significantly enhanced ActD-induced apoptosis in vitro and suppressed xenograft tumor growth in vivo. CONCLUSIONS: The present study demonstrated for the first time that combination of U0126 and LY294002 can augment the cytotoxicity of ActD against Ewing sarcoma cells in vitro and in vivo. Our results indicate that further study on combination of conventional chemotherapies with MEK and PI3K inhibitors may be considered for innovative treatments of Ewing sarcoma patients.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Neoplasms/drug therapy , Dactinomycin/therapeutic use , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Sarcoma, Ewing/drug therapy , Animals , Apoptosis , Bone Neoplasms/enzymology , Butadienes/therapeutic use , Cell Death , Cell Line, Tumor , Chromones/therapeutic use , Drug Resistance, Neoplasm , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Mitogen-Activated Protein Kinases/metabolism , Morpholines/therapeutic use , Nitriles/therapeutic use , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Protein c-fli-1 , RNA, Small Interfering/metabolism , RNA-Binding Protein EWS , Sarcoma, Ewing/enzymology , Signal Transduction/drug effects , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , TransfectionABSTRACT
EWS-Fli1, a fusion gene resulting from the chromosomal translocation t(11;22, q24;q12), encodes a transcriptional activator, promotes cellular transformation, and is often found in Ewing sarcoma and primitive neuroectodermal tumor. The Aurora A and Aurora B kinases belong to a highly conserved family of serine/threonine protein kinases, are tightly regulated during the cell cycle, and are overexpressed in many carcinomas. Because the relationship between the Aurora A and/or Aurora B genes and the EWS-Fli1 fusion gene is unknown, we investigated the regulatory mechanism(s) by which Aurora kinases are controlled. Knockdown of EWS-Fli1 by small interfering RNA reduced mRNA levels not only of EWS-Fli1 but also of Aurora A and Aurora B. Luciferase assay using Aurora A and Aurora B promoters showed up-regulated activities compared with those of an empty vector. Experiments with deletion and point mutants showed positive regulatory Ets-binding sites located -84 and -71 bp upstream of the transcription initiation sites in Aurora A and Aurora B, respectively. Moreover, chromatin immunoprecipitation assay revealed that EWS-Fli1 gene products interact with both the Aurora A and Aurora B promoters. These results strongly suggest that the mitotic kinases Aurora A and Aurora B are regulated by EWS-Fli1 fusion protein in Ewing sarcoma cells.