Synthetic siRNA targeting the breakpoint of EWS/Fli-1 inhibits growth of Ewing sarcoma xenografts in a mouse model.

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.

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

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.

Simultaneous inhibition of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways augment the sensitivity to actinomycin D in Ewing sarcoma.

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.

EWS-Fli1 up-regulates expression of the Aurora A and Aurora B kinases.

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.

Symptomatic lumbar mobile segment with spinal canal stenosis in a fused spine associated with diffused idiopathic skeletal hyperostosis.

BACKGROUND CONTEXT: Chronic, continuous stress at the junction of a stable/unstable site of the spine in diffuse idiopathic skeletal hyperostosis (DISH) has been reported to cause a nonunion. Back pain resulting from the nonunion has been rarely reported and few operative treatments have been suggested. PURPOSE: To report and discuss the pathogenesis, treatment, and surgical outcome of a rare cause of back pain. STUDY DESIGN: Case report of back pain caused by a single lumbar segment is lacking bony union at the caudal end of a fused spine associated with diffuse idiopathic skeletal hyperostosis. METHODS: Back pain in a 66-year-old man who had suffered for 10 years worsened. The back pain and thigh pain became intolerable, and the left buttock and thigh became numb. Radiographs and computed tomography images showed continuous hyperostosis in the anterior aspect of the vertebral bodies from C2 to L2. At the caudal adjacent level of these fused segments, L2/3 level was mobile and had canal stenosis. Decompression and posterior lumbar interbody fusion (PLIF) were performed. RESULTS: The pain disappeared soon after the operation. The nonunited segment showed bony union at the 5-year follow-up. CONCLUSIONS: PLIF may be an option for surgically treating symptomatic nonunited lumbar segments at the caudal end of a fused spine with DISH in cases unresponsive to conservative treatment.

Solitary fibrous tumor in the cervical spine with destructive vertebral involvement: a case report and review of the literature.

INTRODUCTION: Recently, solitary fibrous tumors occurring in spine-related lesions have been reported. However, the destruction of vertebral bodies by this type of tumor has not been reported. MATERIALS AND METHODS: A 71-year-old female presented with pain from a mass on the right side of her neck. Plain radiographs of the cervical spine showed collapse of the C5 vertebral body and dislocation of the C4 vertebral body. The MRI image showed a large mass surrounding C4 and C5, which had low signal intensity in the T1W image and high signal intensity in the T2W image. At first, resection of the tumor and spinal fusion was performed by anterior approach. RESULTS: Histology revealed a solitary fibrous tumor with proliferating spindle cells. Immunohistochemistry showed positive stains for vimentin and CD34. One year postoperatively, a local recurrence manifested extensive destruction of the C4 and C5 vertebral bodies. Then, palliative surgery with posterior cervical instrumentation and radiation therapy were performed. Because the destruction proceeded and the rods were broken 2 years after, she underwent additional occipito-cervical instrumentation. CONCLUSIONS: This is the first report of a solitary fibrous tumor that involves the destruction of the spinal structure. An extensive destruction of the vertebral body by the solitary fibrous tumor needs to be aware in treating this tumor with spinal involvement.