Combined transcriptional and translational targeting of EWS/FLI-1 in Ewing's sarcoma.

PURPOSE: To show the efficacy of targeting EWS/FLI-1 expression with a combination of specific antisense oligonucleotides and rapamycin for the control of Ewing's sarcoma (EWS) cell proliferation in vitro and the treatment of mouse tumor xenografts in vivo. EXPERIMENTAL DESIGN: EWS cells were simultaneously exposed to EWS/FLI-1-specific antisense oligonucleotides and rapamycin for various time periods. After treatment, the following end points were monitored and evaluated: expression levels of the EWS/FLI-1 protein, cell proliferation, cell cycle distribution, apoptotic cell death, caspase activation, and tumor growth in EWS xenografts implanted in nude mice. RESULTS: Simultaneous exposure of EWS cells in culture to an EWS/FLI-1-targeted suppression therapy using specific antisense oligonucleotides and rapamycin resulted in the activation of a caspase-dependent apoptotic process that involved the restoration of the transforming growth factor-beta-induced proapoptotic pathway. In vivo, individual administration of either antisense oligonucleotides or rapamycin significantly delayed tumor development, and the combined treatment with antisense oligonucleotides and rapamycin caused a considerably stronger inhibition of tumor growth. CONCLUSIONS: Concurrent administration of EWS/FLI-1 antisense oligonucleotides and rapamycin efficiently induced the apoptotic death of EWS cells in culture through a process involving transforming growth factor-beta. In vivo experiments conclusively showed that the combined treatment with antisense oligonucleotides and rapamycin caused a significant inhibition of tumor growth in mice. These results provide proof of principle for further exploration of the potential of this combined therapeutic modality as a novel strategy for the treatment of tumors of the Ewing's sarcoma family.     

A link between basic fibroblast growth factor (bFGF) and EWS/FLI-1 in Ewing's sarcoma cells

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301     

Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation

Ewing's sarcoma (ES) is the prototype of a family of tumors (ESFT) of neuroectodermal origin formed by small, round cells with limited neural differentiation, which arise most frequently within bones in children or adolescents. The proliferation of ESFT cells is highly dependent on the establishment of, and signaling through several growth factor-mediated autocrine loops. The mammalian target of rapamycin (mTOR) is a central regulator of translation and cell proliferation, involved in the cellular response to various nutritional, stress and mitogenic effectors. As mTOR has recently been associated with certain human cancers, we investigated the possibility that mTOR played a role in the regulation of ES cell proliferation. Results showed that ES cell lines carrying EWS/FLI-1 alleles of different types expressed different levels of total and phosphorylated mTOR protein. We demonstrate that rapamycin, an mTOR inhibitor, efficiently blocked the proliferation of all cell lines by promoting cell cycle arrest at the G1 phase. This was paralleled by the downregulation of the levels of the EWS/FLI-1 proteins, regardless of their fusion type, and the concomitant restoration of the expression of the TGF-beta type 2 receptor (TGFbeta RII), which is known to be repressed by several EWS-ETS fusion proteins. The expression of a rapamycin-resistant mTOR construct prevented both the proliferation blockade and the EWS/FLI-1 downregulation. These data demonstrate that mTOR signaling plays a central role in ES cell pathobiology and strongly suggest that the use of rapamycin as a cytostatic agent may be an efficient tool for the treatment of ES patients.     

High expression of neuropeptide Y1 receptors in ewing sarcoma tumors

PURPOSE: Peptide receptors are frequently overexpressed in human tumors, allowing receptor-targeted scintigraphic imaging and therapy with radiolabeled peptide analogues. Neuropeptide Y (NPY) receptors are new candidates for these applications, based on their high expression in specific cancers. Because NPY receptors are expressed in selected sarcoma cell lines and because novel treatment options are needed for sarcomas, this study assessed the NPY receptor in primary human sarcomas. EXPERIMENTAL DESIGN: Tumor tissues of 88 cases, including Ewing sarcoma family of tumors (ESFT), synovial sarcomas, osteosarcomas, chondrosarcomas, liposarcomas, angiosarcomas, rhabdomyosarcomas, leiomyosarcomas, and desmoid tumors, were investigated for NPY receptor protein with in vitro receptor autoradiography using (125)I-labeled NPY receptor ligands and for NPY receptor mRNA expression with in situ hybridization. RESULTS: ESFT expressed the NPY receptor subtype Y1 on tumor cells in remarkably high incidence (84%) and density (mean, 5,314 dpm/mg tissue). Likewise, synovial sarcomas expressed Y1 on tumor cells in high density (mean, 7,497 dpm/mg; incidence, 40%). The remaining tumors expressed NPY receptor subtypes Y1 or Y2 at lower levels. Moreover, many of the sarcomas showed Y1 expression on intratumoral blood vessels. In situ hybridization for Y1 mRNA confirmed the autoradiography results. CONCLUSIONS: NPY receptors are novel molecular markers for human sarcomas. Y1 may inhibit growth of specific sarcomas, as previously shown in an in vivo mouse model of human ESFT. The high Y1 expression on tumor cells of ESFT and synovial sarcomas and on blood vessels in many other sarcomas represents an attractive basis for an in vivo tumor targeting.     

FLI-1在星形细胞瘤组织中的表达及与预后的关系

摘 要：［目的］ 探讨FLI-1在星形细胞瘤手术患者肿瘤组织中的表达及其与患者预后的关系。［方法］ 选取104例星形细胞瘤手术患者作为研究对象,采用免疫组化技术分析患者肿瘤组织及癌旁组织的FLI-1表达水平,分析FLI-1表达水平与临床病理特征及预后的相关性,并分析星形细胞瘤患者的预后影响因素。［结果］ 星形细胞瘤组织中FLI-1高表达比例（74.0%）显著高于癌旁组织（11.5%）（P<0.001）。FLI-1表达水平与星形细胞瘤患者的WHO分级显著相关（P<0.001）。FLI-1低表达的星形细胞瘤患者中位生存期（35.3±5.6个月）和3年生存率（44.4%）均高于FLI-1高表达患者（8.9±2.1个月,13.0%;P均＜0.05）。Cox单因素和多因素分析结果均显示WHO分级及FLI-1表达水平是星形细胞瘤患者的预后影响因素（P＜0.05）。［结论］ 星形细胞瘤组织中FLI-1表达水平显著升高,且与患者预后相关,是一个潜在的预后指标。     

Expression of human CSF-1 receptor induces CSF-1-dependent proliferation in murine myeloid but not in T-lymphoid cells.

The receptor for human macrophage colony stimulating factor (CSF-1R) was introduced into hematopoietic cell lines of myeloid and T-lymphoid origin, both of which normally do not express the CSF-1R. Infection of an interleukin-3 (IL-3)-dependent mouse myeloid cell line (FDC-P1) with a high titer retroviral vector expressing the human c-fms c-DNA, enabled CSF-1-dependent proliferation in short-term liquid culture assays as well as in clonal culture systems. CSF-1-dependent cell lines could be established after sorting for CSF-1R positive cells. In contrast to FDC-P1 cells, expression of the CSF-1R in CTLL cells, an IL-2-dependent mouse cytotoxic T-cell line, and in T-cell growth factor III/P40-dependent helper T-cells, ST2/K9.4a2, did not lead to CSF-1-dependent proliferation. These observations lead to the conclusion that ectopically expressed CSF-1R may function on certain myeloid cells where it is normally not expressed, suggesting the presence of signal transduction pathways which can be utilized by that foreign receptor. In contrast, it appears that T-lymphoid cells lack such a signalling mechanism, indicating that quite different modes of transducing mitogenic signals from the cell membrane to the nucleus must have developed during myeloid and T-lymphoid differentiation.     

FLI-1、p63蛋白在肺癌中表达的研究

目的研究FLI-1、p63蛋白在肺鳞状细胞癌、腺癌及小细胞癌中表达情况及其意义.方法收集52例肺癌（鳞癌24例、腺癌18例、小细胞癌10例）及20例正常肺组织,采用免疫组化（EnVision-plus法）进行FLI-1、p63抗体标记.结果 FLI-1抗体阳性细胞定位在细胞核,在鳞癌、腺癌和小细胞癌的阳性率分别为9.1%、5.9%和90.0%,肺小细胞癌分别与肺鳞癌及腺癌相比差异具有显著性（P＜0.01）.FLI-1抗体诊断小细胞癌的敏感性为90.0%,特异性为92.8%;p63抗体阳性细胞定位在细胞核,在正常支气管黏膜、肺鳞癌、腺癌和小细胞癌阳性率分别为20.0%、91.7%、16.7%和80.0%,肺鳞癌与正常支气管上皮及腺癌比较差异有显著性（P＜0.05）,与小细胞癌相比差异无显著性（P＞0.05）.结论 FLI-1为诊断小细胞肺癌有用的抗体,p63在肺鳞状细胞癌及小细胞癌中表达上调,两者联合运用可以对肺癌鉴别诊断.