EWS-Fli1 fusion gene as a diagnostic and therapeutic molecule for Ewing's sarcoma and PNET

易位t(11；22)(q24：q12)是尤文肉瘤(ES)和原始神经外胚层肿瘤(PNET)中发现的一种特殊的染色体异常。易位导致了EWS-FLI1融合基因，该融合基因由22号染色体上EWS基因5‘端的一半与11号染色体上的FLI1基因的3’端融合而成。最近的研究评估了融合基因产物作为异常转录因子的转化潜力。然而，EWS-FLI1的生物学意义仍不清楚。利用竞争性聚合酶链式反应技术，我们发现EWS-FLI1融合基因的表达水平可能与ES/PNET细胞的增殖活性有关。当针对融合基因的反义寡核苷酸(AS)抑制EWS-FLI1的表达时，ES/PNET细胞在体内外的生长均显著降低。流式细胞仪分析表明，AS对细胞的生长抑制作用是通过G1arres…介导的。在细胞周期进程中有更多的T。在本研究中，我们研究了AS对细胞周期调控因子的影响。我们发现EWS-FLI1上调了G1期细胞周期蛋白，包括细胞周期蛋白D1和细胞周期蛋白E，而下调了CDK抑制剂p21和p27的表达。荧光素酶分析和凝胶漂移实验表明，EWS-FLI1直接与p21基因启动子中的ETS共有序列结合并抑制其活性。组蛋白去乙酰酶抑制剂(HDACi)通过诱导癌细胞中p21的表达抑制ES/PNET细胞的生长。P27在ES/PNET组织中的低表达与患者的临床预后密切相关。P27表达载体的导入可明显抑制ES/PNET细胞的生长。我们还利用基因芯片技术筛选了EWS-FLI1在G1-S细胞周期调控中的靶基因。基因表达分析表明，AS处理后有12个基因显著上调，而E2F1等6个基因表达下调。在这些可能的EWS-Fli-1靶基因中，我们重点研究了E2F1，因为它在G1-S转换过程中起着核心作用。Western印迹和RT-PCR分析证实，AS可显著抑制ES/PNET细胞中E2F1的表达。将E2F-诱骗寡核苷酸导入ES/PNET细胞后，细胞的生长受到显著抑制，仅为对照组和突变诱骗处理细胞的40%以下。这些结果提示细胞周期蛋白D1、细胞周期蛋白E、p21、p27和E2F1可能是EWS-FLI1嵌合转录因子的靶基因。抑制EWS-FLI1下游信号可能导致ES/PNET患者的分子靶向治疗。较少

The translocation t(11;22)(q24:q12) is a specific chromosomal abnormality detected in Ewing's sarcoma (ES) and Primitive Neuroectodermal Tumor (PNET). The translocation results in an EWS-Fli1 fusion gene, made up of the 5' half of the EWS gene on chromosome 22 fused to the 3' half of the Fli1 gene on chromosome 11. Recent studies have evaluated transforming potentials of the fusion gene products acting as an aberrant transcription factor. However, the biological significance of EWS-Fli1 is still unknown. Using a competitive PCR technique, we have found that there might be a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of ES/PNET cells. When the EWS-Fli1 expression was inhibited by an antisense oligonucleotide (AS) against the fusion gene, the growth of the ES/PNET cells was significantly reduced both in vitro and in vivo. The flow cytometry analysis indicated that the growth inhibition of the cells by AS was mediated by G1 arres … More t in the cell cycle progression. In the present study, we examined the effects of AS on the cell cycle regulatory factors. We found that G1 cyclins including cyclin D1 and cyclin E were upregulated by EWS-Fli1, whereas CDK inhibitors, p21 and p27, were downregulated. The luciferase analysis and gel shift assay revealed that EWS-Fli1 directly bound to the Ets consensus sequences in p21 gene promoter and inhibited its activity. Histone deacetylase inhibitors (HDACI) which are known to induce p21 expression in cancer cells inhibited ES/PNET cell growth via induction of p21 expression. The low level expression of p27 in ES/PNET specimen had strong correlation with worse clinical prognosis of the patients. Introduction of p27 expression vector markedly inhibited the growth of ES/PNET cells. We also used cDNA microarray analysis to identify target genes of EWS-Fli1 in G1-S cell cycle regulation. The gene expression analysis showed that 12 genes were significantly up-regulated after the AS treatment, whereas 6 genes including E2F1 were down-regulated by the treatment. Among these possible EWS-Fli1-target genes, we focused on E2F1 since it play central role in the regulation of G1-S transition. Western blot and RT-PCR analyses confirmed that E2F1 expression in ES/PNET cells was significantly inhibited by AS treatment. When E2F-decoy oligonucleotides were transfected into ES/PNET cells, growth of the cells was significantly inhibited to less than 40 % of control and mutant-decoy treated cells. These results suggest that cyclin D1, cyclin E, p21, p27 and E2F1 might be the target genes of EWS-Fli1 chimeric transcription factor. Inhibition of EWS-Fli1 downstream signals might lead to the molecular target therapy for the treatment of ES/PNET patients. Less