Analysis on oncogenic mechanisms and therapeutic targets in Ewing's sarcoma

易位t(11;22)（q24:q12）是在尤文氏肉瘤（ES）中发现的一种特异性染色体异常。易位产生了EWS-Fli1融合基因，由22号染色体上EWS基因的5‘一半与11号染色体上Fli1基因的3’一半融合而成。最近的研究评估了作为异常转录因子的融合基因产物的转化潜力。然而，EWS-Fli1的生物学意义尚不清楚。我们已经报道了G1周期蛋白，包括cyclin D1和cyclin E被EWS-Fli1上调，而CDK抑制剂p21和p27则下调。这些分子位于视网膜母细胞瘤肿瘤抑制基因Rb的上游，因此EWS-Fli1可能影响ES中Rb通路导致肿瘤的发生。另一方面，在ES中p53通路的异常尚未得到很好的分析。在本研究中，我们研究了EWS-Fli1对ES细胞中p53通路及其功能的影响，特别是其诱导凋亡的作用。免疫沉淀实验显示EWS-Fli1与p53蛋白结合，并抑制其在p53野生型ES细胞中的功能。而EWS-Fli1对p53转录因子下游凋亡相关基因的表达无显著影响。EWS-Fli1通过抑制p21基因启动子活性，抑制p53靶基因之一p21的表达。因此，我们假设组蛋白去乙酰化酶抑制剂（HDACI）在癌细胞中诱导p21表达可能对ES细胞有效。HDACI通过诱导p21在体内和体外表达抑制ES细胞生长。然而，某种类型的HDACI对耐药ES细胞表现出交叉耐药。因此，在临床试验中应高度重视胚胎干细胞对HDACIs的耐药性。我们还研究了siRNA敲低EWS-Fli1表达对诱导胚胎干细胞凋亡的影响。siRNA攻击EWS-Fli1对胚胎干细胞没有诱导凋亡，但诱导胚胎干细胞衰老。这一发现提示了EWS-Fli1的新功能，即EWS-Fli1可能抑制ES细胞的衰老诱导，从而导致ES的癌变。这些结果表明，抑制EWS-Fli1的这些功能可能有助于开发治疗ES患者的分子靶向疗法。少

The translocation t(11;22)(q24:q12) is a specific chromosomal abnormality detected in Ewing's sarcoma (ES). 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. We have reported that G1 cyclins including cyclin D1 and cyclin E were upregulated by EWS-Fli1, whereas CDK inhibitors, p21 and p27, were downregulated. These molecules are located in the upstream of retinoblastoma tumor suppressor gene Rb, therefore EWS-Fli1 might affect Rb pathway in ES leading to oncogenesis of the tumor. On the other hand, the abnormality in p53 pathway has not been well analyzed in ES yet. In the present study, we investigated the effects of EWS-Fli1 on p53 pathway and its function, especially the inductio … More n of apoptosis in ES cells. The immunoprecipitation assay revealed that EWS-Fli1 bound to p53 protein, and inhibited its function in p53 wild type ES cells. However, EWS-Fli1 did not significantly affect the expression of apoptosis-related genes located in the downstream of p53 transcription factor. The expression of p21, one of the target genes of p53, was inhibited by EWS-Fli1 via the suppression of the activity of p21 gene promoter. Therefore, we hypothesized that histone deacetylase inhibitors (HDACI) which are known to induce p21 expression in cancer cells might be effective on ES cells. HDACI inhibited ES cell growth via induction of p21 expression both in vitro and in vivo. However, a certain type of HDACI showed cross-resistance to the drug-resistant ES cells. Thus we should pay great attention to the resistance of ES cells to HDACIs in clinical trials. We also investigated the effects of the knockdown of EWS-Fli1 expression by siRNA on apoptosis induction in ES cells. The challenge of siRNA against EWS-Fli1 to ES cells did not induce apoptosis, but senescence in ES cells. This observation indicate the new function of EWS-Fli1, i.e., EWS-Fli1 might inhibit the induction of senescence in ES cells which might lead to the oncogenesis of ES. These results suggest that inhibition of these functions of EWS-Fli1 might promise the development of the molecular target therapy for the treatment of ES patients. Less