Regulation of Apoptosis in Ewing Sarcoma

尤文肉瘤是儿童和青少年中第二常见的骨和软组织实体瘤。目前的治疗方案只能治愈50%到70%的局限性尤文肉瘤患者和不到30%的转移性疾病患者。强化化疗并未提高患者的存活率。此外，由于缺乏侵袭性的生物学指标，对患者进行分层以进行更积极的治疗是困难的。我们已经产生了支持Survivin在尤文肉瘤细胞对化疗的耐药性中的作用的实验证据，并发现非转移性尤文肉瘤组织中Survivin的表达与患者总体生存有关(手稿正在准备中)。我们的数据支持Survivin是尤文肉瘤的预后标志物和治疗靶点。NCI的Widemann博士正在考虑一种Survivin的小分子抑制剂进行临床试验，我们将参与评估组织中Survivin的表达。我们还探索了使用TRAIL作为配体，通过其他途径杀死ESFT细胞的可能性，例如受体介导的凋亡途径。我们以前的数据和其他人的数据表明，尤文肉瘤细胞在体外对TRAIL通常是敏感的(Mitsiade，N，等，癌症研究，2001)。由于TRAIL只杀死肿瘤而不是正常细胞，它是治疗耐药肿瘤的理想替代药物，我们参与的NCI的一项临床阶段研究现已完成(在ASCO 2010上公布)，并表明TRAIL耐受性良好，儿童和年轻人没有重大毒性。然而，正如我们和我们的合作者所展示的那样(Lissat，A.，et al AmJ.帕索尔。2007年)，caspase 8低表达的尤文肉瘤细胞对TRAIL耐药，我们已经证明这主要是由于它们高水平的Survivin。我们最近的实验支持，Survivin抑制TRAIL诱导的细胞凋亡的机制是通过结合和抑制促凋亡蛋白Smac以及结合和保护抗凋亡蛋白XIAP不被caspase 3切割。我们发现Smac模拟物和Survivin RNA干扰联合处理可以逆转TRAIL耐药(手稿正在准备中)。我们的数据表明，联合应用Survivin的小分子抑制剂和Smac类似物可以改善接受TRAIL治疗的尤文肉瘤患者的预后。最近，我们发现Survivin被EWS/FLI-1转录上调，EWS/FLI-1是一种尤文特异的异常转录因子，对肿瘤的维持是必要的，并且这种调节涉及到核因子-kB途径。药物抑制NF-kB途径或用siRNA下调EWS/Fli-1转录本，导致Survivin下调，并使尤文肉瘤细胞对TRAIL增敏。这些数据表明，核因子-kB抑制剂可能在尤文肉瘤与TRAIL或化疗药物的联合治疗中发挥作用。

Ewing sarcoma is the second most frequently encountered bone and soft tissue solid tumor in children and adolescents. Current therapeutic regimens cure only 50% to 70% of patients with localized Ewing sarcoma and less than 30% of patients with metastatic disease. Intensified chemotherapy has not improved the patients survival. Also, due to absence of biologic indicators of aggressiveness, stratification of patients for more aggressive treatments is difficult. We have generated experimental evidence that supports a role for survivin in the resistance of Ewing sarcoma cells to chemotherapy and have found association of survivin expression in non metastatic Ewing sarcoma tissues with overall patient survival (manuscript in preparation). Our data support that survivin is a prognostic marker and a therapeutic target in Ewing sarcoma. A small molecule inhibitor of survivin is considered for a clinical trial by Dr. Widemann at the NCI and we will be involved in the evaluation of the tissues for expression of survivin. We have also explored the possibility of engaging alternative pathways to kill ESFT cells, such the receptor-mediated apoptotic pathway, using TRAIL as a ligand. Our previous data and those of others have shown that Ewing sarcoma cells are generally sensitive to TRAIL in vitro (Mitsiades, N., et al, Cancer Res. 2001). Because TRAIL kills only tumor and not normal cells, it is a desirable alternative agent for drug-resistant tumors and a clinical phase I study at the NCI in which we participated has now been completed (presented at ASCO 2010) and showed that TRAIL is tolerated well without major toxicities by children and young adults. However as we and our collaborators have shown (Lissat, A., et al Am. J. Pathol. 2007), Ewing sarcoma cells with low caspase 8 expression are TRAIL resistant and we have since shown that this is primarily due to their high levels of survivin. Our recent experiments support that the mechanism by which survivin inhibits TRAIL-induced apoptosis is by binding and inhibiting the proapoptotic protein SMAC and by binding and protecting the antiapoptotic protein XIAP from cleavage by caspase 3. We showed that combined treatment with Smac mimetics and survivin RNA interference reverses TRAIL resistance (manuscript in preparation). Our data indicate that combined treatment with small molecule inhibitors of survivin and Smac mimetics can improve the outcome of Ewing sarcoma patients treated with TRAIL. More recently, we found that survivin is transcriptionally upregulated by EWS/FLI-1, a Ewing-specific aberrant transcription factor which is necessary for the maintenance of the tumor, and that this regulation involves the NF-kB pathway. Pharmacological inhibition of the NF-kB pathway or downregulation of the EWS/FLI-1 transcript with siRNA results in downregulation of survivin and sensitization of Ewing sarcoma cells to TRAIL. These data suggest that NF-kB inhibitors may have a role in the treatment of Ewing sarcoma in combination with TRAIL or chemotherapuetic agents.