Regulation of Apoptosis in Ewing Sarcoma

尤文氏肉瘤是儿童和青少年中第二常见的骨骼和软组织实体瘤。目前的治疗方案仅治愈50%至70%的局部尤文氏肉瘤患者和不到30%的转移性疾病患者。强化化疗并没有提高患者的生存率。此外，由于缺乏侵袭性的生物学指标，很难对患者进行分层以进行更积极的治疗。我们已经产生了实验证据，支持survivin在尤文氏肉瘤细胞对化疗的耐药性中的作用，并发现了survivin在非转移性尤文氏肉瘤组织中的表达与患者总体生存率之间的关联（手稿正在准备中）。我们的数据支持survivin是尤文氏肉瘤的预后标志物和治疗靶点。NCI的Widemann博士考虑将survivin的一种小分子抑制剂用于临床试验，我们将参与对survivin表达的组织进行评估。我们还探索了使用TRAIL作为配体的其他途径来杀死ESFT细胞的可能性，例如受体介导的凋亡途径。我们之前的数据和其他人的数据表明，体外尤因肉瘤细胞通常对TRAIL敏感（Mitsiades， N., et al ., Cancer Res. 2001）。由于TRAIL只杀死肿瘤细胞而不杀死正常细胞，因此它是耐药肿瘤的理想替代药物，我们参与的NCI临床I期研究现已完成（在ASCO 2010上提交），并表明TRAIL对儿童和年轻人的耐受性良好，无主要毒性。然而，正如我们和我们的合作者所表明的那样（Lissat， A.等人）。J. Pathol. 2007)，低caspase 8表达的Ewing肉瘤细胞具有TRAIL抗性，我们已经证明这主要是由于它们的高存活素水平。我们最近的实验表明，survivin抑制trail诱导的细胞凋亡的机制是通过结合并抑制促凋亡蛋白SMAC和结合并保护抗凋亡蛋白XIAP免受caspase 3的切割。我们发现Smac模拟物和survivin RNA干扰联合治疗可逆转TRAIL耐药性（手稿正在准备中）。我们的数据表明，survivin小分子抑制剂和Smac模拟物联合治疗可以改善TRAIL治疗的Ewing肉瘤患者的预后。最近，我们发现存活素在EWS/FLI-1的转录调控下上调，EWS/FLI-1是一种ewing特异性的异常转录因子，是维持肿瘤所必需的，并且这种调控涉及NF-kB途径。药理抑制NF-kB通路或用siRNA下调EWS/ fl -1转录物可导致survivin的下调和Ewing肉瘤细胞对TRAIL的敏化。这些数据表明NF-kB抑制剂可能在与TRAIL或化疗药物联合治疗尤文氏肉瘤中发挥作用。

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.