Regulation of apoptosis in Ewings sarcoma and neuroblast

尤文氏肉瘤家族肿瘤（ESFT）和神经母细胞瘤（NB）是儿童和青少年中最常见的两种颅外实体肿瘤。尽管在了解这些肿瘤的生物学方面取得了重大进展，但复发和转移性疾病患者的预后仍然令人沮丧。因此，寻找新的药物或策略来根除化疗耐药的肿瘤细胞是很重要的。化疗药物，不论其最初的靶标是什么，都是通过趋同于一个共同的凋亡通路来发挥作用的。我们的研究旨在确定导致ESFT和NB细胞凋亡失调的机制，以了解治疗失败的原因，并发现克服这些肿瘤耐药的替代途径。两种主要的信号通路启动细胞凋亡：(a)线粒体通路，由细胞窘迫和DNA损伤触发；(b)死亡受体介导的通路，由死亡受体与其配体相互作用触发。这两种途径都是通过半胱天冬酶的激活进行的。在死亡受体介导的途径中，肿瘤坏死因子凋亡诱导配体（TRAIL）途径引起了极大的兴趣，因为TRAIL和TRAIL受体激动剂作为杀瘤剂的功效以及它们对正常细胞缺乏毒性，这为开发新的癌症治疗策略提供了令人兴奋的机会。我们之前已经证明TRAIL是ESFT细胞凋亡的有效诱导剂，并且ESFT细胞表达TRAIL受体DR4和DR5。然而，在我们的体外研究中，我们观察到一些ESFT细胞表现出对TRAIL的抗性，并且在与Mackall博士实验室使用ESFT异种移植的合作研究中，我们发现ESFT细胞对TRAIL的抗性与TRAIL受体的下调有关，并且可以通过干扰素治疗来克服。我们还发现caspase 8的低表达有助于ESFT的TRAIL耐药，并且存在于24%的ESFT组织中。我们实验室最近的数据表明，耐药ESFT细胞具有高水平的抗凋亡蛋白survivin。通过下调survivin，我们能够在体外恢复ESFT细胞对化疗药物的敏感性。此外，我们发现，与未表达survivin的ESFT患者相比，表达高水平survivin的局部ESFT患者总体预后较差，这在统计学上具有显著意义。我们目前正在探索survivin在trail耐药ESFT细胞中的作用以及ESFT特异性融合基因EWS/ fl -1对其的调控。关于NB细胞，我们已经证明它们有几个调节性凋亡缺陷，导致线粒体和死亡受体途径失活。据报道，NB细胞表达低水平的caspase 8，但这可能不是唯一的缺陷，因为正如我们与Thiele博士的实验室合作所表明的那样，即使在用干扰素上调caspase 8后，TRAIL诱导的细胞凋亡也是由于TRAIL受体水平低或缺失。我们的实验室还通过直接结合抗凋亡蛋白Bcl-2在NB细胞中发现了caspase 8失活的新机制。我们实验室最近的数据表明，一种名为双功能凋亡调节因子（BAR）的新报道蛋白在trail抗性NB细胞中高水平表达，并与bcl -2和caspase 8形成复合物。它的下调导致caspase 8与bcl-2分离，并逆转NB细胞的TRAIL抗性。这些数据支持Bcl-2与caspase 8的关联是通过BAR完成的，BAR是trail诱导的NB细胞凋亡的重要抑制剂，因此可能是一个治疗靶点。

Ewing sarcoma family tumors (ESFT) and neuroblastoma (NB) are two of the most frequently encountered extracranial solid tumors in children and adolescents. Despite major advances in the understanding of the biology of these tumors, the prognosis of patients with recurrent and metastatic disease remains dismal. Therefore, it is important to identify novel agents or strategies, which will eradicate chemotherapy-resistant tumor cells. Chemotherapeutic agents, irrespective of their initial targets, exert their action by converging into a common apoptotic pathway. Our studies seek to identify mechanisms that lead to dysregulation of apoptosis in ESFT and NB in order to understand the reasons for therapy failures, and discover alternative pathways to overcome drug resistance in these tumors. Two main signaling pathways initiate apoptosis: (a) the mitochondrial pathway, triggered by cell distress and DNA damage and (b) the death receptor-mediated pathway, triggered by the interaction of the death receptors with their ligands. Both pathways proceed through activation of caspases. Among the death receptor-mediated pathways, the Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL) pathway has generated a great deal of interest, because of the efficacy of TRAIL and TRAIL receptor agonists as tumoricidal agents and their lack of toxicity to normal cells, which provide exciting opportunities for development of novel therapeutic strategies in cancer.We have previously shown that TRAIL is a potent inducer of apoptosis in ESFT cells, and that ESFT cells express the TRAIL receptors DR4 and DR5. However, in our in vitro studies, we observed that some ESFT cells exhibit resistance to TRAIL and in collaborative studies with Dr. Mackall's laboratory using ESFT xenografts, we found that ESFT cell resistance to TRAIL is associated with downregulation of the TRAIL receptors, and can be overcome with interferon treatment. We also found that low caspase 8 expression contributes to TRAIL resistance in ESFT and is present in 24% of the ESFT tissues. Recent data in our laboratory have shown that drug-resistant ESFT cells have high levels of the antiapoptotic protein survivin. By downregulating survivin, we were able to restore the sensitivity of ESFT cells to chemotherapeutic agents in vitro. Furthermore, we found that patients with localized ESFT expressing high levels of survivin had an overall poor prognosis when compared to those with no survivin expression and this was statistically significant. We are currently exploring the role of survivin in TRAIL-resistant ESFT cells and its regulation by the ESFT-specific fusion gene EWS/FLI-1.With regard to NB cells, we have shown that they have several regulatory apoptotic defects, which lead to the inactivation of the mitochondrial and the death receptor pathways. NB cells have been reported to express low levels of caspase 8, but this may not be the only defect, because as we have shown in collaboration with Dr. Thiele's laboratory, even after upregulation of caspase 8 with interferon, TRAIL-induced apoptosis is defective due to low or absent levels of TRAIL receptors. Our laboratory has also shown a novel mechanism of caspase 8 inactivation in NB cells through direct binding with the antiapoptotic protein Bcl-2. More recent data from our laboratory have shown that a newly reported protein under the name of Bifunctional Apoptosis Regulator (BAR) is expressed at high levels in TRAIL-resistant NB cells and forms complexes with Bccl-2 and caspase 8. Its downregulation results in dissociation of caspase 8 from bcl-2 and reverses TRAIL resistance in NB cells. These data support that association of Bcl-2 and caspase 8 is accomplished through BAR, which is an important inhibitor of TRAIL-induced apoptosis in NB cells and therefore, a possible therapeutic target.