Regulation of apoptosis in Ewings sarcoma and neuroblast

尤文肉瘤家族肿瘤(ESFT)和神经母细胞瘤(NB)是儿童和青少年最常见的颅外实体瘤。尽管对这些肿瘤的生物学了解取得了重大进展，但复发和转移性疾病患者的预后仍然很差。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，无论它们最初的靶点是什么，都会通过汇聚成一条共同的凋亡途径来发挥作用。我们的研究试图确定导致ESFT和NB细胞凋亡失调的机制，以了解治疗失败的原因，并找到克服这些肿瘤耐药的替代途径。两条主要的信号通路启动细胞凋亡：(A)线粒体途径，由细胞窘迫和DNA损伤触发；(B)死亡受体介导的途径，由死亡受体与其配体相互作用而触发。这两条途径都是通过激活caspase来进行的。在死亡受体介导的途径中，肿瘤坏死因子凋亡诱导配体(TRAIL)途径因其作为肿瘤杀伤剂的有效性和对正常细胞的无毒性而引起人们的极大兴趣，这为开发新的癌症治疗策略提供了令人兴奋的机会。我们先前已经证明TRAIL是一种有效的诱导ESFT细胞凋亡的因子，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细胞对TRAIL表现出耐药性，在与Mackall博士的实验室使用ESFT异种移植进行的合作研究中，我们发现ESFT细胞对TRAIL的耐药性与TRAIL受体的下调有关，并可以通过干扰素治疗来克服。我们还发现，caspase8的低表达与ESFT的TRAIL耐药有关，并且在24%的ESFT组织中存在。我们实验室的最新数据表明，耐药的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，与不表达Survivin的患者相比，局部ESFT高表达Survivin的患者总体预后较差，这在统计学上具有统计学意义。我们目前正在探索Survivin在耐TRAIL的ESFT细胞中的作用及其受ESFT特异性融合基因EWS/FLI-1的调控。对于NB细胞，我们已经发现它们存在几个调节凋亡的缺陷，导致线粒体和死亡受体途径的失活。已有报道称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，即使在干扰素上调caspase 8之后，由于TRAIL受体的低水平或缺失，TRAIL诱导的细胞凋亡也是有缺陷的。我们的实验室还发现了一种新的机制，通过直接与抗凋亡蛋白Bcl-2结合，使caspase 8在NB细胞中失活。本实验室最近的研究表明，在TRAIL耐药的NB细胞中高水平表达一种新的蛋白，命名为双功能凋亡调节因子(BAR)，并与BCCL-2和caspase 8形成复合体。它的下调导致caspase 8从bCL-2中解离，从而逆转了NB细胞对TRAIL的抵抗。这些数据支持bcl2和caspase8的结合是通过BAR完成的，BAR是TRAIL诱导的NB细胞凋亡的重要抑制物，因此可能成为治疗的靶点。

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.