Apoptotic Pathway Defects in Neuroblastoma

晚期神经母细胞瘤，尤其是那些MYCN基因扩增的肿瘤，预后很差，主要原因是 他们在接受多种化疗药物/放射治疗后存活的能力。这样做的持续目标是 研究计划是为了了解遗传变化，如MYCN基因扩增和染色体lp36 杂合性缺失(LOH)，导致这种肿瘤表型。在上一个资助期内，我们发现 关键的凋亡信号分子caspase-8在阶段4的60%中优先被甲基化沉默 有MYCN扩增的神经母细胞瘤患者肿瘤，而1-4期无MYCN扩增的肿瘤中有4% CASP8基因的沉默表达。现在在N-Myc诱导的神经母细胞瘤中也进行了类似的观察 老鼠身上的肿瘤。我们还发现，在正常情况下，人NB细胞中caspase-8的重新编程表达 Caspase-8基因缺失使其对化疗药物阿霉素和顺铂诱导的细胞凋亡再敏感。这 在细胞培养和体内异种移植小鼠模型中均观察到。最后，我们和其他人一样，报告说 Caspase-8既可以作为启动子，也可以作为执行者，使其能够放大某些 线粒体介导的细胞死亡信号。该功能在迄今识别的半胱氨酸天冬氨酸酶中有些独特， 这可能是它在某些肿瘤中选择性沉默的原因之一。根据这些数据，我们假设 通过甲基化沉默CASP8可能会提供一个更宽松的细胞环境，可以耐受 在不经历细胞死亡的情况下过表达N-Myc，可能有助于这些肿瘤细胞 在某些化疗药物治疗下存活下来。为了验证这一假设，我们建议通过以下方法开发小鼠模型 Caspase-8的非活性、显性负性形式的基因敲除或转基因表达 消除或下调酶活性，并确定它是否有助于加速肿瘤细胞的生长 N-Myc的过度表达或这些肿瘤对化疗药物的反应。这些实验 将包括使用互补的方法；即在培养的神经中诱导MYCN等癌基因 从这些小鼠分离的CREST细胞，以及各种细胞、异种移植瘤和体内肿瘤对 心理治疗。最后，我们将检查这些不同的小鼠NB肿瘤和正常肾上腺组织，以及人类 通过微阵列分析不同阶段的患者样本和配对治疗/未治疗样本以确定可能的 除CASP8外，其表达因N-Myc过表达和/或药物治疗而显著改变的基因。 这样的研究将为这些肿瘤细胞如何避开凋亡并延长其寿命提供重要的洞察力。

Late stage neuroblastoma tumors, particularly those with amplified MYCN genes, have a poor prognosis, primarily due to their ability to survive treatment with multiple chemotherapeutic agents/irradiation. The continuing goal of this research program is to understand how genetic alterations, such as MYCN gene amplification and chromosome lp36 loss-of-heterozygosity (LOH), contribute to this tumor phenotype. During the last funding period we found that a critical apoptotic signaling molecule, caspase-8, is preferentially silenced by methylation in >60% of the stage 4 neuroblastoma patient tumors with smplified MYCN, whereas <4% of those stage 1-4 tumors without amplified MYCN silence expression of the CASP8 gene. A similar observation has now been made in N-Myc-induced neuroblastoma tumors from mice. We also found that reprogramed expression of caspase-8 in human NB cells that are normally caspase-8 null resensitized them to apoptosis induced by the chemotherapeutic drugs doxorubicin and eisplatin. This was observed in both cell culture and in vivo xenograft mouse models. Finally, we have reported, as have others, that caspase-8 is capable of functioning as both an initiator and executioner caspase, allowing it to amplify certain mitochondrial-mediated cell death signals. This function is somewhat unique among the caspases identified thus far, and could be one reason it is selectively silenced in certain tumors. Based upon this data we hypothesize that the silencing of CASP8 by methylation may provide a more permissive cellular environment that can tolerate the overexpression of N-Myc without undergoing cell death, and perhaps contribute to the ability of these tumor cells to survive treatment with certain chemotherapeutic drugs. To test this hypothesis we propose to develop mouse models by gene knockout or transgenie expression of an inactive, dominant negative form of caspase-8 that either totally eliminate, or down-regulate enzyme activity, and determine whether it contributes to accelerated tumor cell growth in the presence of N-Myc overexpression or the response of these tumors to chemotherapeutic drugs. These experiments will include the use of complementary approaches; namely the induction of oncogenes such as MYCN in cultured neural crest cells isolated from these mice, as well as the response of the various cells, xenografts, and tn vivo tumors to therapy. Finally, we will examine these different mouse NB tumors and normal adrenal gland tissue, as well as human patient samples of various stages and matched-treated/untreated samples by mieroarray analysis to identify possible genes, other than CASP8, whose expression is significantly altered by N-Myc overexpression and/or drug treatment. Such studies will provide significant insight into how these tumor cells circumvent apoptosis and prolong their life.