Apoptotic Pathway Defects in Neuroblastoma

晚期神经母细胞瘤，特别是MYCN基因扩增的肿瘤，预后差，主要是由于 它们在多种化疗剂/放射治疗下存活的能力。这个持续的目标 研究计划是了解遗传改变，如MYCN基因扩增和染色体lp 36 杂合性缺失（洛）导致这种肿瘤表型。在上一个融资期间，我们发现， 关键的凋亡信号分子caspase-8在>60%的第4阶段中优先被甲基化沉默， MYCN扩增的神经母细胞瘤患者肿瘤，而MYCN扩增的1-4期肿瘤中<4% CASP 8基因的沉默表达。现在在N-Myc诱导的神经母细胞瘤中也有类似的观察结果 小鼠的肿瘤我们还发现，在正常的人NB细胞中，caspase-8的重编程表达 半胱天冬酶-8无效使它们对化疗药物阿霉素和顺铂诱导的细胞凋亡重新敏感。这 在细胞培养物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， 半胱天冬酶-8能够作为启动者和执行者半胱天冬酶发挥作用，使其能够扩增某些 细胞介导的细胞死亡信号这种功能在迄今为止鉴定的半胱天冬酶中有些独特， 这可能是它在某些肿瘤中被选择性沉默的原因之一。根据这些数据，我们假设， 通过甲基化使CASP 8沉默可能提供一种更宽容的细胞环境， N-Myc的过度表达而不经历细胞死亡，可能有助于这些肿瘤细胞的能力， 在某些化疗药物的治疗下存活下来。为了验证这一假设，我们建议开发小鼠模型， 基因敲除或转基因表达失活的显性负性形式的胱天蛋白酶-8， 消除或下调酶活性，并确定它是否有助于加速肿瘤细胞生长， N-Myc过表达的存在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，即在培养的神经细胞中诱导MYCN等癌基因， 从这些小鼠中分离的嵴细胞，以及各种细胞、异种移植物和体内肿瘤对 疗法最后，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品和匹配的治疗/未治疗样品，以鉴定可能的 除CASP 8以外的基因，其表达被N-Myc过表达和/或药物治疗显著改变。 这些研究将为这些肿瘤细胞如何规避凋亡并延长其寿命提供重要的见解。

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.