Apoptotic Pathway Defects in Neuroblastoma

描述(申请人提供)：晚期神经母细胞瘤，特别是那些MYCN基因扩增的肿瘤，预后很差，主要是因为他们在接受多种化疗药物/放射治疗后存活的能力。这项研究计划的持续目标是了解遗传改变，如MYCN基因扩增和染色体lp36杂合性缺失(LOH)是如何导致这种肿瘤表型的。在上一次资助期间，我们发现，在患有简化MYCN的4期神经母细胞瘤患者中，有60%的细胞凋亡信号分子caspase-8优先被甲基化沉默，而在那些没有扩增MYCN沉默CASP8基因的1-4期肿瘤中，&lt；4%的肿瘤细胞中CASP8基因的表达被甲基化抑制。现在在N-Myc诱导的小鼠神经母细胞瘤中也进行了类似的观察。我们还发现，在正常情况下caspase-8缺失的人NB细胞中，caspase-8的重新编程表达使其对化疗药物阿霉素和顺铂诱导的细胞凋亡重新敏感。这在细胞培养和体内异种移植小鼠模型中都能观察到。最后，我们已经报道，和其他人一样，caspase-8能够同时作为起始者和执行者发挥作用，使其能够放大某些线粒体介导的细胞死亡信号。这一功能在迄今发现的caspase中是独一无二的，这可能是它在某些肿瘤中选择性沉默的原因之一。基于这些数据，我们推测，通过甲基化沉默CASP8可能提供了一个更宽松的细胞环境，可以在不经历细胞死亡的情况下耐受N-Myc的过度表达，并可能有助于这些肿瘤细胞在某些化疗药物的治疗下存活。为了验证这一假设，我们建议通过基因敲除或转基因表达一种不活跃的、显性的负型caspase-8来建立小鼠模型，该模型完全消除或下调酶活性，并确定在N-Myc过度表达或这些肿瘤对化疗药物的反应存在时，它是否有助于加速肿瘤细胞的生长。这些实验将包括使用互补的方法；即在从这些小鼠分离的培养的神经脊细胞中诱导癌基因，如MYCN，以及各种细胞、异种移植和体内肿瘤对治疗的反应。最后，我们将通过微阵列分析检测这些不同的小鼠NB肿瘤和正常肾上腺组织，以及不同阶段的人类患者样本和匹配处理/未处理的样本，以确定除CASP8外，其表达因N-Myc过度表达和/或药物治疗而显著改变的可能基因。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。

DESCRIPTION (provided by applicant): 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 simplified 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 cisplatin. 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 transgenic 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 in 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 microarray 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.