Apoptotic Pathway Defects in Neuroblastoma

神经母细胞瘤的凋亡途径缺陷

• 批准号: 6582100
• 负责人: VINCENT J. KIDD
• 金额: $ 33.66万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6582100
• 关键词: apoptosis; cysteine endopeptidases; enzyme activity; gene induction /repression; gene targeting; genetic models; genetically modified animals; human tissue; laboratory mouse; loss of heterozygosity; methylation; microarray technology; model design /development; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplastic growth; neural crest; neuroblastoma; oncogenes; xenotransplantation; apoptosis; cysteine endopeptidases; enzyme activity; gene induction /repression; gene targeting; genetic models; genetically modified animals; human tissue; laboratory mouse; loss of heterozygosity; methylation; microarray technology; model design /development; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplastic growth; neural crest; neuroblastoma; oncogenes; xenotransplantation

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.

描述（由申请人提供）：晚期神经母细胞瘤肿瘤，尤其是那些放大MYCN基因的肿瘤的预后较差，这主要是由于它们能够使用多种化学治疗剂/辐照来生存治疗。该研究计划的持续目标是了解遗传学改变（例如MyCN基因扩增和染色体LP36）的遗传丧失（LOH）如何有助于这种肿瘤表型。在上一个资金期间，我们发现，临界凋亡信号分子Caspase-8优先在> 60％的4阶段4神经母细胞瘤患者肿瘤中被甲基化而被简化的MYCN沉默，而<4％的1-4阶段的tumer均不在没有扩增Casp8基因的mycn silence表达。现在在N-MYC诱导的小鼠神经母细胞瘤肿瘤中也有类似的观察结果。我们还发现，通常是caspase-8的人NB细胞中caspase-8的表达，将其归因于化学治疗药物阿霉素和顺铂诱导的细胞凋亡。在细胞培养和体内异种移植小鼠模型中都观察到了这一点。最后，我们和其他人一样报告说，caspase-8能够充当引发剂和execution子caspase，从而可以扩增某些线粒体介导的细胞死亡信号。该功能在迄今为止确定的caspase中有些独特，这可能是在某些肿瘤中选择性沉默的原因之一。基于这些数据，我们假设通过甲基化对CASP8的沉默可能会提供更宽松的细胞环境，可以忍受N-MYC的过表达而无需经过细胞死亡，并且可能有助于这些肿瘤细胞使用某些化学治疗药物的能力。为了检验这一假设，我们建议通过基因敲除或转基因表达caspase-8的不活跃的负面形式的转基因表达来开发小鼠模型，该形式可以完全消除或下调酶活性，并确定它是否有助于N-MYC过度表达或这些肿瘤对化学疗法药物的反应中加速肿瘤细胞的生长。这些实验将包括使用互补方法；也就是说，从这些小鼠分离的培养神经rest细胞中诱导癌基因，例如MyCN，以及各种细胞，异种移植物和体内肿瘤对治疗的反应。最后，我们将通过微阵列分析检查这些不同的小鼠NB肿瘤和正常的肾上腺组织，以及各种阶段的人类患者样品，并通过微阵列分析进行了匹配的/未处理的样品，以鉴定可能的基因，而除了CASP8以外，它们的表达被N-MYC过表达和/或药物治疗显着改变。这样的研究将为这些肿瘤细胞如何绕过细胞凋亡并延长其寿命提供重大见解。