Role of Polyamines in MYCN-Amplified Neuroblastoma

多胺在 MYCN 扩增神经母细胞瘤中的作用

基本信息

批准号：
7262515
负责人：
ANDRE S BACHMANN
金额：
$ 21.47万
依托单位：
UNIVERSITY OF HAWAII AT MANOA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7262515
关键词：
Accounting Adenosylmethionine Decarboxylase Affect Apoptosis Biological Assay Cell Cycle Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Line Cell Proliferation Cell Survival Cells Cessation of life Child Childhood Cloning Co-Immunoprecipitations Communication DL-alpha-Difluoromethylornithine DNA Development Disease Employee Strikes Enzymes Evaluation Event Flow Cytometry Gene Expression Genetic Transcription Half-Life High Pressure Liquid Chromatography Hydrogen Peroxide In Vitro Individual Investigation Laser Microscopy MYCN gene Malignant Childhood Neoplasm Malignant Neoplasms Mammalian Cell Measures Messenger RNA Metabolic Metabolism Molecular Mutation Neuroblastoma Nitric Oxide Northern Blotting Nuclear Oncogenes Ornithine Decarboxylase Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Physiological Play Polyamines Production Protein Overexpression Proteins Putrescine Rate Regulation Research Retinoblastoma Protein Reverse Transcriptase Polymerase Chain Reaction Risk Role Running SAM486A SAT gene Sepiapterin reductase Series Signal Pathway Signal Transduction Signaling Protein Site-Directed Mutagenesis Spermidine Spermidine/Spermine N1-Acetyltransferase Spermine Staging System Techniques Testing Tetracycline Tetracyclines Translating Western Blotting Work Yeasts base enzyme activity human FRAP1 protein inhibitor/antagonist insight migration novel novel therapeutics outcome forecast response tumor tumorigenesis urea cycle yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Neuroblastoma (NB) is the third most common malignancy in childhood and accounts for about 15% of cancer-related deaths in children. One of the most striking abnormalities found in NB tumors is the amplification of the MYCN oncogene, which strongly correlates with more aggressive tumors and poor prognosis of late stage disease. The complete absence of effective treatments for high-risk (late stage) NB patients indicates the need for novel therapeutic approaches, including drugs that block MYCN expression. The primary objective of our previous work has been to evaluate the polyamine biosynthetic pathway as an alternative target for NB therapy. Central to our investigation was the evaluation of two clinically tested polyamine inhibitors, DFMO and SAM486A, which specifically inhibit the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), respectively. Since MYCN has been shown to activate ODC gene expression, we hypothesized that ODC and polyamines might play a role in MYCN-amplified NB tumorigenesis. Our initial results showed that the depletion of polyamines in MYCN-amplified NB cells caused G1 cell cycle arrest and affected p27Kip1 levels, retinoblastoma protein Rb phosphorylation, and most intriguingly, blocked MYCN expression, thus providing a rationale for the potential use of polyamine inhibitors in late-stage NB patients. In the current proposal we outline studies that examine how MYCN, ODC, and polyamines regulate important cell cycle and cell survival pathways in NB cells. In Specific Aim 1 we will study whether MYCN overexpression in NB cells impacts endogenous ODC and other metabolic enzymes of polyamine metabolism, and whether this translates into changes in polyamine pools, cell cycle progression, and cell proliferation. These studies will be performed using three unique NB cell lines, in which MYCN expression can be switched on/off by adding tetracycline. In Specific Aim 2 we will examine the role of polyamines in cell cycle regulation and PI3K/Akt cell survival activation in NB cells. Specifically, we will determine the differential effects of individual polyamines (putrescine, spermidine, and spermine) on the regulation and phosphorylation/activation of key signaling proteins including MYCN, p27Kip1, Cdk2, Akt, and mTOR. In Specific Aim 3 we will elucidate the role of ODC in cell signaling by examining its interaction with two new cellular proteins. The physiological relevance of these interactions will be verified by mutational analyses and a series of cell-based biological assays. The proposed methods and techniques to pursue this work are: Northern blot, RT-PCR, nuclear run-ons, messenger RNA half-life studies, Western blot, yeast two-hybrid, co-immunoprecipitation, GST-pull down, site-directed mutagenesis, DNA cloning, confocal laser microscopy, enzyme and in vitro kinase assays, flow cytometry, and HPLC. At the conclusion of this research, we will have uncovered how ODC and individual polyamines regulate important molecular events that control cell cycle and cell survival pathways of NB cells. Our research will be useful in the development of novel treatments for the most aggressive forms of the childhood cancer neuroblastoma.

描述（由申请人提供）：神经母细胞瘤（NB）是童年中第三大常见的恶性肿瘤，约占儿童癌症相关死亡的15％。 NB肿瘤中发现的最引人注目的异常之一是MYCN癌基因的扩增，这与晚期疾病的更具侵略性肿瘤和预后不良密切相关。完全没有针对高危（晚期）NB患者的有效治疗方法表明需要采用新型治疗方法，包括阻断MYCN表达的药物。我们先前工作的主要目的是评估多胺生物合成途径，作为NB治疗的替代靶标。我们研究的核心是对两种临床测试的多胺抑制剂DFMO和SAM486A的评估，它们专门抑制了多胺生物合成酶鸟氨酸脱羧酶（ODC）和S-腺苷甲基二氨基氨基氨基氨基氨基二羧化酶（ODC）。由于已显示MYCN激活ODC基因表达，因此我们假设ODC和多胺可能在MYCN扩增的NB肿瘤发生中起作用。我们的最初结果表明，MYCN放大NB细胞中多胺的耗竭引起了G1细胞周期停滞，并影响了P27KIP1水平，视网膜细胞瘤蛋白RB磷酸化以及最有趣的MYCN表达，从而为多胺抑制剂在后期NB患者中的潜在使用提供了依据。在当前的建议中，我们概述了研究MYCN，ODC和多胺如何调节NB细胞中重要的细胞周期和细胞存活途径的研究。在特定目标1中，我们将研究NB细胞中的MYCN过表达是否会影响多胺代谢的内源性ODC和其他代谢酶，以及这是否转化为多胺池的变化，细胞周期进展和细胞增殖。这些研究将使用三种独特的NB细胞系进行，其中可以通过添加四环素来打开/关闭MYCN表达。在特定目标2中，我们将检查多胺在NB细胞中多胺在细胞周期调节和PI3K/AKT细胞存活激活中的作用。具体而言，我们将确定单个多胺（perrescine，permidine和Permine）对关键信号蛋白的调节和磷酸化/激活（包括MYCN，P27KIP1，CDK2，AKT和MTOR）的差异作用。在特定目标3中，我们将通过检查其与两个新细胞蛋白的相互作用来阐明ODC在细胞信号传导中的作用。这些相互作用的生理相关性将通过突变分析和一系列基于细胞的生物学测定来验证。 The proposed methods and techniques to pursue this work are: Northern blot, RT-PCR, nuclear run-ons, messenger RNA half-life studies, Western blot, yeast two-hybrid, co-immunoprecipitation, GST-pull down, site-directed mutagenesis, DNA cloning, confocal laser microscopy, enzyme and in vitro kinase assays, flow cytometry, and HPLC.在这项研究的结论中，我们将发现ODC和单个多胺如何调节控制NB细胞的细胞周期和细胞存活途径的重要分子事件。我们的研究将有助于开发针对儿童癌症神经母细胞瘤最具侵略性形式的新疗法。