Identifying New Glioma-Associated Tumor Suppressors and Oncogenes

鉴定新的神经胶质瘤相关肿瘤抑制因子和癌基因

基本信息

批准号：
10486899
负责人：
Mark Gilbert
金额：
$ 42.53万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10486899
关键词：
10q 11p 11q 12q13 13q 14q13 19q 1p34 1q32 20p 20q 22q 3q26 8q24 Animals Apoptosis Area Astrocytoma Automobile Driving Binding Proteins Bioinformatics Biological Biological Assay Biology Biotechnology Brain Brain Neoplasms CDKN2A gene Cancer Genome Anatomy Project Candidate Disease Gene Cell Line Cellular biology Central Nervous System Neoplasms Chromatin Chromosome abnormality Classification Clinical Collaborations Complex DNA DNase I hypersensitive sites sequencing Data Data Analyses Decarboxylation Deoxyribonuclease I Development Dioxygenases Disease Enzymes Epidermal Growth Factor Receptor Epigenetic Process Exhibits Gene Chips Gene Expression Gene Expression Profile Gene Expression Profiling Gene Expression Regulation Gene Silencing Genes Genetic Genetic Transcription Genome Genomics Glioblastoma Glioma Gliomagenesis Goals Heterogeneity Human Human Genome Project Hypersensitivity In Vitro Isocitrate Dehydrogenase Isocitrates Laboratories Learning Link Location Loss of Heterozygosity Malignant - descriptor Methods Methylation Microarray Analysis Molecular Molecular Target Morphology Mutation Neuroglia Neurons Nuclear Nucleic Acid Regulatory Sequences Oncogenes Operative Surgical Procedures PTEN gene Pathologic Patients Process Protocols documentation Receptor Biology and Gene Expression Recurrence Role SNP array Sampling Signal Transduction Pathway Site Specimen Structure TP53 gene Testing Therapeutic Intervention Tissues Transplantation Tumor Biology Tumor Suppressor Genes Tumor Suppressor Proteins Tumor Tissue Work Xenograft procedure alpha ketoglutarate base cDNA Arrays chromosome 5q loss enzyme activity experimental study expression vector falls gene cloning gene discovery genome-wide immunosuppressed in vivo insight malignant phenotype new technology new therapeutic target next generation sequencing novel self-renewal stem cells transcription factor transcriptome tumor tumorigenesis tumorigenic

项目摘要

Previously we have initiated a large cDNA microarray effort in collaboration with the Human Genome Project and the Cancer Genome Anatomy Project (CGAP) to develop a comprehensive and novel molecular classification schema for human gliomas based on a gene expression profile using cDNA microarray technology. We have constructed our own cDNA microarray "chips" which will be enhanced for new and selective genes thought to be important in glioma biology. This project will include hundreds of tumor specimens and offer an unprecedented opportunity for gene discovery, dissecting signal transduction pathways, and learning this exciting new technology. Glioma stem cell is a tumor subpopulation that can self-renew in culture, perpetuate a tumor in orthotopic transplant in vivo, and generate diversified neuron-like and glia-like postmitotic progeny in vivo and in vitro. Recently, conventional and array-based CGH (aCGH) profiling of human gliomas have shown a significant number of copy number alterations (CNAs) including gain/amplification (1p34-36, 1q32, 3q26-28, 5q, 7q31, 8q24, 11q, 12q13, 13q, 15p15, 17q22- 25,19q, 20p, and 20q), and deletion/loss (3q25-26, 4q, 6q26-27, 9p, 10p, 10q, 11p, 12q22, 13q, 14q13, 14q23-31, 15q13-21, 17p11-13, 18q22-23, 19q, and 22q) (Kotliarov et al., 2006; Nigro et al., 2005; Phillips et al., 2006). The large number of chromosomal aberrations, and the large number of genes contained therein, have to date made it impossible to identify which genes are in part responsible for driving the biology of these tumors. We have analyzed a large number glioma samples for genetic characterization of recurring CNAs using Affymetrix 100K single-nucleotide polymorphism (SNP) array chips and Genechip HumanGenome U133 Plus 2.0 Expression array (Kotliarov et al. 2006). Based on our bioinformatics data from these array and gene expression profiling experiments, we have found novel genes frequently altered in gliomas. Furthermore, we have explored the new biotechnology such as next generation sequencing, for this project. We have generated sequence-verified gene Gateway entry clones of these genes and cloned them into pLenti/UbC/V5 expression vectors for transduction of various target cell lines. With our candidate gene constructs, we will identify whether candidate genes change the biology of these cells in such a way that may be consistent with a role in tumorigenesis (i.e. clonogenecity, proliferation, apoptosis, tumorigenic potential in immunosuppressed animals). The NOB Laboratory recently began collaborating with Dr. Gordon Hager and the Laboratory of Receptor Biology and Gene Expression. Dr. Hager's work has focused on the reorganization of the nuclear chromatin and the impact of these changes on gene regulation. In the context of brain tumor biology, there are a variety of primary central nervous system tumors that despite a malignant phenotype have few mutations. Therefore, it is possible that alterations in the transcriptional profile may help explain this apparent disparity. The NOB laboratory is using the DHS-seq method to profile genome-wide transcriptional changes in glioma patient samples. As described above, the DHS-seq will reveal dynamic changes in the chromatin, which are important in the development and progression of brain tumors and allow us to identify novel molecular targets to treat this disease. We have tested the DHS-seq protocol on two glioma stem cell lines (827P12 and 923P9) and corresponding xenograft tissues. Preliminary analyses of these data suggest that in combination with gene expression and copy number data, we will obtain novel insights into the genomics underlying brain tumor biology. To this end, the NOB laboratory has begun testing this method on patient samples, using tumor tissues and adjacent normal brain directly from surgical specimens. The plan is to continue processing additional patient samples as they become available with the ultimate goal of incorporating the "transcriptome" analysis into the comprehensive genomic analysis that is being planned as a component of the molecular tumor board, described in the Clinical Project.

以前，我们已经与人类基因组项目和癌症基因组解剖项目（CGAP）合作开始了巨大的cDNA微阵列工作，以基于使用cDNA微阵列技术的基因表达谱，开发了人类神经胶质瘤的全面和新颖的分子分类模式。我们已经构建了自己的cDNA微阵列“芯片”，对于新的和有选择性的基因在神经胶质瘤生物学中很重要。该项目将包括数百个肿瘤标本，并为基因发现，解剖信号转导途径以及学习这项令人兴奋的新技术提供前所未有的机会。神经胶质瘤干细胞是一种肿瘤亚群，可以在培养中自我更新，在体内永存，使肿瘤永存，并在体内和体外产生多样化的神经元样和神经胶质后的神经胶状后代。 Recently, conventional and array-based CGH (aCGH) profiling of human gliomas have shown a significant number of copy number alterations (CNAs) including gain/amplification (1p34-36, 1q32, 3q26-28, 5q, 7q31, 8q24, 11q, 12q13, 13q, 15p15, 17q22- 25,19q, 20p, and 20q), and deletion/loss （3Q25-26，4Q，4Q，6Q26-27，9p，10q，10q，10p，11p，12q22，12q22，13q，14Q13，14Q223-31，15Q13-21，17P11-13，17P11-13，18Q22222-23，19Q，19Q和22Q）（Kotliarov等）（Kotliarov等人，2006年，2006年； Nigro等人，2005年； 2005年）。迄今为止，大量的染色体畸变以及其中包含的大量基因不可能确定哪些基因部分负责驱动这些肿瘤的生物学。我们已经使用Affymetrix 100K单核苷酸多态性（SNP）阵列芯片和Genechip Humangememememenome U133加2.0表达阵列（Kotliarov etal。2006），分析了大量胶质瘤样品，用于使用Affymetrix 100K单核苷酸多态性（SNP）阵列芯片（SNP）阵列芯片和Genechip Humangem阵列芯片和Genechip Humangem阵列芯片和Genechip Humangem阵列芯片和Genechip Humangem阵列芯片和Genechip Humangem阵列（2006）。基于我们来自这些阵列和基因表达分析实验的生物信息学数据，我们发现新颖的基因经常在神经胶质瘤中改变。此外，我们已经为该项目探索了新的生物技术，例如下一代测序。我们已经生成了这些基因的序列验证的基因网关进入克隆，并将它们克隆到plenti/ubc/v5表达矢量中，以转导各种靶细胞系。借助我们的候选基因构建体，我们将确定候选基因是否会以这种方式改变这些细胞的生物学，以与肿瘤发生中的作用一致（即克隆生成，增殖，凋亡，肿瘤性在免疫抑制动物中的潜力）。 NOB实验室最近开始与Gordon Hager博士以及受体生物学和基因表达实验室合作。哈格博士的工作集中在核染色质的重组以及这些变化对基因调节的影响。在脑肿瘤生物学的背景下，尽管恶性表型很少突变，但有多种原发性中枢神经系统肿瘤。因此，转录曲线的变化可能有助于解释这种明显的差异。 NOB实验室正在使用DHS-SEQ方法来介绍神经瘤患者样品中全基因组转录变化。如上所述，DHS-Seq将揭示染色质的动态变化，这些变化在脑肿瘤的发育和进展中很重要，并使我们能够鉴定出新的分子靶标以治疗该疾病。我们已经在两种神经胶质瘤干细胞系（827p12和923p9）和相应的异种移植组织上测试了DHS-SEQ方案。对这些数据的初步分析表明，与基因表达和拷贝数数据结合使用，我们将获得对脑肿瘤生物学基因组学的新见解。为此，NOB实验室已开始使用肿瘤组织和直接从手术样本中的邻近正常大脑对患者样品进行测试。该计划是继续处理其他患者样本，因为它们可以使用，其最终目标是将“转录组”分析纳入临床项目中描述的分子肿瘤板的组成部分，以将“转录组”分析纳入全面的基因组分析。