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

• 批准号: 10014745
• 负责人: Mark Gilbert
• 金额: $ 22.91万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10014745
• 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; 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 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; Nucleic Acid Regulatory Sequences; Oncogenes; Operative Surgical Procedures; PTEN gene; Pathologic; Patients; Process; Protocols documentation; Recurrence; Role; Sampling; Signal Transduction Pathway; Single Nucleotide Polymorphism; Site; Specimen; Stem cells; Structure; TP53 gene; Testing; Therapeutic Intervention; Tissues; Transplantation; Tumor Biology; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor Tissue; 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; new technology; new therapeutic target; next generation sequencing; novel; self-renewal; transcription factor; transcriptome; tumor; tumorigenesis; tumorigenic

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 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.

此前，我们与人类基因组计划和癌症基因组解剖计划 (CGAP) 合作启动了一项大型 cDNA 微阵列工作，以使用 cDNA 微阵列技术基于基因表达谱为人类神经胶质瘤开发全面且新颖的分子分类方案。我们已经构建了自己的 cDNA 微阵列“芯片”，该芯片将针对被认为在神经胶质瘤生物学中重要的新的选择性基因进行增强。该项目将包括数百个肿瘤标本，并为基因发现、剖析信号转导途径和学习这项令人兴奋的新技术提供前所未有的机会。胶质瘤干细胞是一种肿瘤亚群，可以在培养物中自我更新，在体内原位移植中使肿瘤永久存在，并在体内和体外产生多样化的神经元样和神经胶质样有丝分裂后后代。最近，人类神经胶质瘤的传统和基于阵列的 CGH (aCGH) 分析显示了大量的拷贝数改变 (CNA)，包括增益/扩增 (1p34-36、1q32、3q26-28、5q、7q31、8q24、11q、12q13、13q、15p15、17q22- 25,19q、20p 和 20q) 以及删除/丢失 （3q25-26、4q、6q26-27、9p、10p、10q、11p、12q22、13q、14q13、14q23-31、15q13-21、17p11-13、18q22-23、19q 和 22q）（Kotliarov 等）尼格罗等人，2006；菲利普斯等人，2006）。迄今为止，大量的染色体畸变以及其中包含的大量基因使得无法确定哪些基因在一定程度上驱动了这些肿瘤的生物学特性。我们使用 Affymetrix 100K 单核苷酸多态性 (SNP) 阵列芯片和 Genechip HumanGenome U133 Plus 2.0 表达阵列（Kotliarov 等，2006）分析了大量神经胶质瘤样本，以了解复发性 CNA 的遗传特征。根据这些阵列和基因表达谱实验的生物信息学数据，我们发现神经胶质瘤中经常发生改变的新基因。此外，我们还为这个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 Gateway 入门克隆，并将它们克隆到 pLenti/UbC/V5 表达载体中，用于转导各种靶细胞系。通过我们的候选基因构建体，我们将确定候选基因是否以可能与肿瘤发生中的作用一致的方式改变这些细胞的生物学（即免疫抑制动物中的克隆发生、增殖、凋亡、致瘤潜力）。 NOB 实验室正在使用 DHS-seq 方法来分析神经胶质瘤患者样本中的全基因组转录变化。如上所述，DHS-seq 将揭示染色质的动态变化，这对于脑肿瘤的发生和进展非常重要，并使我们能够确定治疗这种疾病的新分子靶点。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，结合基因表达和拷贝数数据，我们将获得对脑肿瘤生物学基础基因组学的新见解。为此，NOB 实验室已开始在患者样本上测试这种方法，使用直接来自手术标本的肿瘤组织和邻近的正常大脑。该计划是继续处理更多的患者样本，最终目标是将“转录组”分析纳入全面的基因组分析中，该分析正计划作为分子肿瘤板的组成部分，如临床项目中所述。