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: 10926255
• 负责人: Mark Gilbert
• 金额: $ 52.21万
• 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/10926255
• 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 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; Disparity; 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; 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; Proliferating; Protocols documentation; Receptor Biology and Gene Expression; Recurrence; Reproducibility; 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; 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; permissiveness; postmitotic; self-renewal; stem cells; 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 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.

此前，我们与人类基因组计划（Human Genome Project）和癌症基因组解剖计划（Cancer Genome Anatomy Project， CGAP）合作开展了一项大型cDNA微阵列研究，利用cDNA微阵列技术基于基因表达谱，开发了一种全面而新颖的人类胶质瘤分子分类模式。我们已经构建了自己的cDNA微阵列“芯片”，该芯片将增强对胶质瘤生物学中重要的新基因和选择性基因的识别。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，可以在培养中自我更新，在体内原位移植中使肿瘤永存，并在体内和体外产生多样化的神经元样和胶质样有丝分裂后后代。最近，传统的和基于阵列的人类胶质瘤CGH （aCGH）分析显示了大量拷贝数改变（CNAs），包括增益/扩增（1p34- 36,1q32, 3q26- 28,5q, 7q31, 8q24, 11q, 12q13, 13q, 15p15, 17q22- 25,19q， 20p和20q）和缺失/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等人，2006；Nigro等人，2005；Phillips等人，2006）。到目前为止，大量的染色体畸变和其中包含的大量基因使人们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 et al. 2006）分析了大量胶质瘤样本，以确定复发性CNAs的遗传特征。基于我们从这些阵列和基因表达谱实验中获得的生物信息学数据，我们发现了在胶质瘤中经常改变的新基因。此外，我们还为这个项目探索了新的生物技术，如下一代测序。我们已经建立了这些基因的序列验证基因入口克隆，并将其克隆到pLenti/UbC/V5表达载体上，用于各种靶细胞系的转导。通过我们的候选基因构建，我们将确定候选基因是否以一种可能与肿瘤发生作用一致的方式改变这些细胞的生物学特性（即克隆发生、增殖、凋亡、免疫抑制动物的致瘤潜力）。NOB实验室最近开始与Gordon Hager博士和受体生物学和基因表达实验室合作。Hager博士的工作重点是核染色质的重组以及这些变化对基因调控的影响。在脑肿瘤生物学的背景下，有各种各样的原发性中枢神经系统肿瘤，尽管有恶性表型，但很少有突变。因此，转录谱的改变可能有助于解释这种明显的差异。NOB实验室正在使用DHS-seq方法来分析神经胶质瘤患者样本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，这在脑肿瘤的发生和发展中是重要的，并使我们能够确定新的分子靶点来治疗这种疾病。我们在两种胶质瘤干细胞系（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，我们将对脑肿瘤生物学的基因组学基础获得新的见解。为此，NOB实验室已经开始在患者样本上测试这种方法，使用肿瘤组织和邻近的正常大脑直接从手术标本中提取。该计划是继续处理更多的患者样本，最终目标是将“转录组”分析纳入综合基因组分析，作为临床项目中描述的分子肿瘤板的组成部分。

项目成果

Association of Circadian Clock Gene Expression with Glioma Tumor Microenvironment and Patient Survival.

• DOI: 10.3390/cancers13112756
• 发表时间: 2021-06-02
• 期刊: Cancers
• 影响因子: 5.2
• 作者: De La Cruz Minyety J;Shuboni-Mulligan DD;Briceno N;Young D Jr;Gilbert MR;Celiku O;Armstrong TS
• 通讯作者: Armstrong TS