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: 7733494
• 负责人: Howard Fine
• 金额: $ 27.35万
• 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/7733494
• 关键词: 10p; 10q; 11p; 11q; 12q13; 12q22; 13q; 14q13; 14q23; 19q; 1p34; 1q32; 20p; 20q; 22q; 3q26; 6q26; 7q31; 8q24; Animals; Antibiotics; Apoptosis; Area; Astrocytes; Astrocytoma; Automobile Driving; Bioinformatics; Biological Assay; Brain; Breeding; CDKN2A gene; Candidate Disease Gene; Cell Cycle; Cell Death; Cell Line; Cell Proliferation; Cell Size; Cells; Cellular biology; Chromosome abnormality; Complex; Data; Embryo; Epidermal Growth Factor Receptor; Exhibits; Gene Expression Profiling; Genes; Genetic; Genotype; Glioblastoma; Glioma; Gliomagenesis; Heterogeneity; Human; Human Genome; In Vitro; Knock-out; Knockout Mice; Laboratories; Lesion; Loss of Heterozygosity; Morphology; Mus; Mutant Strains Mice; Mutation; Neurofibromatosis 1; Neuroglia; Neurons; Numbers; Oncogenes; PTEN gene; Plasmids; Purpose; Range; Role; Sampling; Single Nucleotide Polymorphism; Staging; Stem cells; TP53 gene; Therapeutic Intervention; Transplantation; Tumor Biology; Tumor Stem Cells; Tumor Suppressor Genes; Week; Western Blotting; base; chromosome 5q loss; expression vector; gene cloning; immunosuppressed; in vivo; nerve stem cell; novel; relating to nervous system; research study; stem; tumor; tumorigenesis; tumorigenic

Glioblastoma is the most common and biologically aggressive type of glioma and they exhibit high cellular heterogeneity, heterogeneous morphology and complex chromosome aberrations (Furnari et al., 2007). Primary glioblastomas, the majority of cases (> 90%), are genetically characterized by loss of heterozygosity 10q (70% of cases), EGFR amplication (36%), p16 INK4A deletion (31%), and PTEN mutation (25%). TP53 mutations are the most frequent and already present in 60% of precursor low-grade astrocytomas (Ohgaki et al., 2007). 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. Mice lacking PTEN exhibited enlarged, histoarchitecturally abnormal brains, which resulted from increased cell proliferation, decreased cell death, and enlarged cell size. Also Pten -/- stem/progenitor cells display a greater proliferation capacity at least in part, to a shortened cell cycle (Croszer et al., 2001). The combined p16INK4A/ARF knockout and constitutively active EGFR expression in mature astrocytes led to the formation of glioma like lesions following intracranial transplantation (Bachoo et al., 2002). Trp53 and Nf1 (neural-specific neurofibromatosis type 1) double mutant mice show a range of astrocytoma stages, from low-grade astrocytoma to glioblastoma multiforme (GBM) (Reilly et al., 2000). Recently, conventional and array-based CGH (aCGH) profiling of human gliomas have shown a significant number of copy number alterations (CNAs) including gain/amplication (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 Human Genome 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. 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. The target cells we will use will include not only established tumor stem cell lines such as 0308 and 1228 glioma lines, but also mouse embryonic neural stem cells (mNSCs) from various genetically modified mice lines such as PTEN loxP/loxP & p16INK4A/ARF -/-, PTEN loxP/loxP, PTEN loxP/+, p16INK4A/ARF -/- and wildtype mice. To generate double knockout mNSCs for this study, we had bred PTEN loxP/loxP (Croszer et al., 2001) (The Jackson Laboratory, ME) and p16INK4A/ARF -/- (Sharpless et al., 2001) (NCI-Frederick, MD) mice and generated PTEN loxP/loxP & p16INK4A/ARF -/-, PTEN loxP/loxP, PTEN loxP/+, p16INK4A/ARF -/- and wildtype mice. Then we had established E14 mouse embryonic neural stem cells (mNSCs) from these mice.To make PTEN loxP/loxP & p16INK4A/ARF -/-, PTEN loxP/loxP, PTEN loxP/+ mNSCs, we had transfected CRE-EGFP plasmid in these E14 mNSCs for PTEN deletion and selected transfected cells with antibiotics during two weeks. After selecting, we screened clones of PTEN knockout by CRE. We confirmed PTEN knockout through Western Blot analysis and genotyping. With our candidate gene constructs and mNSCs, 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). It is expected that through these in vitro and in vivo screens, we will discover candidate genes which are related to gliomagenesis and which may offer potentially new targets for therapeutic intervention.

胶质母细胞瘤是最常见和生物学上最具侵袭性的胶质瘤类型， 表现出高度的细胞异质性、形态异质性和复杂的染色体 畸变（Furnari等人，2007年）。原发性胶质母细胞瘤，大多数病例（> 90%），遗传特征为10 q杂合性缺失（70%的病例），EGFR 扩增（36%），p16 INK 4A缺失（31%）和PTEN突变（25%）。TP 53突变是 最常见并且已经存在于60%的前体低级别星形细胞瘤中（Ohgaki等， 2007年）。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， 肿瘤在体内原位移植，并产生多样化的神经元样和胶质样 体内和体外的有丝分裂后后代。缺乏PTEN的小鼠表现出增大， 组织结构异常的大脑，这是由于细胞增殖增加， 减少细胞死亡和增大细胞尺寸。Pten -/-干/祖细胞也显示出 更大的增殖能力至少部分地导致细胞周期缩短（Croszer等， 2001年）的报告。p16 INK 4A/ARF基因敲除和EGFR组成型活性表达的联合作用在成熟乳腺癌中的表达。 星形胶质细胞导致颅内移植后形成胶质瘤样病变 （Bachoo等人，2002年）。Trp 53和Nf 1（神经特异性神经纤维瘤病1型）双突变体 小鼠的星形细胞瘤分期范围从低级别星形细胞瘤到多形性胶质母细胞瘤 (GBM)（Reilly等人，2000）。最近，常规的和基于阵列的CGH（aCGH）分析显示， 人类神经胶质瘤已经显示出大量的拷贝数改变（CNA），包括 增益/放大（1 p34 -36，1 q32，3q 26 -28，5 q，7 q31，8 q24，11 q，12 q13，13 q，15 p15，17 q22 -25， 19 q，20 p和20 q）和缺失/丢失（3q 25 -26，4 q，6 q26 -27，9 p，10 p，10 q，11 p，12 q22，13 q， 14 q13、14 q23 -31、15 q13 -21、17 p11 -13、18 q22 -23、19 q和22 q）（Kotliarov等人，2006; Nigro et 例如，2005;菲利普斯等人，2006年）。大量的染色体畸变， 其中包含的基因数量，到目前为止已经无法确定哪些基因是 在一定程度上负责驱动这些肿瘤的生物学。我们分析了大量的 用于使用Affytron 100 K对复发性CNA进行遗传表征的胶质瘤样本 单核苷酸多态性（SNP）芯片和Genechip Human Genome U133 Plus 2.0 表达阵列（Kotliarov等人，2006）。根据我们从这些阵列中获得的生物信息学数据， 和基因表达谱实验，我们发现新的基因经常改变， 神经胶质瘤我们已经产生了这些基因的序列验证的基因网关入口克隆， 将它们克隆到pLenti/UbC/V5表达载体中，用于转导各种靶细胞 线我们将使用的靶细胞不仅包括已建立的肿瘤干细胞系 例如0308和1228神经胶质瘤细胞系，而且还包括来自 各种遗传修饰的小鼠系如PTENloxP/loxP和p16 INK 4A/ARF -/-， PTEN loxP/loxP、PTEN loxP/+、p16 INK 4A/ARF -/-和野生型小鼠。生成双顶出的步骤 对于该研究，我们培育了PTEN loxP/loxP（Croszer等人，2001）（《杰克逊》 Laboratory，ME）和p16 INK 4A/ARF -/-（Sharpless等人，2001）（NCI-Frederick，MD）小鼠和 生成的PTEN loxP/loxP & p16INK4A/ARF -/-、PTEN loxP/loxP、PTEN loxP/+、p16INK4A/ARF -/-和野生型小鼠。建立了E14小鼠胚胎神经干细胞（mNSCs） 为了从这些小鼠中制备PTEN loxP/loxP和p16 INK 4A/ARF -/-、PTEN loxP/loxP、PTEN loxP/+ 在这些E14 mNSC中，我们转染CRE-EGFP质粒用于PTEN缺失， 在两周内用抗生素转染细胞。在选择之后，我们筛选了 通过CRE敲除PTEN。我们通过蛋白质印迹分析和基因分型证实了PTEN敲除。 通过我们的候选基因构建体和mNSCs，我们将确定候选基因是否改变 这些细胞的生物学特性可能与肿瘤发生中的作用一致 (i.e.免疫抑制细胞克隆形成、增殖、凋亡、致瘤性 动物）。预计通过这些体外和体内筛选，我们将发现 与胶质瘤发生相关的候选基因，可能提供潜在的新靶点 进行治疗干预。