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具有侵略性的神经胶质瘤类型，它们表现出高细胞异质性，异质形态和复杂的染色体畸变（Furnari等，2007）。原发性胶质母细胞瘤，大多数病例（> 90％），其特征是杂合性丧失10q（占病例的70％），EGFR扩增（36％），P16 Ink4a缺失（31％）和PTEN突变（25％）。 TP53突变是最常见的，并且已经存在于60％的前体低级星形胶质细胞瘤中（Ohgaki等，2007）。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。缺乏PTEN的小鼠表现出肿大的组织结构异常的大脑，这是由于细胞增殖增加，细胞死亡减少和细胞大小增大所致。 PTEN - / - 茎/祖细胞至少部分地显示出更大的增殖能力，即缩短细胞周期（Croszer等，2001）。成熟星形胶质细胞中的P16INK4A/ARF敲除和组成性活跃的EGFR表达导致颅内移植后的胶质瘤形成如病变（Bachoo等，2002）。 TRP53和NF1（神经特异性神经纤维瘤病1型）双突变小鼠显示出一系列星形胶质细胞瘤阶段，从低级星形胶质细胞瘤到多形胶质母细胞瘤（GBM）（Reilly等，2000）。最近，人神经胶质瘤的常规和基于阵列的CGH（ACGH）分析显示了大量的拷贝数变化（CNA），包括增益/扩增（1p34-36，1q32，1q32，3q26-28，5q，5q，7q31，7q31，8q24，8Q24，8Q24，8Q13，11q13，12Q13，13Q，15P15，15P15，15P15，17Q2222，10Q22，19Q 2，19Q 2，19Q 2，19Q，19q，19q，19q，19q，19q，19q。 3Q25-26、4Q，4Q，6Q26-27、9P，10Q，10Q，10P，11P，12Q22、12Q，13Q，14Q13、14Q23-31、15Q13-21、17P11-13、17P11-13、18Q222222-23、19Q和22Q和22Q）（Kotliarov等人，2006年，2006年； Nigro等人，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。 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).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。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。 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老鼠。为了在这项研究中生成双重敲除MNSC，我们培养了PTEN LOXP/LOXP（Croszer等，2001）（杰克逊实验室，ME）和p16ink4a/arf - / - （Sharpless等，2001）（2001年）（Nci-frederick，MD）（Nci-frederick，MITE）和生成的Pten Loxp/loxp/loxp/loxp＆p16， loxp/loxp，pten loxp/+，p16ink4a/arf - / - 和野生型小鼠。 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.选择后，我们通过CRE筛选了PTEN淘汰的克隆。我们通过蛋白质印迹分析和基因分型证实了PTEN敲除。 借助我们的候选基因构建体和MNSC，我们将确定候选基因是否会改变这些细胞的生物学，以与肿瘤发生的作用相一致（即克隆生成，增殖，凋亡，肿瘤菌种潜在免疫抑制动物中的潜在）。可以预期，通过这些体外和体内筛选，我们将发现与神经胶质作用有关的候选基因，并可能为治疗干预提供潜在的新靶标。