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: 8349326
• 负责人: Howard Fine
• 金额: $ 63.46万
• 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/8349326
• 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; Lesion; Loss of Heterozygosity; Morphology; Mus; Mutant Strains Mice; Mutation; Neurofibromatosis 1; Neuroglia; Neurons; Oncogenes; PTEN gene; Plasmids; Role; Sampling; Single Nucleotide Polymorphism; Staging; Stem cells; TP53 gene; The Jackson Laboratory; Therapeutic Intervention; Transplantation; Tumor Biology; Tumor Stem Cells; Tumor Suppressor Genes; Western Blotting; base; chromosome 5q loss; expression vector; gene cloning; immunosuppressed; in vivo; nerve stem cell; novel; relating to nervous system; research study; self-renewal; stem; tumor; tumorigenesis; tumorigenic

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。缺乏 PTEN 的小鼠表现出增大的、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尺寸增大所致。此外，Pten -/- 干/祖细胞至少部分地表现出更大的增殖能力，从而缩短细胞周期（Croszer 等人，2001）。 p16INK4A/ARF 敲除和成熟星形胶质细胞中持续活跃的 EGFR 表达相结合，导致颅内移植后神经胶质瘤样病变的形成（Bachoo 等，2002）。 Trp53 和 Nf1（神经特异性神经纤维瘤病 1 型）双突变小鼠表现出一系列星形细胞瘤阶段，从低级别星形细胞瘤到多形性胶质母细胞瘤 (GBM) (Reilly 等，2000)。最近，人类神经胶质瘤的传统和基于阵列的 CGH (aCGH) 分析显示出大量的拷贝数改变 (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 等人，2006；Nigro 等人，2005；Phillips 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 人类基因组 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。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。我们将使用的靶细胞不仅包括已建立的肿瘤干细胞系，如 0308 和 1228 神经胶质瘤系，还包括来自各种转基因小鼠系的小鼠胚胎神经干细胞 (mNSC)，如 PTEN loxP/loxP 和 p16INK4A/ARF - /-、PTEN loxP/loxP、PTEN loxP/+、p16INK4A/ARF -/- 和野生型小鼠。为了为本研究产生双敲除 mNSC，我们培育了 PTEN loxP/loxP (Croszer et al., 2001) (The Jackson Laboratory, ME) 和 p16INK4A/ARF -/- (Sharpless et al., 2001) (NCI-Frederick) , MD) 小鼠并生成 PTEN loxP/loxP & p16INK4A/ARF -/-, PTEN loxP/loxP、PTEN loxP/+、p16INK4A/ARF -/- 和野生型小鼠。然后我们从这些小鼠中建立了 E14 小鼠胚胎神经干细胞 (mNSC)。为了制备 PTEN loxP/loxP & p16INK4A/ARF -/-、PTEN loxP/loxP、PTEN loxP/+ mNSC，我们将 CRE-EGFP 质粒转染于对这些 E14 mNSC 进行 PTEN 删除，并在两周内用抗生素选择转染细胞。选择后，我们通过CRE筛选PTEN敲除的克隆。我们通过蛋白质印迹分析和基因分型证实了 PTEN 敲除。通过我们的候选基因构建体和mNSC，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）。预计通过这些体外和体内筛选，我们将发现与神经胶质瘤发生相关的候选基因，并可能为治疗干预提供潜在的新靶点。