Identifying New Glioma-Associated Tumor Suppressors and Oncogenes

胶质瘤干细胞是一种在体外培养条件下能够自我更新，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神经胶质样有丝分裂后后代的肿瘤亚群。缺乏PTEN的小鼠表现出扩大的、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。此外，Pten-/-干细胞/祖细胞表现出更大的增殖能力，至少在一定程度上是因为细胞周期缩短(Croszer等人，2001年)。在成熟的星形胶质细胞中，p16INK4A/ARF基因敲除和成分活性的EGFR表达导致了颅内移植后胶质瘤样病变的形成(Bachu等人，2002年)。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等人，2000年)。最近，人脑胶质瘤的常规和基于阵列的CGH(ACGH)分析已经显示出相当数量的拷贝数改变(CNA)，包括增加/放大(1p34-36、1q32、3q26-28、5q、7q31、8q24、11q、12q13、13q、15p15、17q22-25、19q、20p和20q)以及缺失/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年；尼格罗等人，2005年；菲利普斯等人，2006年)。到目前为止，大量的染色体异常和其中包含的大量基因使得人们无法确定哪些基因在一定程度上推动了这些肿瘤的生物学。我们使用Affymetrix 100K单核苷酸多态性(SNP)芯片和基因芯片人类基因组U133 Plus 2.0表达阵列(Kotliarov等人)分析了大量胶质瘤样本的复发CNAs的基因特征。2006)。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频繁改变的新基因。我们已经构建了这些基因的序列验证基因Gateway Entry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(MNSCs)，如PTEN loxP/loxP&p16INK4A/ARF-/-、PTEN loxP/loxP、PTEN loxP/+、p16INK4A/ARF-/-和野生型小鼠。为了为这项研究产生双基因敲除mNSCs，我们培育了PTEN loxP/loxP(Croszer等，2001)(杰克逊实验室，ME)和p16INK4A/ARF-/-(夏普莱斯等，2001)(NCI-Frederick，MD)小鼠，并产生了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(MNSCs)。为了获得PTEN loxP/loxP和p16INK4A/ARF-/-、PTEN loxP/loxP、PTEN loxP/+mNSCs，我们将CRE-EGFP质粒导入这些E14小鼠神经干细胞中进行PTEN缺失，并用抗生素筛选转染细胞。筛选后，用Cre法筛选PTEN基因敲除克隆。我们通过Western Blot分析和基因分型证实了PTEN基因的敲除。通过我们的候选基因构建和mNSCs，我们将确定候选基因是否以一种可能与肿瘤发生中的角色一致的方式改变这些细胞的生物学特性(即克隆形成、增殖、凋亡、免疫抑制动物的致瘤潜力)。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，并可能为治疗干预提供潜在的新靶点。

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.