RNAi screen of the glioblastoma stem cell kinome under hypoxia and normoxia

缺氧和常氧条件下胶质母细胞瘤干细胞激酶组的RNAi筛选

• 批准号: 8640989
• 负责人: BRENT H. COCHRAN
• 金额: $ 35.73万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8640989
• 关键词: Astrocytes; Brain Glioblastoma; Brain Neoplasms; Cause of Death; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Clinical; Data; Diagnosis; Genes; Glioblastoma; Growth; Human; Hypoxia; Mean Survival Times; Methods; Modeling; Mus; Neurons; Phospho-Specific Antibodies; Phosphotransferases; RNA Interference; Research; Resistance; Serum; Specificity; Stem cells; Tumor Stem Cells; Xenograft Model; Xenograft procedure; base; cancer stem cell; cell growth; glioma cell line; high throughput screening; improved; inhibitor/antagonist; knock-down; screening; small hairpin RNA; success; traditional therapy; tumor; tumor growth; tumorigenesis; tumorigenic

DESCRIPTION (provided by applicant): Glioblastomas are highly aggressive brain tumors with a mean survival time of only 14 months. There is increasing evidence that brain tumors and glioblastomas in particular are driven by a subpopulation of cancer stem cells. It has recently been possible to isolate and maintain continuously in culture these putative tumor stem cells. These cells grow as neurospheres and retain the ability to differentiate into neurons and astrocytes despite being highly tumorigenic mouse xenografts. We have developed methods for high throughput screening of these GBM stem cells using RNAi. Here we propose to comprehensively identify kinases that are required for the growth and survival of glioblastoma stem cells under normoxic and hypoxic conditions. We will focus on kinases since they are druggable targets for which a great many inhibitors are already available and for which several have already been clinical successes. Our first aim will be to perform shRNA screens of the human kinome to identify kinases commonly required for the growth and survival of glioblastoma stem cell lines. Our preliminary results indicate that the kinases required for tumor stem cell growth are distinct from the kinases required for growth of traditional serum grown cell lines. Thus, these screens are likely to uncover new kinase targets for glioblastoma therapy. Moreover, glioblastomas are known to be tumors that have hypoxic microenvironments that can limit the efficacy of traditional therapies. Our evidence indicates that glioblastoma stem cells actually thrive under hypoxia. Our preliminary data also indicates that there are kinases that are required for tumor stem cell growth under normoxic conditions, but not hypoxic conditions and vice versa. Identifying the kinases that are required for growth and survival under hypoxic conditions is thus critical for elimination of cancer stem cells in the tumor. Thus, we will screen these GBM stem cells under both hypoxic and normoxic conditions. In the second aim, we will investigate the cellular functions of kinases identified in the RNAi screens. In addition, we will determine whether key kinases identified in the screen are in fact activated in primary human glioblastomas, and determine whether inhibition of these kinases in mouse xenografts will inhibit tumor growth. Results of these studies should identify and prioritize new kinase targets for treatment of glioblastoma.

描述（由申请人提供）：胶质母细胞瘤是一种高度侵袭性的脑肿瘤，平均生存时间仅为14个月。越来越多的证据表明，脑肿瘤和胶质母细胞瘤尤其是由癌症干细胞亚群驱动的。最近已经可以分离并持续培养这些假定的肿瘤干细胞。这些细胞作为神经球生长，并保留分化成神经元和星形胶质细胞的能力，尽管是高度致瘤性的小鼠异种移植物。我们已经开发了使用RNAi高通量筛选这些GBM干细胞的方法。在这里，我们建议全面确定所需的激酶，在常氧和缺氧条件下的胶质母细胞瘤干细胞的生长和生存。我们将集中在激酶，因为它们是可药用的目标，已经有很多抑制剂，其中一些已经在临床上取得了成功。 我们的第一个目标是对人类激酶组进行shRNA筛选，以鉴定胶质母细胞瘤干细胞系生长和存活所需的激酶。我们的初步结果表明，肿瘤干细胞生长所需的激酶是从传统的血清生长的细胞系生长所需的激酶不同。因此，这些筛选可能会发现胶质母细胞瘤治疗的新激酶靶点。此外，胶质母细胞瘤是已知的具有缺氧微环境的肿瘤，其可以限制传统疗法的功效。我们的证据表明，胶质母细胞瘤干细胞实际上在缺氧条件下茁壮成长。我们的初步数据还表明，在常氧条件下，肿瘤干细胞生长需要激酶，但在缺氧条件下则不需要，反之亦然。因此，鉴定在缺氧条件下生长和存活所需的激酶对于消除肿瘤中的癌症干细胞至关重要。因此，我们将在低氧和常氧条件下筛选这些GBM干细胞。 在第二个目标中，我们将研究在RNAi筛选中鉴定的激酶的细胞功能。此外，我们将确定在筛选中鉴定的关键激酶是否在原发性人胶质母细胞瘤中实际上被激活，并确定在小鼠异种移植物中抑制这些激酶是否会抑制肿瘤生长。这些研究的结果应确定和优先考虑新的激酶靶点治疗胶质母细胞瘤。