Diverse roles of Notch signaling in glioblastoma

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是一种无法治愈的脑恶性肿瘤，治疗选择有限。在GBM中，具有干细胞样特性的细胞（GBM干细胞或GSC）引发并增殖肿瘤，并且对常规放化疗具有高度抗性。理解GSC的生物学和开发直接靶向它们的疗法的主要障碍是缺乏普遍识别它们的分子标记物。先前的观察表明，抑制Notch信号传导（一种调节神经胶质细胞发育中的命运决定的途径）减弱但不完全阻断GSC的自我更新。这些发现提高了Notch激活可能对 一些但不是所有的GSC，表明GSC隔室中的功能和分子异质性。在飓风桑迪之前，我们启动了一系列实验，旨在阐明Notch信号被激活的GBM细胞的身份，并了解它们在肿瘤生长期间和对化疗的反应中对肿瘤异质性的贡献。使用经基因工程改造以响应Notch信号传导的激活而表达荧光报告基因的原代人GBM培养物，我们发现，在有利于GSC自我更新的体外条件下，Notch在不表达CD133（一种公认的GSC细胞表面标记物）的细胞中被激活。此外，我们发现体外诱导分化增加了具有活化的Notch信号传导的细胞的分数。这些发现提出了关于Notch通路在GBM细胞层次中的作用的重要问题：Notch信号是否识别具有肿瘤起始特性的干细胞或GBM中不同类型的祖细胞？Notch信号在体内活跃的细胞有哪些谱系？这些细胞对化疗有何反应？不幸的是，这场风暴对我们的实验室造成了重大破坏，包括我们的原始人类GBM培养物的损失。此外，我们必须在纽约大学的一个新的动物设施中重新建立我们的小鼠群体。通过这个资助机会，我们正在申请资金来扩展我们的初步工作，以进一步了解Notch途径在GBM中的功能。我们的实验将评估具有活性Notch信号传导的细胞是否具有肿瘤起始性质以及它们在体内产生什么样的细胞谱系，包括在用化疗剂替莫唑胺（GBM临床管理的主要药物）治疗后。我们预计，我们的研究结果将产生一个临界质量的数据，这将导致一个R01提案的出版和成功提交。重要的是，我们的发现将揭示神经肿瘤学研究的一个重要领域，并将促进新的和明智的治疗策略的设计。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is an incurable brain malignancy with limited treatment options. Within GBM, cells with stem-like properties (GBM stem cells or GSCs) initiate and propagate tumors, and are highly resistant to conventional chemoradiotherapy. A major obstacle in understanding the biology of GSCs and developing therapies that directly target them has been the lack of molecular markers that universally identify them. Previous observations indicated that inhibition of Notch signaling, a pathway that regulates fate decisions in neuroglial development, attenuates but does not completely block the self-renewal of GSCs. These findings raise the possibility that Notch activation may be critical to some but not all GSCs, suggesting functional and molecular heterogeneity within the GSC compartment. Prior to Hurricane Sandy, we initiated a set of experiments aiming to clarify the identity of GBM cells in which Notch signaling is activated and understand their contribution to tumor heterogeneity during tumor growth and in response to chemotherapy. Using primary human GBM cultures genetically engineered to express fluorescent reporters in response to activation of Notch signaling, we discovered that, under in vitro conditions that favor GSC self-renewal, Notch is activated in cells that do not express CD133, a well-established cell surface marker of GSCs. Furthermore, we found that in vitro induction of differentiation increases the fraction of cells with activated Notch signaling. These findings raise important questions about the role of the Notch pathway in the cellular hierarchy of GBM: Does Notch signaling identify stem cells with tumor initiating properties or a different type of progenitor cells within GBM? What are the lineages that descend from cells in which Notch signaling is active in vivo? And how do these cells respond to chemotherapy treatment? Unfortunately, the storm inflicted substantial damage to our laboratory, including loss of our primary human GBM cultures. In addition, we had to re-establish our mouse colony in a new animal facility within NYU. Through this funding opportunity, we are requesting funds to extend our preliminary work to further understand the function on the Notch pathway in GBM. Our experiments will assess whether cells with active Notch signaling have tumor- initiating properties and what cell lineages they generate in vivo, including after treatment with the chemotherapeutic agent temozolamide, a mainstay in clinical management of GBM. We anticipate that our findings will generate a critical mass of data that will lead to a publication and a successful submission of an R01 proposal. Importantly, our findings will shed light on an important area of research within neuro-oncology and will facilitate the design of novel and informed therapeutic strategies.