Diverse roles of Notch signaling in glioblastoma

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是一种无法治愈的脑部恶性肿瘤，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GSCs)发起和繁殖肿瘤，并且对传统的放化疗具有高度的抵抗力。在理解GSCs的生物学和开发直接针对它们的治疗方法方面的一个主要障碍是缺乏通用的分子标记来识别它们。先前的观察表明，抑制Notch信号通路，调节神经胶质细胞发育过程中的命运决定，可以减弱但不完全阻止GSCs的自我更新。这些发现增加了Notch激活可能对 一些而不是所有的GSC，提示GSC在功能和分子上的异质性。在飓风桑迪之前，我们启动了一系列实验，旨在澄清在其中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。利用基因工程的原代人GBM培养物来表达荧光报告基因，以响应Notch信号的激活，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的细胞中被激活，CD133是GSC公认的细胞表面标志。此外，我们发现，在体外诱导分化增加了激活Notch信号的细胞比例。这些发现提出了关于Notch通路在GBM细胞层次结构中的作用的重要问题：Notch信号是否识别具有肿瘤启动特性的干细胞或GBM中不同类型的前体细胞？在活体中Notch信号活跃的细胞有哪些血统？这些细胞对化疗有什么反应？不幸的是，风暴对我们的实验室造成了实质性的破坏，包括失去了我们的原代人类GBM培养。此外，我们不得不在纽约大学内的一个新的动物设施中重新建立我们的老鼠殖民地。通过这一资助机会，我们正在申请资金，以扩大我们的前期工作，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跃Notch信号的细胞是否具有肿瘤启动特性，以及它们在体内产生什么细胞系，包括在化疗药物替莫唑胺治疗后，替莫唑胺是GBM临床治疗的主要药物。我们预计，我们的发现将产生大量的数据，这些数据将导致发表和成功提交R01提案。重要的是，我们的发现将阐明神经肿瘤学中一个重要的研究领域，并将促进设计新的和知情的治疗策略。

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.