Role of SerpinB3 in glioblastoma cancer stem cells

项目摘要 SERPINB3在胶质母细胞瘤癌细胞中的作用 胶质母细胞瘤(GBM)是最具侵袭性的原发恶性脑肿瘤，中位生存期为18-20 月份。尽管包括手术、放射和化疗在内的治疗干预措施，但多个克隆的 化疗和放疗耐药细胞重新填充肿瘤，导致复发和患者高比率 死亡率。这些细胞被称为癌症干细胞(CSCs)，因为它们具有自我更新和生成的能力 肿瘤中存在细胞异质性。我们实验室鉴定出交界性黏附分子-A(JAM-A) 并通过功能研究证明，JAM-A是自我更新的必要条件和充分条件 和肿瘤的生长。我们确定JAM-A信号通过Akt在GBM CSCs中维持多能性转录 因子的活性；然而，中间信号网络还没有完全阐明。为了进一步描述这一点 途径，我们从GBM CSCs中免疫沉淀JAM-A并进行质谱分析 JAM-A直接与之结合的蛋白质。这一分析导致了丝氨酸/半胱氨酸蛋白酶的鉴定 抑制剂SerpinB3作为结合伙伴。有趣的是，SerpinB3不包含保守的PDZ结构域 几乎存在于其他所有已知的JAM-A结合伙伴上。尽管有多种促肿瘤机制， 包括调节转化生长因子-β1和抑制细胞凋亡，已经在以下背景下被提出 在其他癌症中，人们对GBM CSCs中该蛋白的功能及其与JAM-A的关系知之甚少 目前尚不清楚。使用体外CSC功能分析，我积累了SerpinB3是 CSCs维持所必需的，SerpinB3的减少可减弱转化生长因子-β-1的表达。基座 根据这些观察，我假设SerpinB3与JAM-A的相互作用对于维持 GbM CSCs通过调节转化生长因子-β-1和抑制细胞凋亡。目标1将检验SerpinB3的假设 通过抑制细胞凋亡和上调转化生长因子-β1来维持干细胞的状态。 用西拉美辛作用于细胞膜，阐明SerpinB3在抑制细胞凋亡中的作用。另外，我会 探讨丝裂原B3在神经干细胞转化生长因子-β-1信号调控中的作用。目标2将检验这一假设 以JAM-A/SerpinB3相互作用为目标将损害自我更新和GBM增长。我将使用DSSO 用来确定两种蛋白质相互作用区域的交联物。最后，我将确定 用小干扰肽破坏JAM-A/SerpinB3相互作用对CSC状态的影响。 这个项目的成功完成将促进我们对CSCS状态是如何在 GBM通过特定的JAM-A细胞内结合域，连接细胞通讯和细胞信号。这个 这项奖学金中概述的研究将为我提供一个在脑瘤研究和治疗方面获得经验的机会 允许我通过科学会议和指导机会继续我的培训，为职业生涯做准备 作为一名内科科学家。

Project Summary ROLE OF SERPINB3 IN GLIOBLASTOMA CANCER STEM CELLS Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of 18-20 months. Despite therapeutic interventions including surgery, radiation, and chemotherapy, multiple clones of chemo- and radiotherapy-resistant cells repopulate the tumor, resulting in recurrence and a high rate of patient mortality. These cells are referred to as cancer stem cells (CSCs) due to their ability to self-renew and generate the cellular heterogeneity present in the tumor. Our lab identified junctional adhesion molecule-A (JAM-A) on CSCs and, through functional studies, demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. We determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the intermediate signaling network is yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from GBM CSCs and performed mass spectrometry to determine the proteins to which JAM-A directly binds. This analysis led to the identification of the serine/cysteine protease inhibitor SerpinB3 as a binding partner. Interestingly, SerpinB3 does not contain the conserved PDZ domain that is present on nearly every other known JAM-A binding partner. Although multiple pro-tumorigenic mechanisms, including regulation of TGF-β1 and inhibition of apoptosis, have been proposed for SerpinB3 in the context of other cancers, very little is known about the function of the protein in GBM CSCs, and its relationship to JAM-A is yet to be elucidated. Using in vitro CSC functional assays, I have accumulated evidence that SerpinB3 is necessary for the maintenance of CSCs and that reduction of SerpinB3 attenuates TGF-β1 expression. Based on these observations, I hypothesize that SerpinB3 interaction with JAM-A is essential for the maintenance of GBM CSCs through regulation of TGF-β1 and inhibition of apoptosis. Aim 1 will test the hypothesis that SerpinB3 maintains the CSC state through inhibition of apoptosis and upregulation of TGF-β1. I will disrupt the lysosomal membrane with siramesine to elucidate the role of SerpinB3 in the inhibition of apoptosis. Additionally, I will investigate the role of SerpinB3 in the regulation of TGF-β1 signaling in CSCs. Aim 2 will test the hypothesis that targeting the JAM-A/SerpinB3 interaction will compromise self-renewal and GBM growth. I will utilize DSSO crosslinking to determine the region of interaction between the two proteins. Finally, I will determine the consequence of disrupting the JAM-A/SerpinB3 interaction on the CSC state with small interfering peptides. Successful completion of this project will advance our understanding of how the CSCs state is maintained in GBM via specific JAM-A intracellular binding domains, bridging cellular communication and cell signaling. The studies outlined in this fellowship will provide me an opportunity to gain experience in brain tumor research and allow me to continue my training though scientific meetings and mentorship opportunities, preparing for a career as a physician scientist.