Role of SerpinB3 in glioblastoma cancer stem cells

项目摘要 SerpinB 3在胶质母细胞瘤干细胞中的作用 胶质母细胞瘤（GBM）是最具侵袭性的原发性恶性脑肿瘤，中位生存期为18-20 个月尽管有包括手术、放疗和化疗在内的治疗干预， 化疗和放疗抗性细胞重新填充肿瘤，导致复发和高患者死亡率。 mortality.这些细胞被称为癌症干细胞（CSC），因为它们具有自我更新和生成肿瘤细胞的能力。 肿瘤中存在的细胞异质性。我们的实验室鉴定了连接粘附分子-A（JAM-A）， 通过功能研究，证明JAM-A对于自我更新是必要的，也是充分的 和肿瘤生长。我们确定JAM-A通过GBM CSC中的Akt信号来维持多能性转录， 因子活性;然而，中间信号网络尚未完全阐明。为了进一步描述这一点， 通过免疫沉淀GBM CSC中的JAM-A，并进行质谱分析以确定GBM CSC中的JAM-A。 JAM-A直接结合的蛋白质。该分析导致鉴定了丝氨酸/半胱氨酸蛋白酶 抑制剂SerpinB 3作为结合伴侣。有趣的是，SerpinB 3不包含保守的PDZ结构域， 存在于几乎所有其它已知的JAM-A结合配偶体上。尽管有多种促肿瘤发生机制， 包括调节TGF-β1和抑制细胞凋亡，已经提出SerpinB 3在 在其他癌症中，对GBM CSC中蛋白质的功能及其与JAM-A的关系知之甚少 尚待阐明。使用体外CSC功能测定，我已经积累了SerpinB 3是 SerpinB 3是维持CSC所必需的，SerpinB 3的减少会减弱TGF-β1的表达。基于 根据这些观察，我假设SerpinB 3与JAM-A的相互作用对于维持 GBM CSC通过调节TGF-β1和抑制凋亡。目的1将检验SerpinB 3 通过抑制细胞凋亡和上调TGF-β1维持CSC状态。我会破坏溶酶体 用siramesine处理细胞膜以阐明SerpinB 3在抑制细胞凋亡中的作用。另外，我将 探讨SerpinB 3在肿瘤干细胞TGF-β1信号转导中的作用。目标2将检验以下假设： 靶向JAM-A/SerpinB 3相互作用将损害自我更新和GBM生长。我将使用DSSO 交联以确定两种蛋白质之间的相互作用区域。最后，我将确定 这是用小干扰肽破坏CSC状态上的JAM-A/SerpinB 3相互作用的结果。 成功完成这个项目将促进我们的理解如何在CSC的状态是保持在 GBM通过特异性JAM-A细胞内结合结构域，桥接细胞通讯和细胞信号传导。的 在这个奖学金概述的研究将为我提供一个机会，获得在脑肿瘤研究的经验， 允许我通过科学会议和指导机会继续我的训练，为职业生涯做准备 作为一个医生科学家。

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.