Role of SerpinB3 in glioblastoma cancer stem cells

SerpinB3 在胶质母细胞瘤干细胞中的作用

• 批准号: 10845572
• 负责人: Adam Lauko
• 金额: $ 5.27万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10845572
• 关键词: Apoptosis; Attenuated; Binding; Biological Assay; Brain Neoplasms; Cell Death; Cell Maintenance; Cell Proliferation; Cells; Chemotherapy and/or radiation; Co-Immunoprecipitations; Communication; Cysteine Proteinase Inhibitors; Data; Development; Fellowship; Foundations; Future; Glioblastoma; Growth; Heterogeneity; Immunoprecipitation; In Vitro; Inhibition of Apoptosis; Invaded; LIF gene; Laboratories; Lysine; Lysosomes; Maintenance; Malignant Neoplasms; Malignant neoplasm of brain; Mass Spectrum Analysis; Membrane; Mentorship; Molecular; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Phenotype; Phosphorylation Site; Physicians; Population; Proliferating; Protein Secretion; Proteins; Proto-Oncogene Proteins c-akt; Radiation therapy; Recombinants; Recurrence; Regulation; Reporting; Research; Resistance; Role; Scientist; Serine; Serpins; Signal Transduction; Site; Testing; Therapeutic; Therapeutic Intervention; Training; Transforming Growth Factor beta; Up-Regulation; cancer stem cell; career; chemotherapy; crosslink; disease prognostic; disuccinimidyl suberate; experience; inhibitor; insight; junctional adhesion molecule; knock-down; liquid chromatography mass spectrometry; meetings; mortality; nerve stem cell; neutralizing antibody; new therapeutic target; next generation; novel; overexpression; pluripotency; protein expression; protein function; self-renewal; standard of care; stem cell function; stem cell model; stem cell self renewal; stem cells; therapeutic development; therapy resistant; transcription factor; treatment response; tumor; tumor growth; tumor initiation; tumorigenic

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.

项目概要 SERPINB3 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 几个月。尽管采取了包括手术、放疗和化疗在内的治疗干预措施，但多个克隆 化疗和放疗耐药的细胞重新填充肿瘤，导致复发和患者的高发病率 死亡。这些细胞由于具有自我更新和生成能力而被称为癌症干细胞（CSC） 肿瘤中存在的细胞异质性。我们的实验室鉴定出连接粘附分子-A (JAM-A) CSCs 并通过功能研究证明 JAM-A 对于自我更新来说是必要且充分的 和肿瘤生长。我们确定 JAM-A 在 GBM CSC 中通过 Akt 发出信号以维持多能性转录 因子活动；然而，中间信号网络尚未完全阐明。为了进一步明确这一点 途径，我们免疫沉淀 GBM CSC 中的 JAM-A，并进行质谱分析以确定 JAM-A 直接结合的蛋白质。该分析导致丝氨酸/半胱氨酸蛋白酶的鉴定 抑制剂 SerpinB3 作为结合伴侣。有趣的是，SerpinB3 不包含保守的 PDZ 结构域， 几乎存在于所有其他已知的 JAM-A 结合伴侣上。尽管有多种促肿瘤机制， SerpinB3 的作用包括调节 TGF-β1 和抑制细胞凋亡 对于其他癌症，人们对 GBM CSC 中该蛋白的功能及其与 JAM-A 的关系知之甚少 尚待阐明。通过体外 CSC 功能测定，我积累了 SerpinB3 的证据 SerpinB3 的减少会减弱 TGF-β1 的表达。基于 根据这些观察结果，我假设 SerpinB3 与 JAM-A 的相互作用对于维持 GBM CSCs 通过调节 TGF-β1 并抑制细胞凋亡。目标 1 将检验 SerpinB3 的假设 通过抑制细胞凋亡和上调 TGF-β1 维持 CSC 状态。我会破坏溶酶体 膜与西拉美辛阐明 SerpinB3 在抑制细胞凋亡中的作用。另外，我将 研究 SerpinB3 在 CSC 中 TGF-β1 信号传导调节中的作用。目标 2 将检验以下假设： 针对 JAM-A/SerpinB3 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/SerpinB3 相互作用的结果。 该项目的成功完成将加深我们对 CSC 状态如何在 GBM 通过特定的 JAM-A 细胞内结合域，桥接细胞通讯和细胞信号传导。这 该奖学金中概述的研究将为我提供获得脑肿瘤研究经验的机会 让我通过科学会议和指导机会继续接受培训，为职业生涯做好准备 作为一名医学科学家。