Constitutive Integrin Beta1 Signaling in Lung Cancer

肺癌中的组成型整合素 Beta1 信号传导

• 批准号: 10376760
• 负责人: Scott Haake
• 金额: $ 19.37万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10376760
• 关键词: Adenocarcinoma Cell; Agar; Binding; CD29 Antigen; Cell Adhesion; Cell Line; Cell Proliferation; Cells; Complex; Cytoplasmic Tail; Data; Development; Drug Targeting; ECM receptor; Epithelial; Extracellular Domain; Extracellular Matrix; Funding; Future; Gene Expression Profile; Gene Expression Profiling; Genes; Growth; Histological Techniques; Human; Integral Membrane Protein; Integrin Binding; Integrins; Knock-out; Ligands; Lung; Lung Adenocarcinoma; Lysine; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Mentors; Mutate; Neoplasm Metastasis; Nude Mice; Oncology; Pathway Analysis; Phosphorylation; Physicians; Polyubiquitination; Proteins; Proto-Oncogene Proteins c-akt; Receptor Cell; Receptor Signaling; Role; Scientist; Signal Transduction; TRAF6 gene; Tail; Testing; The Cancer Genome Atlas; Therapeutic; Tissue-Specific Gene Expression; Training; Tumor Cell Line; Ubiquitin; base; cancer clinical trial; cell motility; dimer; drug development; epithelial to mesenchymal transition; human data; in vitro Model; in vivo; lung cancer cell; mechanotransduction; mouse model; neglect; prevent; receptor; response; single-cell RNA sequencing; skills; transcriptome sequencing; tumor; tumor growth; tumor initiation; tumorigenesis

Project Summary: Integrins are transmembrane extracellular matrix (ECM) receptors composed of α and β subunits that form heterodimers. Integrins regulate many functions including cell adhesion and migration as well as signal and mechano-transduction. Integrins require both an ECM-binding ectodomain and a cytoplasmic tail that binds a variety of intracellular proteins to promote signaling. Integrins are implicated in tumor initiation, progression, and metastasis. However, integrin-targeted drugs, which focused on antagonizing the ectodomain, have largely failed in oncology clinical trials. These results suggest that the cytoplasmic tail may drive tumorigenesis independent of the ECM-binding ectodomain, though the mechanisms remain undefined. The principal integrins in the lung contain the b1 subunit. I have shown that integrin b1-knock out (KO) human lung adenocarcinoma cells injected into the lungs of athymic mice fail to form tumors. These data suggest that integrin b1 is required for lung adenocarcinoma development. Next, I developed integrin b1-KO cells that express either wildtype (WT) integrin b1, integrin b1 with a non-functional/mutated tail (b1 mt-tail) or integrin b1 with a non-functional ECM-binding ectodomain (Tac-b1). The WT and Tac-b1 cells formed soft agar colonies whereas the b1 mt-tail cells formed none. These data suggest that the integrin b1 tail is sufficient to permit tumor growth. RNA-seq gene expression analysis identified decreased epithelial-to-mesenchymal transition (EMT) and TGFb signaling signatures in integrin b1-KO cells. This correlation between integrin b1-KO and decreased expression of EMT-related genes was corroborated in human tumors. When the integrin b1-KO cells were further examined, I observed that while canonical TGFb signaling was unaffected, TGFb1-induced AKT phosphorylation was decreased. This observation is consistent with my mentor’s previous data that integrin b1 regulates AKT activation in a PI3K-independent manner that involves AKT lysine-63 polyubiquitination mediated by TRAF6. In summary, integrin b1 tail is required for tumor growth, and loss of integrin b1 is associated with loss of EMT and TGFb signal transduction. Based on these findings, I hypothesize that ECM-independent integrin b1 tail signaling regulates EMT and permits lung cancer growth by regulating TGFb1-induced AKT activation. I have developed two aims to test this hypothesis. In Aim 1, I will test the hypothesis that integrin b1 promotes tumor growth by facilitating EMT. I will use lung cancer mouse models and evaluate EMT in tumors with and without integrin b1 using histologic techniques and single cell RNA-seq. In Aim 2, I will test the hypothesis that the integrin b1 tail is sufficient for tumor development, and tail interactions with TRAF6 are required for TGFb-dependent AKT activation, EMT, and cell proliferation. I will use b1 mt-tail and Tac-b1 cells to define the role of the integrin b1 tail and the mechanisms whereby it facilitates TGFb signaling to promote lung cancer. Moving forward, I expect to leverage this training and data for R01-funding focused on integrin- targeted cancer therapeutics.

项目摘要：整合素是由α和β组成的跨膜细胞外基质受体 形成异二聚体的亚基。整合素还调节许多功能，包括细胞黏附和迁移 作为信号和机械转导。整合素需要细胞外基质结合的胞外区和细胞质尾巴 这结合了多种细胞内蛋白来促进信号传递。整合素与肿瘤的发生有关， 进展和转移。然而，专注于拮抗胞外结构域的整合素靶向药物， 在肿瘤学临床试验中基本上失败了。这些结果表明，细胞质尾巴可能驱动 肿瘤的发生独立于ECM结合的胞外区，尽管其机制仍不明确。 肺中的主要整合素含有b1亚基。我已经证明了整合素b1-敲除(KO)人 注入无瘤小鼠肺部的肺腺癌细胞未能形成肿瘤。这些数据表明 整合素b1是肺腺癌发生所必需的。接下来，我开发了整合素b1-ko细胞，它能表达 野生型(WT)整合素b1、具有无功能/突变尾巴的整合素b1(b1 mt-ail)或具有 非功能性ECM结合胞外结构域(Tac-b1)。WT和Tac-b1细胞形成软琼脂集落，而 B1mt-Tail细胞无一形成。这些数据表明整合素b1的尾巴足以使肿瘤生长。 RNA-seq基因表达分析证实上皮向间充质转化(EMT)减少 整合素b1-KO细胞中的TGFb信号特征。整合素b1-kO与降低的这种相关性 EMT相关基因在人类肿瘤中的表达得到证实。当整合素b1-KO细胞进一步 我观察到，虽然规范的TGFb信号没有受到影响，但TGFb1诱导了AKT的磷酸化 是减少的。这一观察结果与我导师之前的数据一致，即整合素b1调节AKT 以PI3K非依赖的方式激活，涉及TRAF6介导的AKT赖氨酸-63多泛素化。在……里面 总结，整合素b1尾巴是肿瘤生长所必需的，整合素b1的丢失与EMT和Tail的丢失有关。 TGFb信号转导。基于这些发现，我假设不依赖于ecm的整合素b1尾部信号。 通过调节TGFb1诱导的AKT激活来调节EMT并允许肺癌生长。 为了检验这一假设，我提出了两个目标。在目标1中，我将检验整合素b1的假设 通过促进EMT促进肿瘤生长。我将使用肺癌小鼠模型，评估EMT在肿瘤中的应用 使用组织学技术和单细胞RNA-seq.在目标2中，我将测试 假设整合素b1的尾巴足以促进肿瘤的发展，并且尾巴与TRAF6的相互作用是 依赖于TGFb的AKT激活、EMT和细胞增殖所必需的。我会用b1Mt-Tail和Tac-b1电池 确定整合素b1Tail的作用及其促进TGFb信号转导的机制 肺癌。展望未来，我预计将利用此培训和数据用于R01-专注于整合素的资金- 有针对性的癌症治疗。