Constitutive Integrin Beta1 Signaling in Lung Cancer

项目概述：整合素是由α和β组成的跨膜细胞外基质（ECM）受体 形成异二聚体的亚基。整合素调节许多功能，包括细胞粘附和迁移 作为信号和机械传导。整合素需要ECM结合胞外域和胞质尾区 结合多种细胞内蛋白质以促进信号传导。整合素与肿瘤的发生有关， 进展和转移。然而，整合素靶向药物，其重点是拮抗胞外域， 在肿瘤临床试验中基本上失败了。这些结果表明，细胞质尾可能驱动 肿瘤发生独立于ECM结合胞外域，尽管机制仍不明确。 肺中的主要整合素含有b1亚基。我已经证明，整合素b1敲除（KO）人 注射到无胸腺小鼠肺中的肺腺癌细胞不能形成肿瘤。这些数据表明 整联蛋白B1是肺腺癌发展所必需的。接下来，我开发了整合素b1-KO细胞， 野生型（WT）整联蛋白b1、具有非功能性/突变尾的整联蛋白b1（b1 mt-尾）或具有非功能性/突变尾的整联蛋白b1 非功能性ECM结合胞外域（Tac-b1）。WT和Tac-b1细胞形成软琼脂集落， B1 MT-尾细胞没有形成。这些数据表明，整合素b1尾部足以允许肿瘤生长。 RNA-seq基因表达分析鉴定了上皮向间质转化（EMT）减少， 整合素b1-KO细胞中的TGFb信号传导特征。整合素b1-KO与 EMT相关基因的表达在人类肿瘤中得到证实。当整合素b1-KO细胞被进一步 通过检查，我观察到虽然经典的TGF β信号不受影响，但TGF β 1诱导的AKT磷酸化 减少了。这一观察结果与我的导师之前的数据一致，即整合素b1调节AKT 以PI 3 K非依赖性方式激活，涉及TRAF 6介导的AKT赖氨酸-63聚泛素化。在 总之，整联蛋白b1尾是肿瘤生长所必需的，整联蛋白b1的缺失与EMT的缺失相关， TGF β信号转导。基于这些发现，我假设ECM非依赖性整合素b1尾部信号传导 调节EMT并通过调节TGFb 1诱导的AKT活化而允许肺癌生长。 我提出了两个目标来检验这个假设。在目标1中，我将检验整合素b1 通过促进EMT促进肿瘤生长。我将使用肺癌小鼠模型并评估肿瘤中的EMT 使用组织学技术和单细胞RNA-seq在有和没有整合素b1的情况下。在目标2中，我将测试 假设整合素b1尾部足以促进肿瘤的发展，并且与TRAF 6的尾部相互作用是 TGFb依赖性AKT活化、EMT和细胞增殖所需。我将使用b1 mt-tail和Tac-b1细胞 确定整合素b1尾的作用及其促进TGF β信号传导的机制， 肺癌展望未来，我希望利用这一培训和数据的R 01-资金集中在整合素- 靶向癌症治疗。

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.