Protein kinase C and lung carcinogenesis

蛋白激酶C与肺癌发生

基本信息

批准号：
10733467
负责人：
David Feldser
金额：
$ 67.44万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-06 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10733467
关键词：
Actins Adenocarcinoma Cell Affect Animals Benzo(a)pyrene Biology Biosensor CRISPR/Cas technology Cancer Etiology Cancer Model Carcinogens Carcinoma Cell Separation Cells Cellular biology Cessation of life Clinical Clustered Regularly Interspaced Short Palindromic Repeats Competence Cytoskeletal Modeling Data Defect Development Dimensions Disease Environmental Carcinogens Epithelial Cells Epithelium Event Exposure to Extracellular Matrix Family Fluorescence Fluorescence Resonance Energy Transfer Gene Expression Genetic Genetically Engineered Mouse Goals Grant Guanine Nucleotide Exchange Factors Guanosine Triphosphate Hematopoietic Human In Vitro Invaded Joints KRAS oncogenesis KRAS2 gene KRASG12D Knock-out Link Lipids LoxP-flanked allele Lung Lung Adenocarcinoma Lung Neoplasms Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of prostate Mass Spectrum Analysis Mediating Membrane Mesenchyme Modeling Molecular Monomeric GTP-Binding Proteins Mus Mutation Neoplasm Metastasis Non-Small-Cell Lung Carcinoma Nude Mice Oncogenic Outcome Patients Peptide Hydrolases Phenotype Phorbol Esters Phosphorylation Phosphotransferases Play Positioning Attribute Production Protein Analysis Protein Deficiency Protein Kinase C Publishing RAS driven cancer Rationalization Receptor Protein-Tyrosine Kinases Recurrence Role Second Messenger Systems Series Signal Pathway Signal Transduction Stromal Cells Techniques Testing Tumor Promoters Work adenoma cancer cell cancer initiation candidate identification cell motility cell type experience in vivo innovation insight lung cancer cell lung carcinogenesis lung metastatic lung tumorigenesis malignant breast neoplasm member mouse model mutant neoplastic cell novel permissiveness protein activation protein kinase C epsilon protein kinase C kinase rac1 GTP-Binding Protein receptor screening targeted treatment tumor tumor initiation tumor microenvironment tumorigenesis

项目摘要

ABSTRACT Lung cancer is the leading cause of cancer-related deaths in the U.S., with ~236,700 new cases and ~130,200 deaths estimated for 2022. A joint effort by both PIs in this grant led to the identification of the oncogenic kinase protein kinase C epsilon (PKCe) as a key player in lung carcinogenesis. PKCe is aberrantly up-regulated in lung adenocarcinoma and is associated with poor outcome in patients specifically harboring KRAS mutations. Using genetically engineered mouse (GEM) models, we demonstrated that PKCe is required for both carcinogen- and Kras-driven lung tumorigenesis. In addition to this role in cancer initiation, our work established novel roles for PKCe in cellular events associated with late cancer stages. Indeed PKCe is a major player in lung cancer cell motility and invasion via activation of the small G-protein Rac1, linking this kinase to metastatic dissemination. That PKCe plays critical permissive roles first during adenoma initiation and then again later during the acquisition of metastatic competency highlights the functional complexities of the PKCe signaling events in lung cancer. Interestingly, CRISPR-mediated inactivation of PKCe in the initiating cell-of-origin does not significantly affect Kras- G12D-induced tumor development, leading us to hypothesize that PKCe does not strictly act in a tumor cell autonomous manner to permit oncogenic KRAS-mediated tumorigenesis. To test this hypothesis, in Aim 1 we will generate and characterize a series of GEM models to restrict genetic deletion of PKCe to either oncogenic Kras- expressing cancer cells or to diverse stromal cell types present in the tumor microenvironment, including non- cancerous epithelial, mesenchyme and hematopoietic cells. Gene expression studies on isolated cells using fluorescence-based lineage tracing techniques will provide significant mechanistic insights. In Aim 2, we will thoroughly dissect the mechanistic basis of motility/invasive signaling activation by PKCe. Our hypothesis is that PKCe activates Rac1-mediated formation of cell ruffles and motility in KRAS mutant lung cancer cells via Rac Guanine nucleotide Exchange Factors (Rac-GEFs). We will identify and characterize candidate Rac-GEFs as PKCe effectors responsible for this phenotype. In Aim 3, we will establish the involvement of PKCe and its effector Rac-GEFs for the development of metastatic lung cancer. We will use combined in vitro and in vivo approaches to pin down mechanistic defects in the metastatic cascade upon CRISPR-mediated deletion of PKCe and Rac-GEFs in lung adenocarcinoma cells. We will establish GEM models and use lentiviral CRISPR-based approaches to determine the permissive contribution of PKCe and its GEF effectors to lung adenocarcinoma metastasis, and identify relevant gene expression and signaling signatures contributing to this phenotype. Thus, by using innovative, state-of-the-art genetic and molecular approaches, our studies should reveal the vast multidimensional complexity of PKCe signaling in lung cancer development as well as underscore unappreciated mechanisms of carcinogen- and KRAS-mediated lung oncogenesis.

抽象的肺癌是美国癌症相关死亡的主要原因，新增病例约 236,700 例，新增病例约 130,200 例预计 2022 年死亡人数。两位 PI 在此资助中的共同努力导致了致癌激酶的鉴定蛋白激酶 C epsilon (PKCe) 在肺癌发生中起关键作用。肺中 PKCe 异常上调腺癌，并且与携带 KRAS 突变的患者的不良预后相关。使用通过基因工程小鼠 (GEM) 模型，我们证明了 PKCe 对于致癌物和致癌物都是必需的。 Kras 驱动的肺部肿瘤发生。除了在癌症发生中的作用之外，我们的工作还为与晚期癌症阶段相关的细胞事件中的 PKCe。事实上 PKCe 是肺癌细胞的主要参与者通过激活小 G 蛋白 Rac1 来实现运动和侵袭，将该激酶与转移扩散联系起来。那 PKCe 首先在腺瘤发生过程中发挥关键的许可作用，然后在获得性腺瘤过程中再次发挥关键作用。转移能力凸显了肺癌中 PKCe 信号事件的功能复杂性。有趣的是，CRISPR介导的起始细胞源中PKCe的失活并不显着影响Kras- G12D 诱导肿瘤发展，使我们推测 PKCe 并不严格作用于肿瘤细胞自主方式允许致癌 KRAS 介导的肿瘤发生。为了检验这个假设，在目标 1 中我们将生成并表征一系列 GEM 模型，以将 PKCe 的遗传删除限制为致癌 Kras- 表达癌细胞或肿瘤微环境中存在的多种基质细胞类型，包括非癌性上皮细胞、间质细胞和造血细胞。使用分离细胞进行基因表达研究基于荧光的谱系追踪技术将提供重要的机制见解。在目标 2 中，我们将彻底剖析 PKCe 运动/侵入信号激活的机制基础。我们的假设是 PKCe 通过 Rac 激活 KRAS 突变肺癌细胞中 Rac1 介导的细胞皱褶形成和运动鸟嘌呤核苷酸交换因子（Rac-GEF）。我们将确定候选 Rac-GEF 并将其描述为 PKCe 效应子负责这种表型。在目标 3 中，我们将建立 PKCe 及其效应器的参与 Rac-GEF 用于发展转移性肺癌。我们将使用体外和体内相结合的方法确定 CRISPR 介导的 PKCe 和 Rac-GEF 删除后转移级联中的机械缺陷在肺腺癌细胞中。我们将建立GEM模型并使用基于慢病毒CRISPR的方法确定 PKCe 及其 GEF 效应子对肺腺癌转移的许可贡献，以及识别导致该表型的相关基因表达和信号特征。因此，通过使用创新、最先进的遗传和分子方法，我们的研究应该揭示巨大的多维 PKCe 信号在肺癌发展中的复杂性，并强调了未被认识到的机制致癌物和 KRAS 介导的肺癌发生。