Protein kinase C and lung carcinogenesis

摘要 肺癌是美国癌症相关死亡的主要原因，新增病例约236，700例， 预计2022年死亡。在这项资助下，两个PI的共同努力导致了致癌激酶的鉴定 蛋白激酶C β（PKCe）在肺癌发生中起关键作用。PKCe在肺内异常上调 在腺癌患者中，KRAS突变与预后不良相关。使用 在基因工程小鼠（GEM）模型中，我们证明了PKCe是致癌物质和 Kras驱动的肺肿瘤发生。除了在癌症发生中的这种作用外，我们的工作还建立了新的作用， PKCe在与晚期癌症阶段相关的细胞事件中的作用事实上，PKCe是肺癌细胞中的主要参与者， 通过激活小G蛋白Rac 1，将这种激酶与转移性播散联系起来，从而促进运动和侵袭。的 PKCe首先在腺瘤发生过程中起关键的允许作用，然后在获得 转移能力突出了肺癌中PKCe信号传导事件的功能复杂性。 有趣的是，CRISPR介导的PKCe在起始细胞中的失活并不显著影响Kras-1。 G12 D诱导的肿瘤发展，使我们假设PKCe并不严格作用于肿瘤细胞 自主的方式允许致癌KRAS介导的肿瘤发生。为了验证这一假设，在目标1中，我们将 生成并表征一系列GEM模型，以将PKCe的遗传缺失限制于致癌Kras- 表达癌细胞或肿瘤微环境中存在的不同基质细胞类型，包括非- 癌性上皮细胞、间充质细胞和造血细胞。使用基因芯片对分离的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理见解。在目标2中，我们 彻底剖析PKCe激活运动/侵袭信号的机制基础。我们的假设是 PKCe通过Rac激活Rac 1介导的KRAS突变肺癌细胞皱褶形成和运动 鸟嘌呤核苷酸交换因子（Rac-GEFs）。我们将确定和描述候选Rac-GEFs， PKCe效应子负责该表型。在目标3中，我们将建立PKCe及其效应物的参与， Rac-GEF用于转移性肺癌的发展。我们将使用体外和体内相结合的方法， 在CRISPR介导的PKCe和Rac-GEF缺失后确定转移级联中的机制缺陷 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效应物对肺腺癌转移的容许贡献，和 鉴定有助于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以及强调了 致癌物和KRAS介导的肺肿瘤发生。

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.