Protein kinase C and lung carcinogenesis

 描述(申请人提供)：本申请侧重于研究肺癌发生的早期事件中涉及的新机制。环境致癌物是肺癌的主要致病因素，因为它们诱导遗传和表观遗传改变，最终导致肺上皮细胞恶性转化。KRAS是非小细胞肺癌(NSCLC)中的一种常见改变，其致癌基因突变是由多环芳烃(PAHs)等肺癌致癌物和许多其他环境致癌物诱发的。蛋白激酶C受体(PKC)是一种促有丝分裂、促进生存和致癌的激酶，已被证实在上皮性癌症中表达上调，包括 非小细胞肺癌。我们实验室的研究表明，PKC是非小细胞肺癌细胞肿瘤形成、侵袭和转移的重要介质。最近，我们建立了一种在PKC缺失背景下诱导KRAS肺特异性表达的小鼠模型(LSL-K-RasG12D；PKC-/-)，并发现PKC基因(PRKCE)的遗传消融显著削弱了由激活的KRAS等位基因驱动的肿瘤的形成。这表明PKC在KRAS肺肿瘤的发生过程中是必需的。此外，在电子计算机数据库中，对KRAS突变的人肺腺癌的分析显示，蛋白激酶C的高表达与患者总生存期较短之间存在显著关联。总之，这导致我们假设PKC是肺癌致癌物作用的必要中介。在具体目标1中，我们将检查PKCKO小鼠(在A/J遗传背景中)是否对已知可导致KRAS突变的肺癌致癌物的影响具有抵抗力，包括多环芳烃苯并[a]芘(B[a]P)和乌拉坦。我们将检查PKC的药理抑制剂(V1-2是否能抑制这种形成 由这些致癌物或由激活的KRAS等位基因引起的肺肿瘤。将继续进行机制研究，以评估致癌物是否上调肺上皮细胞中的蛋白激酶C。在具体目标2中，我们将使用遗传学和药理学方法来确定PKC是否介导了KRAS和致癌物诱导的肿瘤发生所需的肺癌前体细胞(细支气管肺泡干细胞或BASCs)的扩张。为了明确地确定PKC在启动过程中的作用，我们将在细胞模型中使用功能获得方法，并为该激酶产生一个可诱导的肺特异性转基因小鼠系，以确定这是否会导致癌前病变或恶性肺损害的形成。最后，在特定的目标3中，我们将剖析KRAS和PKC在肺癌中功能相互作用的机制基础，重点集中在a)RAS级联的元件的分析，b)在KRAS肿瘤发生的背景下鉴定PKC基因信号和受该激酶调控的转录网络，以及c)评估PKC在上皮-间充质转化中的潜在作用，这是获得肺癌细胞侵袭能力所必需的过程。我们的研究应该为环境致癌物的分子效应提供新的机制见解，并揭示肺癌发生的早期事件的重要方面，从而影响我们对肺癌病因学的理解。

 DESCRIPTION (provided by applicant): This application focuses on the study of novel mechanisms involved in early events of lung carcinogenesis. Environmental carcinogens are major causative agents of lung cancer, as they induce genetic and epigenetic alterations that ultimately lead to the malignant transformation of lung epithelial cells. Oncogenic mutations in KRAS, a common alteration in non-small cell lung cancer (NSCLC), are induced in high frequency by lung carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and many other environmental carcinogens. It has been established that protein kinase C epsilon (PKC), a mitogenic, pro-survival, and tumorigenic kinase, is up-regulated in epithelial cancers, including NSCLC. Studies from our laboratory revealed that PKC is an essential mediator of tumor formation, invasiveness, and metastasis of NSCLC cells. More recently, we developed a mouse model for inducible lung-specific expression of KRas in a PKC-deficient background (LSL- K-rasG12D; PKC-/-), and found that genetic ablation of the PKC gene (PRKCE) markedly impairs the formation of tumors driven by the activated KRas allele. This suggests that PKC is required for the initiation of KRas lung tumorigenesis. Moreover, in silico database analysis in KRAS mutated human lung adenocarcinomas revealed a significant association between high PKC expression and short overall patient survival. Altogether, this led us to hypothesize that PKC is a necessary mediator of the actions of lung carcinogens. In Specific Aim 1 we will examine if PKC KO mice (in A/J genetic background) are resistant to the effects of lung carcinogens known to induce mutations in KRas, including the PAH benzo[a]pyrene (B[a]P) and urethane. We will examine if a pharmacological inhibitor of PKC (V1-2) inhibits the formation of lung tumors induced by these carcinogens or by an activated KRas allele. Mechanistic studies will be pursued to assess if carcinogens up-regulate PKCin lung epithelial cells. In Specific Aim 2, we will use genetic and pharmacological approaches to determine if PKC mediates the expansion of lung cancer progenitor cells (bronchioalveolar stem cells or BASCs) required for KRas- and carcinogen-induced tumorigenesis. To unambiguously establish a role for PKC in initiation we will use gain-of-function approaches in cellular models as well as generate an inducible lung-specific transgenic mouse line for this kinase to determine if this leads to the formation of pre-malignant or malignant lung lesions. Finally, in Specific Aim 3 we will dissect the mechanistic basis for the functional interaction between KRas and PKC in lung cancer, focusing on a) the analysis of elements of the Ras cascade, b) the identification of a PKC gene signature and transcriptional networks regulated by this kinase in the context of KRas tumorigenesis, and c) the assessment of a potential role for PKC in epithelial-mesenchymal transition (EMT), a process required for the acquisition of invasive capacity of lung cancer cells. Our studies should provide novel mechanistic insights into the molecular effects of environmental carcinogens as well as reveal important aspects of early events of lung carcinogenesis, thus impacting on our understanding of lung cancer etiology.