Protein kinase C and lung carcinogenesis

 描述（由申请人提供）：本申请侧重于研究肺癌发生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子，因为它们诱导遗传和表观遗传改变，最终导致肺上皮细胞的恶性转化。KRAS的致癌突变是非小细胞肺癌（NSCLC）中的常见改变，其高频率由肺致癌物如多环芳烃（PAH）和许多其他环境致癌物诱导。已经确定蛋白激酶C β（PKC β），一种促有丝分裂、促存活和致肿瘤的激酶，在上皮癌中上调，包括 NSCLC。我们实验室的研究表明，PKC β是NSCLC细胞肿瘤形成、侵袭和转移的重要介质。最近，我们开发了一种在PKC β-缺陷背景下诱导肺特异性表达KRas的小鼠模型（LSL-K-rasG 12 D; PKC β-/-），并发现PKC β基因（PRKCE）的遗传消除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 β是KRas肺肿瘤发生的起始所必需的。此外，在KRAS突变的人肺腺癌中的计算机数据库分析揭示了高PKC β表达与患者总生存期短之间的显著相关性。总而言之，这使我们假设PKC β是肺致癌物作用的必要介质。在具体目标1中，我们将检查PKC β KO小鼠（A/J遗传背景）是否对已知诱导KRas突变的肺致癌物（包括PAH苯并[a]芘（B[a]P）和氨基甲酸乙酯）的影响具有抗性。我们将研究PKC β 1（PKC β 1 -2）的药理学抑制剂是否抑制了 由这些致癌物或激活的KRas等位基因诱导的肺肿瘤。将进行机制研究以评估致癌物是否上调肺上皮细胞中的PKC β。在特定目标2中，我们将使用遗传和药理学方法来确定PKC β是否介导KRas和致癌物诱导的肿瘤发生所需的肺癌祖细胞（支气管肺泡干细胞或BASC）的扩增。为了明确确定PKC β在启动中的作用，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病变的形成。最后，在具体目标3中，我们将剖析肺癌中KRas和PKC β之间功能性相互作用的机制基础，重点是a）Ras级联的元件分析，B）在KRas肿瘤发生的背景下鉴定PKC β基因签名和由该激酶调节的转录网络，以及c）评估PKC β在上皮-间质转化（EMT）中的潜在作用，获得肺癌细胞侵袭能力所需的过程。我们的研究将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.