Role of JNK Pathway in Lung Tumorigenesis

描述(申请人提供)：K-RAS和突变/过度表达的EGFR家族成员在非小细胞肺癌(NSCLC)中起主导癌基因的作用。然而，这些癌基因介导转化的信号通路仍然不清楚。此外，癌基因既能刺激促进肿瘤发生的信号，也能刺激抗肿瘤信号。在这方面，JNK1、JNK2和JNK3编码的JNKMAPKs被生长因子和癌基因激活，文献证明JNKs在细胞转化中既有积极的作用，也有消极的作用。MKK4是JNKs的双特异性激酶激活剂，已在多种人类癌症中作为肿瘤转移抑制因子出现。与这一发现一致的是，我们对JNK1和JNK2缺陷小鼠的实验显示，与野生型小鼠相比，致癌物诱导的肺肿瘤发生增加。此外，与未转化的肺上皮细胞相比，多个人非小细胞肺癌细胞株显示JNK活性降低，并且转基因功能获得的JNK1抑制非锚定非依赖性非小细胞肺癌的生长。因此，我们认为JNK1和JNK2在肺癌中作为肿瘤抑制通路的组成部分发挥作用。相比之下，对JNK3基因缺陷小鼠的初步研究显示，致癌物诱导的肺肿瘤发生减少。此外，相对于未转化的肺上皮细胞或未受累的肺组织，JNKS的mRNA和蛋白在非小细胞肺癌细胞系和原发肿瘤中也有表达。因此，我们假设JNK3是在肺癌进展过程中被诱导的，并且代表了一种促肿瘤形成的JNK亚型。为了检验这些假设，我们将完成以下具体目标。目的1：探讨特异性JNKs在JNK1、JNK2、JNK3缺失小鼠肺肿瘤发生中的作用。还将测试特定的JNK作为宿主肺微环境信号成分调控肺肿瘤发生的作用。目的2：检测特异性JNKs在体外对肿瘤抑制或细胞转化的作用。将表达JNKs分子抑制剂的未转化的肺上皮细胞系用致癌K-RAS基因转导，并测量转化、分化和凋亡的标准。此外，还将监测转导了功能增益JNKs或显性阴性JNKs的NSCLC细胞的细胞转化标准。目的：探讨非小细胞肺癌细胞株中JNK1、JNK2活性降低的机制(S)。MKPs作为癌基因诱导的JNK1和JNK2活性的负调节因子的作用将被强调。目的：应用免疫组化和定量RT-PCR方法检测已存档的人肺肿瘤组织中JNKs的激活状态和AIMS 1-3中定义的特异性信号分子的表达状态，并与临床行为进行相关分析。完成这些特定的目标将导致全面了解这个MAP激酶家族在肺肿瘤发生中的复杂作用，并为探索这些激酶在其他人类癌症中的作用提供一种形式。此外，对JNK MAP激酶在肺癌中的多方面作用的详细了解是对抗肺癌的这一途径的理论基础和精确的治疗靶点所绝对需要的。

DESCRIPTION (provided by applicant): K-Ras and mutated/over-expressed EGFR family members function as dominant oncogenes in non-small cell lung cancer (NSCLC). Yet, the signal pathways that mediate transformation by these oncogenes remain ill-defined. Moreover, oncogenes stimulate both pro- and anti-tumorigenic signaling. In this regard, the JNK MAPKs, encoded by jnk1, jnk2 and jnk3, are activated by growth factors and oncogenes and the literature documents both positive and negative roles for JNKs in cellular transformation. MKK4, a dual-specificity kinase activator of the JNKs, has emerged as a tumor metastasis suppressor in diverse human cancers. Consistent with this finding, our experiments with JNK1 and JNK2-deficient mice reveal increased carcinogen-induced lung tumorigenesis relative to wild-type littermates. Also, multiple human NSCLC cell lines show decreased JNK activity relative to non-transformed lung epithelial cells and transfected gain-of-function JNK1 inhibits anchorage-independent NSCLC growth. Thus, we propose that JNK1 and JNK2 function as components of a tumor suppressor pathway in lung cancer. By contrast, preliminary studies with JNK3- deficient mice reveal decreased carcinogen-induced lung tumorigenesis. In addition, JNKS mRNA and protein is expressed in NSCLC cell lines and primary tumors relative to non-transformed lung epithelial cells or uninvolved lung tissue. Thus, we hypothesize that JNK3 is induced during lung cancer progression and represents a pro-tumorigenic JNK isoform. To test these hypotheses, we will complete the following specific aims. Aim 1: Determine the in vivo role for specific JNKs in murine lung tumorigenesis with mice lacking jnk1, jnk2 or jnk3. The role of specific JNKs as signal components of the host lung microenvironment regulating lung tumorigenesis will also be tested. Aim 2: Test the in vitro role of specific JNKs in tumor suppression or cellular transformation. Non-transformed lung epithelial cell lines expressing molecular inhibitors of the JNKs will be transduced with oncogenic K-Ras and criteria of transformation, differentiation and apoptosis will be measured. Also, NSCLC cells transduced with gain-of-function JNKs or dominant-negative JNKS will be monitored for criteria of cellular transformation. Aim 3: Define the mechanism(s) accounting for decreased JNK1 and JNK2 activity in NSCLC cell lines. The role of MKPs as oncogene-induced negative regulators of JNK1 and JNK2 activity will be highlighted. Aim 4: The activation state of JNKs and expression status of specific signaling molecules defined in Aims 1-3 will be measured in archived primary human lung tumors with IHC and quantitative RT-PCR and correlated with clinical behaviour. Completion of these specific aims will lead to a comprehensive understanding of the complex role of this family of MAP kinases in lung tumorigenesis and provide a format for exploration of the role of these kinases in other human cancers. Furthermore, a detailed understanding of the multi-faceted role of the JNK MAP kinases in lung cancer is absolutely required for rationale and precise therapeutic targeting of this pathway to combat lung cancer.