Roles of Claudin-7 in Lung Cancer

描述(申请人提供)：肺癌是一种主要的肺部疾病，占所有癌症死亡人数的四分之一以上。因此，迫切需要研究该病的病因，确定遗传和环境风险因素，并发现更好的诊断和治疗策略。对抗肺癌的一种策略是识别通常会抑制肿瘤发展的基因。我们已经开发出第一个基因工程小鼠模型，在该模型中claudin-7基因缺失。用基因打靶方法敲除claudin-7可导致肺上皮细胞的过度增殖。杂合子claudin-7小鼠患自发性肺癌的几率增加。重要的是，在肺癌细胞中，claudin-7的表达在细胞-细胞交界处被干扰或下调，并且claudin-7的过表达抑制了培养的人肺癌细胞的生长。这些研究提出了一种假设，即claudin-7作为一种肿瘤抑制因子在肺癌进展中发挥着重要作用。为了研究claudin-7如何在细胞培养和体内小鼠模型中抑制癌症生长，该项目将解决两个具体目标。目的1：探讨claudin-7在肺癌细胞生长和存活中的作用。我们将使用流式细胞术、TUNEL成像、Matrigel侵袭实验和细胞-细胞分离方法来确定当claudin-7在claudin-7缺陷的人肺癌细胞系NCI-H1299中稳定表达时，细胞周期、细胞凋亡以及细胞黏附和侵袭的性质是否发生改变。这些实验将确定claudin-7如何抑制肺癌细胞的生长。特定目的2：研究环境致癌物对肺癌体内生长及Cln7小鼠体内肿瘤-宿主相互作用的影响。我们将调查环境致癌物，如4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)，是否可以诱导Cln7小鼠比Cln7小鼠更高的肿瘤发病率。我们还将研究Claudin-7表达减少是否会导致宿主细胞-细胞连接和黏附的改变，从而使接种的Lewis肺癌LLC1细胞能够更有利地与肺微环境相互作用，更容易转移。该项目将使我们能够收集初步数据，以确定Cln7小鼠品系是否为研究claudin-7在肺癌发生中的作用的可行模型系统。这些研究还将为未来的NIH RO1项目奠定基础，该项目将研究claudin-7作为肿瘤抑制因子的分子机制。与公共卫生相关：对抗肺癌的一个策略是识别通常会抑制肿瘤发展的基因。Claudin-7在限制肺上皮细胞不受控制的细胞生长方面很重要，正如我们的基因工程Claudin-7基因敲除小鼠模型所揭示的那样。现在重要的是研究claudin-7缺陷的小鼠在暴露于环境致癌物时是否更容易患肺癌，以及claudin-7如何抑制癌症生长。

DESCRIPTION (provided by applicant): Lung cancer is a major lung disease and accounts for more than one-fourth of all cancer deaths. Therefore, research into causes of the disease, identification of genetic and environmental risk factors, and discovery of better diagnosis and treatment strategies are urgently needed. One strategy to combat lung cancer is to identify genes that normally suppress tumor development. We have developed the first genetically engineered mouse model in which claudin-7 gene is deleted. Knockout of claudin-7 using gene targeting approach resulted in hyperproliferation of lung epithelial cells. Heterozygous claudin-7 mice showed an increased incidence of developing spontaneous lung tumors. Importantly, claudin-7 expression is either disrupted or downregulated at the cell-cell junction in lung cancer cells, and the overexpression of claudin-7 reduced human lung cancer cell growth in culture. These studies prompted the hypothesis that claudin-7 plays important roles in lung cancer progression as a tumor suppressor. To investigate how claudin-7 suppresses cancer growth in cell culture as well as in mouse models in vivo, this project will address two specific aims. Specific Aim 1: To investigate the roles of claudin-7 in lung cancer cell growth and survival in culture. We will use flow cytometry, TUNEL imaging, Matrigel invasion assay and cell- cell dissociation methods to determine if the properties of cell cycle, apoptosis, as well as cell adhesion and invasion, are altered when claudin-7 is stably expressed in a claudin-7-deficient human lung cancer cell line NCI-H1299. These experiments will determine how claudin-7 reduces lung cancer cell growth. Specific Aim 2: To investigate environmental carcinogens on the growth of lung carcinoma in vivo and on tumor-host interactions in Cln7 mice. We will investigate if environmental carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), can induce a greater tumor incidence in Cln7 mice compared to Cln7 mice. We will also investigate whether reduced claudin-7 expression results in altered host cell-cell junction and adhesion, allowing inoculated Lewis lung carcinoma LLC1 cells to interact with the pulmonary microenvironment more favorably and metastasize more easily. This project will allow us to collect preliminary data to determine whether Cln7 mouse strain is a viable model system for studies of claudin-7 function in lung carcinogenesis. These studies will also lay the foundation for a future NIH RO1 project to investigate the molecular mechanisms of how claudin-7 functions as a tumor suppressor. PUBLIC HEALTH RELEVANCE: One strategy to combat lung cancer is to identify genes that normally suppress tumor development. Claudin-7 is important in limiting lung epithelia from uncontrolled cell growth, as revealed by our genetically engineered claudin- 7 knockout mouse model. It is now important to investigate whether mice deficient in claudin-7 are more susceptible for lung cancer formation when exposed to environmental carcinogens and how claudin-7 suppresses cancer growth.