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。对抗肺癌的一个策略是找出通常抑制肿瘤发展的基因。我们已经开发了第一个基因工程小鼠模型，其中claudin-7基因被删除。基因靶向法敲除claudin-7可导致肺上皮细胞过度增殖。杂合claudin-7小鼠发生自发性肺肿瘤的几率增加。重要的是，在肺癌细胞中，claudin-7在细胞-细胞交界处的表达被破坏或下调，claudin-7的过表达降低了培养的人肺癌细胞的生长。这些研究提示了claudin-7作为肿瘤抑制因子在肺癌进展中发挥重要作用的假设。为了研究claudin-7如何在细胞培养和小鼠体内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在培养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在缺乏claudin-7的人肺癌细胞系NCI-H1299中稳定表达时，细胞周期、凋亡、细胞粘附和侵袭的特性是否发生改变。这些实验将确定claudin-7是如何减少肺癌细胞生长的。特异性目的2：探讨环境致癌物对肺癌在体内生长的影响及肿瘤-宿主在Cln7小鼠体内的相互作用。我们将研究环境致癌物，如4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）是否能诱导Cln7小鼠比Cln7小鼠更大的肿瘤发生率。我们还将研究claudin-7表达的降低是否会导致宿主细胞-细胞连接和粘附的改变，从而使接种的Lewis肺癌LLC1细胞更有利地与肺微环境相互作用并更容易转移。该项目将使我们能够收集初步数据，以确定Cln7小鼠品系是否是研究Cln7在肺癌发生中的功能的可行模型系统。这些研究也将为未来NIH RO1项目研究claudin-7作为肿瘤抑制因子的分子机制奠定基础。公共卫生相关性：对抗肺癌的一个策略是确定通常抑制肿瘤发展的基因。claudin- 7基因工程敲除小鼠模型显示，claudin- 7在限制肺上皮细胞不受控制的生长方面很重要。现在重要的是研究claudin-7缺失的小鼠在暴露于环境致癌物中时是否更容易形成肺癌，以及claudin-7如何抑制癌症的生长。

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.