COX2-FAK相互作用在食管鳞癌发展及转移中的机制研究

China is a country with a high incidence and mortality of esophageal squamous cell carcinoma (ESCC), which urges enhanced clinical therapy. The needs of basic and clinical research on ESCC are increasing. Our previous studies indicated cyclooxygenase-2 (COX2) was overexpressed in ESCC, COX2 inhibitors inhibited the proliferation and invasion of ESCC cells. Furthermore, COX2 interacted with focal adhesion kinase (FAK) in ESCC cells, which is dependent on the phosphorylated Y384 residue of COX2. Therefore, we hypothesize that COX2-FAK interaction may play an important role in the progression and metastasis of ESCC. In this study, we will first use tyrosine kinase inhibitor and in vitro kinase assay to search the kinase for the phosphorylation of Y384 residue of COX2. Second, we will establish different esophageal epithelial cell lines expressing FAK and COX2, knockouting FAK but expresing COX2, expressing FAK mutant and COX2. We will use ELASA to detect the cytokines in the supernatant and immunocytoflurescent to explore the effect of COX2-FAK interation on biological activity and downstream signaling pathway. Third, to expand the understanding of the influence of COX2-FAK interation on esophageal epithelial cells in vitro, we will observe the difference of the migration and invasion ability of the above cell lines, and the difference of activity and content of matrix metalloproteinases (MMPs) in the supernatant. This study will help to understand the function of COX2-FAK interaction in the progression and metastasis of ESCC.

我国是食管鳞癌的高发国家。我们前期研究发现环氧化酶2（COX2）在食管鳞癌中过表达，COX2抑制剂可抑制食管鳞癌细胞的增殖及迁移。这与COX2在食管鳞癌细胞中与黏着斑激酶(FAK)结合有关，且依赖于COX2 Y384位点的磷酸化状态，但机制不清。本课题首先利用酪氨酸激酶抑制剂及体外激酶实验，寻找磷酸化COX2 Y384位点的激酶，并构建表达COX2但敲除FAK的食管鳞状上皮细胞系，明确其磷酸化是否依赖于FAK；第二分别构建表达FAK和COX2，敲除FAK但表达COX2，以及表达FAK突变体和COX2的食管鳞状上皮细胞系，通过检测细胞因子的变化探讨COX2-FAK相互作用对各自的生物学活性及下游信号通路的影响；第三通过检测上述细胞系的迁移和侵袭，观察COX2-FAK相互作用对食管鳞状上皮细胞生物学功能的影响。本研究将为探讨COX2-FAK相互作用在食管鳞癌的发展和转移中的作用提供实验室依据。

我国是全球食管癌发病率和死亡率最高的国家之一，食管癌临床诊治形势严峻，因此仍然需要包括深入食管癌基础研究、改进早期诊断技术、革新手术方法和完善以手术为主的综合治疗等在内的多方面来进一步提高食管癌的治疗效果。已有多项研究表明环氧化酶Cox2在食管癌中过表达，Cox2特异性抑制剂可以在体外抑制食管癌细胞系增殖，并抑制肿瘤细胞迁移，但是具体的分子机制及临床意义不明。我们的工作发现，Cox2在人食管上皮细胞中与黏着斑激酶FAK结合，并诱导细胞发生EMT及促进细胞迁移和侵袭的能力，并且上述过程均依赖于FAK激酶介导的Cox2 Y384位点的磷酸化状态。Cox2诱导的下游外泌因子TXA2，PGE2和PGI2的表达水平也决定于FAK激酶诱导的Cox2 Y374位点磷酸化状态。Cox2诱导的细胞转移和EMT也都依赖于FAK激酶诱导的Cox2 Y374位点磷酸化状态。这说明FAK介导的EMT是部分经过磷酸化Cox2实现的。因此我们推测Cox2-FAK的相互作用可能在食管癌的转移中发挥重要作用。