ATF1-T184位点磷酸化在鼻咽癌中的作用及机制研究

Activating transcription factor 1 (ATF1) played an important role in tumor progression. We found in the pilot study that Thr184 at ATF1 was a novel phosphorylation site at ATF1. Phosphorylated ATF1-T184 was higher in NPC than adjacent tissues. The trascription activity and cell transformation ability on nasopharyngeal carcinoma (NPC ) cells were altered when mutation was implemented at the Thr184. After literature analysis, we could infer that pATF1-T184 played an important role in its transcription regulation and NPC progression. . The biological function and molecular mechanism of pATF1-T184 in NPC progression would be thoroughly investigated in this project: immunohistochemistry staining would be used to investigate the expression status of pATF1-T184 in NPC patients and clinicopathological association would be further analyzed; ATF1-Thr184 mutation transfected NPC cell would be used to investigate ATF1 affecting colony formation, invasion and metastasis; ChIP,IP, luciferase and Western bolt would be used to characterize the transcription regulation of ATF1-T184;IP-MS method would be used to sreen the kinase for pATF1-T184 and the result would be verified by a set of experiments. . This study would strive to elucidate the biological function and transcription regulation of pATF1-T184 in NPC progression, with the hope of finding a new target for NPC diagnosis or therapy.

活化转录因子ATF1在肿瘤中起着重要作用。我们发现，ATF1第184位Thr位点为其全新磷酸化位点：磷酸化ATF1-T184在鼻咽癌中高表达；该位点突变后，ATF1的转录活性及对鼻咽细胞的转化功能明显改变。推测pATF1-T184在其转录调控及鼻咽癌发生发展中起着重要作用。本课题将深入探讨pATF1-T184在鼻咽癌中的作用及机制：采用免疫组化方法研究pATF1-T184在鼻咽癌中的表达，结合临床以阐明其临床意义；转染ATF1-T184突变体进入鼻咽细胞，观察其克隆形成、成瘤性及转移能力等变化；采用ChIP、IP、报告基因、Western等方法研究ATF1-T184对ATF1的转录调控机制；采用蛋白质免疫共沉淀结合质谱方法（IP-MS）筛选ATF1-T184位点相关的磷酸化激酶并进行各个层面验证。以期阐明pATF1-T184的转录调控机制及在鼻咽癌中的作用，为鼻咽癌诊断和治疗提供新的靶点。

活化转录因子ATF1在肿瘤中起着重要作用。我们发现，ATF1第184位Thr位点为其全新磷酸化位点：磷酸化ATF1-T184在鼻咽癌中高表达；该位点突变后，ATF1的转录活性及对鼻咽细胞的转化功能明显改变。推测pATF1-T184在其转录调控及鼻咽癌发生发展中起着重要作用。项目深入探讨磷酸化ATF1-T184在鼻咽癌中的作用及机制。. 项目已基本按计划完成相应研究，主要研究内容包括：1.鉴定ATF1-T184位点磷酸化相关激酶。具体为通过蛋白质免疫共沉淀结合质谱方法、TCGA数据和文献分析初步鉴定PKA和MAPK为相关激酶。2.探讨磷酸化 ATF1-T184对ATF1转录活性的调控机制。通过表达相关性、染色质免疫共沉淀（ChIP）、荧光素酶报告基因、放线菌酮等实验提示MMP2和MMP9是ATF1的下游调控靶点，其转录表达活性受ATF1-T184磷酸化调控。3.研究磷酸化ATF1-T184在鼻咽癌、胃癌等肿瘤中的作用。通过细胞表型等实验，结合肿瘤组织表达情况和临床预后因素分析表明磷酸化ATF1-T184可促进鼻咽癌、胃癌等肿瘤进展。4.探讨ATF1调节信号通路在鼻咽肿瘤与正常细胞之间的差异。利用全基因表达谱芯片分析了ATF1调节通路的差异。5.分析了ATF1基因SNP多态性与鼻咽癌发病风险相关性。通过病例对照研究结果提示ATF1基因SNP基因型与鼻咽癌发病风险有关。6.MMP9是ATF1的下游调控靶点，且存在T458新磷酸化位点，我们因而制备了磷酸化MMP9-T458抗体，发现在多个肿瘤中该位点磷酸化水平表达上调，该抗体可望用于肿瘤诊断和针对该位点的实验室检测。. 本项目的实施为磷酸化ATF1-T184在鼻咽癌中的作用及机制提供了详实的实验依据，可望为鼻咽癌诊断和治疗提供新的靶点。ATF1基因多态性与鼻咽癌发病风险相关性的发现和磷酸化MMP9-T458抗体的制备也可能为肿瘤的诊断提供新的检测方法。