ADAMs调控PCDH9剪切促进胃癌侵袭转移的机理研究

Cleavaging of molecular on the surface of tumor cells contributes to metastasis of various tumors. Our previous studies revealed that full-length and extracellular domain of PCDH9 decreased with the process of metastasis of gastric cancer and intracellular domain of PCDH9 increased in the nucleus , indicating PCDH9 cleavage and nuclear translocation occurs during metastasis.However, the mechanism underlying this phenomenon remains unclear. Biological analysis showed that the constructure of PCDH9 was easily attacked by ADAMs(a disintegrin and metalloproteases) metalloproteases. ADAMs were upregulated in gastric cancer and resulted in shedding of the extracellular domain of PCDH family. We postulated that ADAMs(ADAM10/17) might be responsible for the downregulation of PCDH9. Interestingly, we found that a novel transcription factor, FoxM1, could regulate the expression of ADAMs(ADAM10/17). Therefore, we postulate that FoxM1 regulates ADAMs expression, which then results in shedding of the extracellular domain of PCDH9. Then metastasis initiated. In the present study, we sought to investigate the role of “FoxM1-ADAMs-PCDH9” axis in the development and progression of human gastric cancer, which will be essential in improving the efficiency of the treatment of gastric cancer and predicting the outcome of patients with gastric cancer.

肿瘤细胞膜表面蛋白（粘附分子等）在胃癌的转移过程中发挥了重要作用。我们前期研究发现原钙黏蛋白全长PCDH9和PCDH9胞外段在胃癌转移过程中表达下调，而胞内段出现在细胞核内，提示转移过程中PCDH9发生了剪切和核转位，然而其具体机制尚不明确。生物学分析和初步研究显示PCDH9是蛋白水解酶ADAMs的作用底物。ADAMs成员在胃癌中表达升高,能够水解PCDH家族成员,但其对PCDH9的剪切调控促进胃癌转移的研究未见报道。进一步研究发现ADAMs成员受到FoxM1的调控，故我们推测“FoxM1调节ADAMs的表达，调控PCDH9的剪切，促进胃癌细胞的转移”。本课题拟通过基因沉默、过表达、ADAMs抑制剂、质谱等技术研究ADAMs对PCDH9剪切作用，通过突变、免疫共沉淀和ChiP等技术研究FoxM1对ADAMs的调节作用，初步阐明“FoxM1-ADAMs-PCDH9”轴在胃癌转移中的作用。

项目背景：.肿瘤细胞膜表面分子的膜剪切和核转位是肿瘤细胞转移的一个重要机制。我们发现PCDH9存在膜剪切和核转位现象，但其具体机制不明。.主要研究内容和重要成果.①PCDH9候选调控轴FoxM1-ADAM在胃癌中的表达及其临床价值.检测了FoxM1和ADAMs在胃癌组织中表达情况，ADAMs在胃癌中表达升高，选择性抑制ADAMs的表达可抑制胃癌细胞的增殖。部分ADAMs有可能成为胃癌治疗的靶点。.②FoxM1调控ADAM17促进胃癌增殖和成瘤.FoxM1能够调控ADAM17的表达，前者直接促进后者的转录活化，导致胃癌细胞功能学改变。FoxM1及ADAM17表达是胃癌患者的重要预后因素。.③PCDH9的功能学研究.构建pcDH-PCDH9-Flag质粒，转染胃癌细胞、行核质分离实验。PCDH9-Flag存在剪切带，其胞内段大小约为70KD。核质分离实验显示PCDH9胞内段存在核定位。构建了PCDH9胞内段突变体（PCDH9-Cter-Flag）,转染细胞。PCDH9-Cter-Flag存在细胞核定位，PCDH9-Cter-Flag过表达显著地抑制了SGC7901细胞的迁移和侵袭。RNA-seq发现CDH2是PCDH9-Cter的靶点之一，PCDH9-Cter过表达抑制CDH2的转录及表达。PCDH9-Cter通过调控CDH2的表达抑制胃癌细胞的迁移。PCDH9-Cter入核后和DNMT1相互作用，促进CDH2启动子区域的DNA甲基化，抑制CDH2的转录。.科学意义或应用前景：.PCDH9存在剪切体，胞内段的PCDH9存在核定位，核内PCDH9通过促进DNMT1介导的CDH2启动子的甲基化抑制CDH2的转录，抑制胃癌细胞迁移。本研究不但利于完善胃癌转移机制，更会对胃癌患者疗效和预后的判断提供新的思路。