Slit3/Robo1/Nek9/VAV2通路调控胃癌细胞骨架重构和转移的机制研究

Neuronal guidance pathway Slit/Robo1 plays critical roles in tumor metastasis. In previous studies, we have revealed the upstream mechanisms of how Slit/Robo1 regulated the metastasis of gastric cancer, but its downstream molecular effectors remained not fully understood. By proteomic analysis, we found that Nek9 could interact with Robo1. Compared with normal gastric tissues, Nek9 was up-regulated in gastric cancer tissues and its expression was also increased in the metastatic lymph nodes and distant metastatic tissues than in the primary cancer tissues. Knockdown of Nek9 could induce cytoskeleton remodeling, inhibit cancer metastasis and suppress Rho GTPase activities. The intrinsic kinase activity is critical for the function of Nek9 and we confirmed by cDNA array and bioinformatics that VAV2 might serve as a effector of Nek9. The phosphorylation of Nek9 could activate Rho GTPase which plays a central role in cytoskeleton remodeling. Therefore, we speculated that Slit3-Robo1-Nek9-VAV2 is the critical pathway in regulating cytoskeleton remodeling and metastasis of gastric cancer. In the present project, we aim to further elucidate the function of Nek9 and its mechanism in the cytoskeleton remodeling and metastasis of gastric cancer. On the basis of this, we will clarify whether Nek9 with intrinsic kinase activity is the key signal transducer of Slit/Robo1 pathway. Combined the studies of tissues and serum from patients with gastric cancer, this project would strengthen the current understanding of the nature of cancer metastases and develop the novel therapeutic targets in gastric cancer.

神经导向相关通路Slit/Robo1是调节肿瘤转移的重要通路。本课题组率先阐明了Slit/Robo1通路调控胃癌转移的上游机制，但其下游分子作用机制尚不完全清楚。在既往基础上，本课题组发现Nek9是Robo1的相互作用分子。Nek9的表达在胃癌转移淋巴结和转移灶内显著增强，且下调Nek9可改变胃癌细胞骨架结构，抑制转移和Rho GTPase活性。Nek9的功能依赖于其激酶活性，而VAV2可能是Nek9的下游靶分子。磷酸化的VAV2可激活Rho GTPase而影响细胞骨架的重构。因此，我们推测Slit3-Robo1-Nek9-VAV2可能是调节胃癌细胞骨架重构和转移的重要通路。本项目拟进一步明确Nek9在胃癌细胞骨架重构和转移中的作用机制，重点阐明具有激酶活性的NEK9是否是Slit/Robo1通路的关键信号转导分子。结合临床标本研究，本项目将深化对胃癌转移本质的认识，提供新的胃癌治疗靶点。

【背景】.神经导向相关分子通路Slit/Robo是调节肿瘤转移的重要通路。本课题组前期已发现Slit/Robo调节胃癌转移并初步阐明了其上游机制，进一步发现NEK9是Robo1的相互作用分子，因此在本项目中围绕NEK9这一丝氨酸/苏氨酸蛋白激酶，展开功能学和机制学研究。.【研究内容】.1. 在胃癌细胞中上调或下调Robo1相互作用分子NEK9的表达，通过体内外功能学实验、免疫印迹、形态学实验观察NEK9对胃癌细胞骨架和转移能力的调节作用；.2. 通过磷酸化蛋白组学及免疫印迹明确NEK9的磷酸化作用靶标分子，通过生物信息学分析获得靶标分子潜在磷酸化位点，采用点突变的方法构建系列质粒，分别转染入胃癌细胞，通过体内外功能学实验、免疫印迹、形态学实验等明确靶标分子受到NEK9磷酸化调控的具体位点；.3. 通过生物信息学分析、RT-PCR、免疫印迹、荧光素酶报告基因、染色质免疫沉淀等方法探索IL-6通过STAT3对下游靶标miRNA的转录调控作用，以及靶标miRNA对其直接靶基因的调节作用；.4. 通过功能学和形态学研究明确ARHGAP35对胃癌转移和细胞骨架重构的作用；.5. 通过公共数据库分析和独立胃癌配对组织样本队列的免疫组化研究，揭示关键节点分子的临床表达水平及临床意义。.【结果】.1. NEK9可直接磷酸化ARHGEF2，提高ARHGEF2催化RhoA进入激活状态的活性，从而导致细胞骨架的重构和胃癌细胞的转移；.2. IL-6-STAT3可转录抑制miR-520f-3p，进一步调节NEK9和GP130的表达，对微环境中炎症信号通路的激活形成反馈调节；.3. p-STAT3、NEK9 和 GP130 的染色强度在淋巴结和远处转移中的染色强度显著增高，在转移性胃癌组织中增加。p-STAT3、NEK9 和 GP130 之间存在正相关，且与胃癌患者预后相关；.4. ARHGAP35通过调节胃癌细胞EMT影响胃癌细胞骨架改变和转移潜能的变化。.【结论及研究意义】.本项目发现NEK9是介导Slit/Robo通路调节胃癌细胞骨架重构的重要分子，是联系炎症微环境和肿瘤细胞相互作用的重要桥梁，对阐明Slit/Robo的下游机制具有重要意义，对寻找胃癌转移的潜在治疗靶标奠定了基础。