HMGA2-FOXL2-ARHGAP5-NEK2反馈通路调节胃癌细胞骨架和转移的机制研究

HMGA2/FOXL2 pathway plays critical roles in EMT and metastasis of gastric cancer. In previous studies, we have revealed that HMGA2 could regulate FOXL2 through Rb-E2F1 axis, but the downstream molecular effectors of FOXL2 remain not fully understood. Our further study showed that knockdown of FOXL2 could induce cytoskeleton remodeling, suppress Cdc42 activity and increase RhoA activity. ARHGAP5 and NEK2 were the potential transcription targets of FOXL2, both of which could affect cell motility. Inhibition of ARHGAP5 down-regulated ARHGEF9, and they could directly modulate the activities of Rho GTPases. NEK2 could regulate FOXL2 through its kinase activity and this effect may be associated with the phosphorylation of HMGA2 by NEK2. Therefore, we speculated that HMGA2-FOXL2-ARHGAP5-ARHGEF9 is the critical pathway in regulating cytoskeleton remodeling and metastasis of gastric cancer. In the present project, we aim to further elucidate the transcriptional regulation of ARHGAP5 and ARHGEF9 by FOXL2. On the basis of this, we will clarify how ARHGAP5 affect cytoskeleton remodeling and whether the effect on HMGA2 phosphorylation by Nek2 is the key to sustained FOXL2. Combined with the studies of gastric cancer tissues, this project would strengthen the current understanding of the nature of cancer metastasis and develop novel therapeutic targets in gastric cancer.

HMGA2/FOXL2是调节胃癌EMT和转移的重要通路。本课题组率先阐明HMGA2通过Rb-E2F1调控FOXL2，但FOXL2下游作用机制尚不清楚。在既往基础上，本课题组发现FOXL2影响胃癌细胞骨架和Rho GTPase活性。ARHGAP5和NEK2是FOXL2潜在的转录靶标，二者均可影响胃癌转移能力。ARHGAP5可影响ARHGEF9，共同作用于Rho GTPase而影响细胞骨架重构。NEK2可通过其激酶活性调节FOXL2，这可能通过磷酸化HMGA2实现。由此，本课题组推测HMGA2-FOXL2-ARHGAP5-NEK2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FOXL2对下游靶标的转录调控，重点阐明ARHGAP5影响细胞骨架的机制，以及具有激酶活性的NEK2是否通过HMGA2维持FOXL2通路的持续激活。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标。

【背景】.细胞骨架的重构是肿瘤转移的分子事件。本课题组前期已发现FOXL2调节胃癌转移并初步阐明了其上游机制，因此在本项目中围绕FOXL2这一关键转录因子，展开功能学和机制学研究，以期阐明FOXL2的下游机制。.【研究内容】.1. 通过蛋白组学、染色质免疫沉淀后测序、生物信息学分析等技术筛选FOXL2的下游靶标分子；.2. 通过转录因子报告基因实验验证FOXL2对NDFPI1的转录活性调控；.3. 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；.4. 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. 符合表达差异倍数大于 1.2 倍且 P value小于0.05 筛选标准的视为差异表达蛋白质。在此次蛋白组学分析中，共发现636个差异蛋白。将之与 4045 个 ChIP 数据的差异基因共有 95 个交集。.2. 使用上述95个基因 Symbol 进行基于 GO、KEGG 通路的富集分析，寻找基因集合内共同的功能及相关通路。共显著富集得到348个GO的功能条目和18条KEGG通路。.3. 通过 string 软件分析 FOXL2 下游 95 个差异表达基因的相互作用关系，显示 MAD1L1-KIFC1、INSR-PRKCE、GNA11-GNA5与UBE3A-NDFIP1等基因之间具有强相关性。NDFIP1 是 Nedd4 家族蛋白之一，参与泛素化修饰过程。进一步通过生物信息学测序和双荧光素酶报告基因证实FOXL2可结合于NDFIP1的启动子区；.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FOXL2可能通过其下游广泛的靶基因参与细胞骨架的调节，具有重要的潜在应用转化价值。