Slit3/Robo1/myo9b通路调控胃癌侵袭和转移的机制研究

Slit/Robo is the classical signaling pathway that could regulate the motility of neurones and recent studies showed that it also played critical roles in tumor metastases. In a previous study, we have revealed that Slit3/Robo1 directly regulated metastases in gastric cancer, but the downstream molecular effectors remained unclear in this pathway. By proteomic analysis, we found that Robo1 could interact with myo9b. Myo9b was a protein with GAP activity and it might directly modulate the activities of Rho GTPases. Rho GTPases were well-known molecules that could regulate the dynamic re-organization of cytoskeleton through modulating the balance between the polymerization and depolymerization of F-actin. The immunohistochemical staining indicated that the expression levels of Robo1 and myo9b were increased in gastric cancer and were both associated with metastasis status of gastric cancer. In addition, myo9b was up-regulated in gastric cancer cells with increased metastatic potential and knockdown of myo9b could inhibit the invasive and migrative potential of cells. Therefore, we speculated that Slit3-Robo1-myo9b-Rho GTPase is the critical pathway in regulating metastasis of gastric cancer. In the present study, we aim to elucidate the function of myo9b and its mechanism in the metastases of gastric cancer. On the basis of this, we will clarify whether the signaling trasducion of Slit3/Robo1 pathway depends on the interaction between Robo1 and myo9b. This program would strengthen the current understanding of tumor metastases and develop the novel theraprutic targets in gastric cancer.

Slit/Robo是调节神经细胞运动的经典信号通路，近年来的研究提示该通路也参与了肿瘤转移的调控。本课题组已阐明了Slit3/Robo1对胃癌转移的调节作用，但其下游机制尚不清楚。在此基础上，本课题组发现myo9b可与Robo1发生相互作用。myo9b具有GAP活性，它可通过改变Rho GTPase的活性而调节细胞骨架的重组。Robo1和myo9b在胃癌中的表达均升高，且二者的表达强度与胃癌是否发生转移具有直接的相关性。此外，下调myo9b可显著抑制胃癌细胞的转移能力。因此，我们推测Slit3-Robo1-myo9b-Rho GTPase可能是调节胃癌转移的重要通路。本项目拟进一步明确myo9b在胃癌转移中的作用及机制，并将重点阐明Slit3/Robo1通路的作用是否依赖于Robo1和myo9b的相互结合。本项目有助于深化对胃癌转移的认识，并提供胃癌转移的治疗新靶点。

神经导向相关分子通路在肿瘤转移中的作用日益受到关注，其中以Slit/Robo通路的研究最为广泛。本课题组通过系列的研究，率先阐明了Slit/Robo1通路在胃癌转移中的调节作用及其上游机制：胃癌中miR-218的表达下调，继而解除对下游靶基因Robo1、POU2F2和IKK-β的表达抑制； IKK-β可通过NF-κB通路进一步上调POU2F2和Robo1，从而放大了Slit/Robo1通路的信号转导效应，最终表现为对胃癌转移和侵袭的促进作用。在此基础上，本课题以Slit/Robo1下游信号通路为研究重点，阐述了Robo1相互作用蛋白myo9b参与Slit/Robo1通路的分子机制。本课题研究内容包括：验证myo9b对胃癌细胞侵袭和转移潜能的直接调控作用；验证myo9b是否通过调节RhoA、Rac1或Cdc42的活性发挥作用；探索Slit/Robo1通过myo9b调节胃癌转移的机制；明确Slit/Robo1/myo9b在胃癌中表达特征与临床意义。通过上述研究内容，获得以下重要结果：1. 上调Myo9b可显著促进胃癌细胞体外及体内转移能力，下调myo9b显著抑制胃癌细胞的体外和体内转移能力；2. Myo9b可促进RhoA由激活状态向失活状态转变，从而改变胃癌细胞祥泰，促进胃癌细胞发生转移；3. Rho GAP结构域是myo9b的关键功能区域，Rho GAP可促进胃癌发生转移；4. Slit/Robo1通路通过myo9b发挥对胃癌转移的调控作用；5. Myo9b在胃癌中表达升高，在淋巴结转移灶中的表达强度高于原发灶，其高表达提示胃癌患者总生存期显著缩短。以上结果对阐明Slit/Robo1的下游机制具有重要意义，有助于深化对Slit/Robo1在肿瘤发生发展过程中所发挥作用的理解，对寻找胃癌转移的潜在治疗靶标奠定了基础。