Rap2a通过TNIK-Akt信号通路促进肾癌转移

Metastasis is the leading cause of death for renal cell carcinoma patients. Rap2a has recently attracted considerable attention as that it is found closely related with tumor metastasis. In our previous study, we identified that forced expression of Rap2a drastically promoted cancer cell invasion and phosphorylation of Akt. However, the underlying molecular mechanism is not clear. It is demonstrated that TNIK is an important regulator of cell adhesion, motility and metastasis. In our preliminary experiments, we found that both Rap2a and TNIK were overexpressed in renal cancer cells. Rap2a overexpression could increase the phosphorylation level of TNIK. Moreover, we found that si-TNIK could suppress renal cell invasion and TNIK promote renal cancer cell invasion through Akt signalling pathway..Consequently, we suppose that Rap2a may regulate renal cell carcinoma metastasis through TNIK-Akt signalling pathway. To confirm this hypothesis, we will investigate whether Rap2a can bind with TNIK or not and verify the binding sites by IP and site mutation. Furthermore, we will study the effect of Rap2a on metastatic biological behavior through TNIK. Then, we will examine the correlation between Rap2a, TNIK expression and metastasis by 271 cases tissue microarray. Lastly, we will verify whether Rap2a can promote tumor metastasis through TNIK-Akt signalling pathway by in vivo nude mouse model. Our study will provide a new target for renal cell carcinoma gene therapy.

转移是肾癌患者死亡主要原因。近年来研究发现小G蛋白Rap2a与肿瘤转移密切相关，我们既往研究发现Rap2a过表达可促进肾癌细胞侵袭和Akt磷酸化，但具体机制尚不清楚。TNIK是调节细胞粘附、运动、转移的关键因子。申请者预实验发现，肾癌细胞中Rap2a和TNIK均高表达，Rap2a能促进TNIK磷酸化激活；下调TNIK表达可抑制肾癌细胞侵袭，TNIK通过调节下游Akt信号促进肾癌细胞侵袭。因此，本课题提出“Rap2a通过TNIK-Akt信号通路影响肾癌转移”。为证明这一假说，我们将在分子水平研究Rap2a与TNIK的结合，确认其结合位点及对TNIK活性的影响；细胞水平研究Rap2a通过TNIK-Akt信号通路对肾癌细胞转移相关生物学行为的影响，并通过肾癌组织芯片分析Rap2a和TNIK表达与肾癌转移预后的关系，最后通过动物实验验证Rap2a通过TNIK-Akt信号通路调控肾癌转移作用及机制。

Rap2a属于Ras超家族小分子量GTP结合蛋白的Rap亚家族，因其效应因子结构域第39位苯丙氨酸的不同，表明其可能通过特异下游信号分子调控独特的生物学功能。研究表明Rap2家族蛋白参与许多生物学过程，其功能主要包括整联蛋白介导的细胞粘附调节，细胞骨架的动态组装，参与胞内运输及细胞极性的建立，出胞和细胞增殖等。由于细胞粘附调节，细胞骨架的动态组装很有可能与细胞的运动迁移有关，因此，Rap2a是否影响肿瘤细胞的迁移侵袭，是一个非常值得探讨的问题。本实验结果表明，Rap2a、TNIK及p-Akt在四种肾癌细胞中高表达。Rap2a通过激活TNIK进而磷酸化Akt促进肾癌转移，而Akt的变化也反过来影响TNIK的蛋白表达，后两者之间形成闭合环路。Rap2a的17位点的丝氨酸对其生物活性的维持至关重要。TNIK也参与肾癌细胞迁移侵袭的调控，其CNH结构域的完整性对TNIK的生物活性，尤其是对Akt的调控起关键作用。并且，Rap2a可通过抑制TNIK泛素化降解进而使TNIK蛋白表达增加。本实验结果在细胞、分子和整体动物水平分析了Rap2a通过TNIK-Akt信号通路促进肾癌转移的作用。最终阐明Rap2a与TNIK在肾癌转移中的作用，为治疗肾癌新的靶点筛选提供理论依据。