KIF15/Rab11介导的α5β1整合素内体循环回膜促进胰腺癌p-FAK-Y397依赖性转移机制研究

The highly metastasis is a major cause of poor response to treatment of pancreatic cancer. The focus of current research is the molecular mechanism in metastasis of pancreatic cancer. The published literature confirmed that the depolymerization of the focal adhesions (FAs) on the ventral and caudal sides of cancer cells by FAK Phosphorylation was the key steps during cancer cell metastasis , and activated Integrin endosomes recyling back to plasma membrane recruited FAK to be phosphorylated in FAs for depolymerization of the FAs. High expression of p-FAK-Y397 in pancreatic cancer was associated with distant metastasis. Our previous study found that the high expressed kinesin 15 (KIF15) in pancreatic cancer can induces up-regulation of p-FAK-Y397 and cause depolymerization of the FAs by p-FAK-Y397 and enhance the abilty of invasion and metastasis of pancreatic cancer cells. Our preliminary experiments were performed to confirm that KIF15 interaction with Rab11 can form a complex and Rab11 is the key protein that mediate α5β1 integrin endosomes recyling back to plasma membrane. But the molecular mechanism how KIF15 up-regulated the expression of p-FAK-Y397 is not clear. Based on these clues, we propose a hypothesis that KIF15 interacts with Rab11 via its C-terminal tail domain, and KIF15/Rab11 complex mediates integrin α5β1 endosomes recyling back to plasma membrane, following by FAK phosphorylation (p-FAK-Y397) and activation in circulating endosome, which mediates depolymerization of focal adhesions in the recruitment sites (FAs), and enhance the abilty of invasion and metastasis of pancreatic cancer cells. Therefore, our project intends to confirm this molecular mechanism through cell and molecular biology researches, and validates its function through studies on clinical specimens and animal, in an attempt to elucidate the key mechanism of pancreatic cancer metastasis and provide relevant intervention targets.

高转移性是胰腺癌疗效差的主要原因，其转移分子机制是当前研究重点。研究表明:激活的黏着斑激酶（FAK）介导的黏着斑（FAs)解体是癌细胞转移的关键环节之一，而活化的整合素在内吞循环回膜到FAs过程中能募集并激活FAK引起FAs解体；胰腺癌中p-FAK-Y397高表达且与远处转移相关。本项目前期研究发现：胰腺癌中高表达的KIF15通过上调p-FAK-Y397，促进FAs解体，增强胰腺癌细胞侵袭转移能力；预实验证实KIF15与介导整合素α5β1循环内体回膜的关键蛋白Rab11相互作用形成复合物；但KIF15上调p-FAK-Y397的分子机制不清楚。基于上述线索，本研究提出研究假说：KIF15通过其C端尾部结构域与Rab11相互作用介导整合素α5β1循环回膜，募集并激活FAK促进FAs解体，增强胰腺癌侵袭转移能力。本项目拟通过相关实验研究技术验证上述胰腺癌转移分子机制，提供可能的治疗靶点。

胰腺癌在早期容易发生远处转移，其关键原因在于肿瘤细胞具有较强的迁移和局部浸润的能力。研究发现细胞尾部黏着斑的解聚是癌细胞在初期发生迁移运动的关键限速步骤，而整合素的循环回膜过程是黏着斑解聚的其中一个关键激活途径。目前已证实整合素β1循环回膜过程所介导的迁移能力上调是胰腺癌发生远处的关键原因。因此，深入研究整合素β1循环回膜的分子机制是解释胰腺癌早期发生侵袭转移的关键所在。为了确定参与整合素β1循环回膜的关键驱动蛋白，我们首先构建了整合素循环回膜的细胞模型，并将驱动蛋白家族的siRNA文库转染至胰腺细胞中筛选了参与整合素β1循环回膜的驱动蛋白，最终发现驱动蛋白家族15（KIF15）是参与整合素回膜一个关键调节因子。通过免疫组化发现KIF15在发生转移的胰腺癌组织中表达上调，并通过体内外实验证实了KIF15促进了胰腺癌细胞的迁移和侵袭能力。进一步发现KIF15是激活整合素β1/FAK信号的关键蛋白，以FAK-Y397依赖的方式加速黏着斑的分解，该过程可能由KIF15促进Integrin β1的循环回膜所介导。同时，通过内体循环阻滞剂Dynasore或干扰内体蛋白RAB11a的表达后，发现整合素β1的循环是KIF15促进胰腺癌细胞黏着斑解聚和转移的必要条件。进一步通过蛋白质谱鉴定发现KIF15可能与PI3K-C2α相互结合，并以循环内体标志物RAB11A依赖的方式促进整合素β1/FAK信号传导和黏着斑的解聚，二者互作可能依赖于KIF15的C端尾部结构域。此外，我们还发现KIF15的磷酸化是驱动蛋白功能活化的关键激活事件，其磷酸化修饰主要依赖于其C端乙酰化修饰过程。通过乙酰化酶家族的筛选发现SIRT1是介导KIF15去乙酰化进而介导其发生磷酸化修饰的关键去乙酰化酶。最后，我们通过SIRT1抑制剂EX527和KIF15抑制剂KIF15-IN作用于胰腺癌细胞，在二者的联合作用下胰腺癌细胞侵袭转移能力在体内外显著被抑制，同时在该药物浓度下，小鼠并未发生其他脏器的毒理作用。这些发现表明，KIF15与PI3K-C2α相互作用，以SIRT1乙酰化修饰依赖的方式控制整合素β1的内体循环，激活Integrinβ1/FAK信号通路进而促进胰腺癌细胞的黏着斑周转和远处转移。