Kibra经抑制aPKCζ介导的极性相关囊泡的运输及PATJ的转运抑制乳腺癌细胞的定向迁移

Breast cancer ranked as the second leading cause of cancer death worldwide. The main reason of death of breast cancer patients is distant migration cell in advanced stage. Our pilot studies showed that Notch3 can inhibit EMT through upregulating the expression of Kibra. However, the underlying mechanism of Kibra supressing EMT is still unclear. The published studies reported that aPKCζ is essential for the exocytosis of apical protein containing vesicles, and Kibra could bind to the aPKCζ protein or PATJ protein directly. Kibra may be an important regulator involving in directly migration by affecting the exocytosis of apical protein containing vesicles and the trafficking of PATJ. In this project, to further investigate the mechanism of Kibra inhibiting metastasis in breast cancer cells,immunofluorescence (IF) and confocal microscopy will be used to detect the effect of Kibra in the location of apical protein containing vesicles and PATJ. Co-immunoprecipitation (Co-IP) will be used to comfirm that Kibra bind to aPKCζ or PATJ directly. Rescue assay will be performed to clarify that Kibra mediated apical protein containing vesicles through aPKCζ. The immunohistochemistry（IHC）will be used to detected the relationship between Kibra expression and breast cancer metastasis in clinic. The result will be comfirmed in vivo and in vitro functional assays such as invasion assay, migration assay and tumor formation assay in mice. This project is of great importance for identification of novel targets, the development of potential therapeutic methods and the achievement of individualized therapies in breast cancer.

乳腺癌致死率在全球癌症中居第二位，远处转移是其死亡的主要原因。我们前期研究发现：Notch3通过上调Kibra抑制EMT，但Kibra如何抑制EMT的分子机制尚不明确。有报道显示：aPKCζ是促进胞内极性相关的囊泡的运输的关键因子；Kibra蛋白可以与aPKCζ蛋白或PATJ蛋白直接结合。Kibra或许因此通过抑制极性相关囊泡的运输和PATJ的转运至细胞运动前缘调节细胞定向迁移。本项目拟用免疫荧光、激光共聚焦显微镜检测Kibra对极性相关囊泡和PATJ定位的影响；通过免疫共沉淀明确Kibra可分别与aPKCζ或PATJ直接结合；通过挽救实验证明Kibra对极性相关运输囊泡的作用是通过aPKCζ实现的，应用IHC法研究Kibra与临床乳腺癌转移的关系，并经侵袭实验、迁移实验和裸鼠成瘤实验在体外和体内加以验证。本课题对寻找新的乳腺癌治疗靶点，开发潜在的治疗方法，实现个体化治疗具有重要意义。

乳腺癌致死率在全球癌症中居第二位，远处转移是其死亡的主要原因。研究乳腺癌远处转移的机制，发现调控乳腺癌远处转移的关键靶分子，对寻找新的乳腺癌治疗靶点、降低乳腺癌患者死亡率具有重要意义。我们前期研究发现：Notch3通过上调Kibra抑制乳腺癌的迁移，但Kibra抑制迁移的分子机制尚不明确。研究表明，Kibra与aPKCζ均是参与乳腺癌迁移过程的重要分子，且与细胞黏附的调节密切相关，因而我们提出假设：在乳腺癌中，Kibra可能通过aPKCζ调控细胞黏附相关分子的表达，进而抑制乳腺癌的迁移。在本项目中，我们建立了稳定高表达Kibra以及稳定敲除Kibra的乳腺癌细胞株，并通过划痕实验、Transwell迁移实验对比Kibra不同表达情况对乳腺癌细胞运动能力的影响，通过Western Blot实验检测稳定高表达Kibra以及稳定敲除Kibra的乳腺癌细胞系中Integrin-actin相关分子的表达情况，通过Western Blot实验检测Kibra与aPKCζ的表达相关性，通过免疫共沉淀实验确定Kibra与aPKCζ的调控作用，通过挽救实验明确Kibra是否通过aPKCζ调控乳腺癌的迁移的。结果显示，Kibra可抑制乳腺癌的迁移功能。Talin、kindlin2、Filamin A等Integrin-actin相关分子在Kibra高表达的乳腺癌细胞株中表达升高，在Kibra敲减的乳腺癌细胞株中表达减少。高表达Kibra可抑制aPKCζ的表达，Kibra对Integrin-actin相关分子的调控是通过aPKCζ起作用的。另外，高表达Kibra可抑制aPKCζ的磷酸化水平。Kibra对aPKCζ的磷酸化水平的影响是通过与其直接结合而起作用的。因此，我们得出结论：Kibra可抑制aPKCζ的磷酸化水平，Kibra通过与aPKCζ直接作用，进而上调整合素-肌动蛋白的表达水平，从而抑制乳腺癌细胞的迁移。