微管负极靶向蛋白CAMSAP2介导的非中心体微管翻译后修饰在肝癌转移中的作用及机制研究

Emerging evidence suggests that noncentrosomal microtubules (MTs) play an essential role in intracellular transport, cell motility and cell polarity. This intresting findings enlight us whether cancer cells can promote the ability of directional migration by maintaining noncentrosomal microtubules, suppressing the microtubule-organizing ability of the centrosome. Calmodulin Regulated Spectrin Associated Protein Family Member 2 (CAMSAP2), a Microtubule Minus-End Binding Protein, can specifically deposit on MT minus-ends, and serve as “seeds” for noncentrosomal MTs outgrowth. However, its role in carcinogenesis and metastasis remains poorly understood. In this study, we investigated the potential mechanism of CAMSAP2 in regulation the assembly pattern of microtubules in hepatocellular carcinoma (HCC) metastasis. Our data showed that upregulation of CAMSAP2 in human HCC tissues was associated with metastasis Moreover, CAMSAP2 expression was positively related to the metastatic potential of HCC cell lines. Knockdown of CAMSAP2 impaired cell directional motility and led to the transformation of the partially noncentrosomal MTs array into a completely radial centrosomal pattern. Interestingly, Both western blotting and immunofluorescent staining showed that CAMSAP2 depletion strongly reduced the abundance of detyrosinated and acetylated MTs in HCC cells. Furthermore, we demonstrated that GEF-H1, a microtubule-regulated Rho-GEF, is a main downstream regulator of CAMSAP2-mediated MTs post-translational modifications in HCC cells. Knockdown of GEFH1 decreased CAMSAP2-mediated HCC invasion and metastasis, whereas overexpression of GEFH1 rescued the decreased invasion and metastasis induced by CAMSAP2 knockdown. RhoA activity assays and microtubule cosedimentation assay showed that CAMSAP2 overexpression upregulated RhoA activity and increased the soluble fraction of GEF-H1 ,whose activity is inhibited by binding to MTs but did not interact with detyrosinated and acetylated MTs. In conclusion, these findings highlight the importance of CAMSAP2 in HCC metastasis and suggest that CAMSAP2 may serve as a molecular target for HCC therapy.

最新研究发现，上皮及神经细胞中部分微管负极并未锚定于中心体，而是游离于细胞质形成CAMSAP2依赖性非中心体微管。微管结构与功能的非对称性是细胞定向运动的先决条件。显然，放射状对称性中心体微管不是细胞运动的最优方式，那么肿瘤细胞是否存在并通过非中心体微管促进侵袭转移呢？本项目预实验显示，肝癌组织CAMSAP2高表达且与转移正相关，肝癌细胞中存在CAMSAP2依赖性非中心体微管；干扰其表达抑制细胞侵袭转移，部分非中心体微管转换成放射状中心体微管；CAMSAP2可提高非中心体微管翻译后修饰水平并调控GEF-H1活性。我们提出假设：CAMSAP2促进非中心体微管形成并提高其翻译后修饰水平，释放GEF-H1，升高GEF-H1/RhoA活性以促进肝癌转移。本项目拟探索CAMSAP2介导的非中心体微管翻译后修饰在肝癌转移中作用及机制，研究其对GEF-H1/RhoA活性调控，为抑制肝癌转移提供新的思路。

最新研究发现，上皮及神经细胞中部分微管负极并未锚定于中心体，而是游离于细胞质形成CAMSAP2依赖性非中心体微管。微管结构与功能的非对称性是细胞定向运动的先决条件。显然，放射状对称性中心体微管不是细胞运动的最优方式，那么肿瘤细胞是否存在并通过非中心体微管促进侵袭转移呢？本研究通过Western blot、免疫荧光、免疫组化、RT-PCR等检测CAMSAP2在肝癌组织中的表达水平，结果显示肝癌组织中CAMSAP2 在肝癌组织中表达显著上调且与临床侵袭转移指征及患者不良预后呈正相关；划痕愈合实验、Transwell 迁移侵袭实验及3D 细胞侵袭模型表明过表达CAMSAP2 后，肝癌细胞迁移侵袭能力显著增强；反之，抑制CAMSAP2 表达后，肝癌细胞迁移侵袭能力减弱。接着，本研究通过免疫荧光及免疫共沉淀实验证实，肝癌细胞中存在CAMSAP2依赖性非中心微管。在此基础上，我们采用免疫荧光、微管洗脱再生试验等证实CAMSAP2 依赖性非中心体微管重排促进肝癌细胞定向迁移；进一步研究结果显示，CAMSAP2 通过激活/JNK/c-Jun 通路抑制HDAC6 表达，促进非中心微管乙酰化修饰及微管骨架重排进而促进肝癌侵袭转移。裸鼠原位种植转移模型证实，CAMSAP2能够促进肝癌肺转移，下调CAMSAP2 表达的基础上进一步抑制HDAC6 表达，裸鼠肝癌肺转移较CAMSAP2敲减组明显增加，且生存期显著缩短。综上，我们的研究证实了CAMSAP2 介导的非中心体微管翻译后修饰在肝癌转移中的作用，为抑制肝癌转移提供了新的思路。