TBK1调控抗原递呈细胞和肿瘤细胞迁移功能的分子机制及其对炎症和肿瘤的影响

Migration capacity is not only required for mobilization and residence of antigen-presenting cells (APC) in vivo, but for initiation of immune response and exertion of their diverse immune functions. Moreover, migration of tumor cells is closely associated with tumor metastasis. It is a key scientific question for both basic immunology and oncology how to regulate the migration of cells in order to control the functions of APC and to resolve the concern of tumor metastasis. It has been reported that the serine threonine kinase TBK1 (Tank-binding kinase 1) can regulate the production of type I interferons (IFN) and thus the innate immune response through phosphorylation and activation of the transcription factors IRF3 (interferon regulatory factor 3) and IRF7. However, it is barely known whether TBK1 also plays crucial roles in physiological and pathological processes other than innate immunity. Our preliminary data showed that RAW264.7 macrophages and 3LL Lewis lung carcinoma cells with TBK1 deficiency using CRISPR-Cas9 were impaired for the migration capacity. So we hypothesized in the current project that TBK1 may control the migration of APC and tumor cells. We aim to explore the effects of TBK1 on cell migration by using APC and tumor cells with TBK1 deficiency or TBK1 knockdown, and the underlying mechanisms by which TBK1 promotes cell migration. Moreover, we plan to confirm the effects and mechanisms for TBK1-mediated regulation of cell migration in inflammation and tumor with the assistance of TBK1 inhibitors. Our study will discover new molecular mechanisms for understanding the control of APC migration, and will identify targets for combating against migration, invasion and metastasis of tumor cells.

迁移功能不仅与抗原递呈细胞（APC）体内游走定位和触发免疫应答、发挥免疫功能相关，而且与肿瘤转移过程密切相关。如何调控细胞迁移功能以控制APC功能和解决肿瘤转移问题，这是基础免疫学和肿瘤学均关注的关键科学问题。研究证明丝苏氨酸蛋白激酶TBK1通过活化转录因子IRF3/7控制多种病原体诱导的I型IFN产生，发挥调控天然免疫应答的作用，但TBK1的其他生理病理功能还有待于进一步研究。前期研究发现TBK1基因敲除的RAW264.7巨噬细胞和小鼠3LL肺癌细胞的迁移功能显著下降，因此我们假设TBK1可能调控APC和肿瘤细胞迁移。我们拟用TBK1缺失或敲低的APC和肿瘤细胞探讨TBK1对细胞迁移功能的影响及其促进细胞迁移的分子机制，并在炎症和肿瘤小鼠模型中结合TBK1抑制剂验证TBK1调节细胞迁移功能的效应和分子机制。该研究将为APC迁移功能的调控找到新机制，也将为肿瘤迁移、侵袭和转移寻找到新靶标。

迁移功能不仅与抗原递呈细胞（APC）体内游走定位和触发免疫应答、发挥免疫功能相关，而且与肿瘤转移过程密切相关。如何调控细胞迁移功能以控制APC功能和解决肿瘤转移问题，这是基础免疫学和肿瘤学均关注的关键科学问题。研究证明丝苏氨酸蛋白激酶TBK1主要发挥调控天然免疫应答的作用，但TBK1的其他生理病理功能还有待于进一步研究。我们拟用TBK1缺失或敲低的APC和肿瘤细胞探讨TBK1对细胞迁移功能的影响及其促进细胞迁移的分子机制，并在炎症和肿瘤小鼠模型中探讨TBK1调节细胞迁移功能的效应和分子机制。我们在人乳腺癌组织芯片上发现TBK1的表达量和活化在乳腺癌中呈现异常增强，于预后呈现负相关。我们利用CRISPR-Cas9技术在小鼠乳腺癌细胞系4T1-Luc和人乳腺癌细胞系MAD-MB-231敲除TBK1的表达，或者在其他肿瘤细胞系内通过siRNA介导的TBK1敲低，或者使用TBK1抑制剂，或者结合TBK1过表达，发现TBK1促进乳腺癌细胞的体外迁移和体内肺转移。基于转录组学、蛋白质磷酸化蛋白组学和TBK1结合蛋白的质谱分析，我们发现TBK1敲除抑制AMP蛋白激酶α亚基（AMPKα）的表达，促进AMPKα的磷酸化；TBK1敲除抑制小G蛋白Rac1/Cdc42的GTP活化状态；使用AMPKα激动剂可以抑制Rac1/Cdc42的活化并增强TBK1敲除导致的肿瘤细胞迁移功能改变和Rac1/Cdc42的活化。使用TBK1敲除的巨噬细胞和树突状细胞，我们证实TBK1亦可调控APC的体外迁移功能。因此我们推测，在肿瘤细胞和APC中，TBK1可以通过调控AMPKα的磷酸化和Rac1/Cdc42的活化，从而影响细胞的迁移。我们的研究提示TBK1可能作为潜在靶标应用于肿瘤靶向治疗和肿瘤免疫治疗的实践中，具有重要的潜在应用价值。