CLASP2调控膀胱癌PD-L1细胞膜定位表达的机制研究

One of the main reasons of infiltration and metastasis of advanced-stage bladder cancer is immune escape. Programmed cell death ligand 1 (PD-L1) on the plasma membrane of bladder cancer cells could bind with programmed cell death protein 1 (PD-1) on inflammatory cells, which would consequently induce the apoptosis of inflammatory cells and immune escape. Therefore, the investigation of regulations of PD-L1 in bladder cancer would be very meaningful for reversing immune escape and treating bladder cancer. However, the mechanisms of regulation of PD-L1 in bladder cancer have not been reported yet. Our previous study demonstrated that cytoplasmic linker-associated protein 2 (CLASP2) was closely related to the malignance of bladder cancer. And the pilot investigations showed that CLASP2 could regulate the function and distribution of microtubules, recruit motor protein KIF1C, and modulate plasma membrane localized expression of PD-L1, which would be a new mechanism of abnormal expression of PD-L1 in bladder cancer. We would further confirm and investigate this mechanism by utilizing various techniques, such as dynamic immunofluorescent observation. The study would be performed on three levels of protocols including clinical samples, cellular experiments in vitro, and mice experiments. The exact mechanism of CLASP2 modulating microtubule functions, the role of KIF1C trafficking PD-L1 and their interactive domains will be revealed. New targets of bladder cancer treatment would be provided via implement of present study.

晚期膀胱癌出现浸润转移的一个重要原因是免疫逃逸。肿瘤细胞膜的程序性死亡分子配体1（PD-L1）可与炎症细胞的程序性死亡分子1（PD-1）结合，促炎症细胞凋亡，从而实现免疫逃逸，所以研究PD-L1的调控机制对逆转免疫逃逸、治疗晚期膀胱癌有重要意义。但国内外尚无膀胱癌PD-L1异常表达的机制报道。我们前期研究发现胞质连接蛋白2（CLASP2）与膀胱癌的恶性程度密切相关；预实验发现CLASP2可调控微管功能及空间分布，招募转运PD-L1的马达蛋白KIF1C，从而影响PD-L1的细胞膜定位表达，这可能是一个膀胱癌异常表达PD-L1的新机制。我们将重点应用“动态”免疫荧光等技术，从临床标本、细胞实验、小鼠模型三个层面，进一步验证及深入研究该机制，以明确CLASP2调控微管功能的具体方式；确证马达蛋白KIF1C的转运作用及其与PD-L1的交互作用结构域。本课题的成果将为膀胱癌的治疗提供新靶点。

程序性死亡分子配体1（PD-L1）在膀胱癌的进展、免疫逃逸中起重要作用。本课题首先明确了CLASP2在膀胱癌中表达的意义，发现高表达CLASP2的膀胱癌患者易出现转移，并且对辅助化疗效果较差。临床上，可以检测CLASP2来判断膀胱癌患者的预后和化疗疗效；然后明确了CLASP2可调控微管功能，影响PD-L1膜蛋白的表达和功能定位的意义，提出了PD-L1调控新机制；关于PD-L1抗体相关的免疫治疗在晚期膀胱癌中地位越来越重要，课题组利用脂质体配合光动力疗法抑制膀胱癌胞膜PD-L1的表达，进而增加肿瘤局部微环境中T细胞的浸润数目和杀伤肿瘤细胞能力，为膀胱癌的治疗提供新思路；同时，课题组设计的新型纳米粒子能够完美地解决了传统光动力受到活化血小板维持的肿瘤血管屏障限制的关键问题，有望为膀胱癌的诊疗带来崭新的突破，具有极大的临床转化价值；近年来，肿瘤微环境相关研究受到重视，本课题组发现了膀胱癌微环境中肿瘤相关成纤维细胞（CAF）不但在肿瘤的进展、转移过程中起关键作用，而且可通过IGF-1/ERβ/Bcl-2信号轴导致顺铂耐药；本课题组筛选、证明了一个膀胱癌进展的新机制，LncRNA MAGI2-AS3可通过靶向miR-31-5p/TNS1轴来抑制膀胱癌进展。项目执行期间共培养毕业硕士研究生5名，招收博士研究生2名。指导获得湖南省优秀硕士学位论文和中南大学优秀硕士学位论文各1次。