MIIP缺失在结直肠癌免疫逃逸中的作用及机制研究

Imbalance of immune system plays a role in the development and progression of colorectal cancer (CRC). PD-1 immune checkpoint inhibitors have been approved to treat stage IV CRC patients whose tumors are microsatellite instable-high (MSI-H). However, the PD-1 inhibitors are not effective in treating CRCs with chromosome instability (CIN). Our previously funded NSFC grant led to the findings that MIIP haploinsufficiency induced CIN and promoted the progression of CRC. Our recent work showed that MIIP affected the immune surveillance in CRCs with CIN. In this proposal, we will perform in vitro experiments, animal models and clinical analysis of CRC patients to demonstrate that MIIP deletion/ downregulation augments the deacetylation activity of HDACs and consequently inhibits the expression of MHC I through increasing the binding of COUP-TFII (transcription repressor) to MHC I enhancer and decreasing the binding of NF- kappa B (transcription activator) to MHC I enhancer. As the same time, the MHC I-related antigen presentation is inhibited through reduction of the expression of TAP1 because of the decreased histone H3 acetylation of the TAP1 promoter. Consequently, the recognition of tumor cells by CD8 + cytotoxic T cells is weakened. This project will deepen the understanding of the function and mechanism of MIIP in the development and progression of CRC, and provide a potential target and screening indicator for the treatment of CRC with CIN.

结直肠癌（CRC）的发生发展与免疫失衡有关，PD-1抑制剂可用于微卫星高度不稳定（MSI-H）的晚期CRC，但不适用于染色体不稳定性（CIN）的CRC。前一项NSFC项目证实MIIP缺失诱导CIN，并促进CRC进展；前期工作提示MIIP可能影响CIN型CRC的免疫监视。本项目拟通过体外实验、动物模型、临床病例分析，证实MIIP缺失在CRC细胞中降低其对HDAC去乙酰化活性的抑制，通过增加转录抑制子COUP-TFII与MHC I增强子的结合、降低转录活化子NF-κB与MHC I增强子的结合进而抑制MHC I的表达；同时通过抑制TAP1启动子组蛋白H3的乙酰化进而降低TAP1表达，抑制MHC I相关的抗原递呈，从而降低CD8+ T细胞对肿瘤细胞的识别，实现肿瘤免疫逃逸。本项目将进一步阐述MIIP在CRC中的作用机制网络，并为CIN型CRC提示新的潜在的治疗靶点和病例筛选指标。

本项目利用Co-IP技术证实MIIP在CRC细胞中与HADC6形成复合物并抑制其去乙酰化功能；利用ChIP实验证实HDAC6与TAP1启动子结合；通过体外CRC细胞稳定过表达或降表达MIIP证实MIIP的水平直接影响HDAC6的酶活性及TAP1和MHC I的水平；并且在MIIP稳定低表达的CRC细胞中同时降表达HDAC6能够对抗MIIP对TAP1、MHC I的影响；人CRC细胞系的裸鼠皮下移植瘤模型进一步证实CRC中存在MIIP-HDAC6-TAP1-MHC I作用轴。然后，我们证实人CRC细胞中的MIIP能够促进共培养的人CD8+ T细胞的增殖和活性；通过免疫健全C57BL/6小鼠MC38移植瘤和BALB/c小鼠CT26移植瘤模型证实MIIP通过HDAC6-TAP1-MHC I轴促进CRC中CD8+ T细胞浸润、抑制肿瘤生长。在C57BL/6和BALB/c小鼠CRC移植瘤模型中给予CD8抗体，进一步证实MIIP对CRC的抑制作用部分地通过MIIP-HDAC6-TAP1-MHC I-CD8+ T细胞的抗肿瘤免疫作用实现。我们在TCGA数据库CRC病例和本中心CRC组织中也证实了上述结论。至此，通过两项国自然的研究，我们提出MIIP在CRC中发挥抑癌基因的作用，MIIP单拷贝缺失/表达下调不仅导致CRC细胞染色体不稳定性、迁移侵袭能力增强，同时通过HDAC6-TAP1-MHC I-CD8+ T细胞抑制抗肿瘤免疫，为阐述CRC中肿瘤细胞与免疫微环境之间的相互作用提供一个新的研究方向。另外，通过在体外细胞和移植瘤模型中给予HDAC6抑制剂TubA，不仅印证了MIIP-HDAC6-TAP1-MHC I-CD8+ T细胞作用轴在CRC发展中作用，而且证实HDAC6抑制剂能够增强MIIP的抗肿瘤免疫作用，提供了HDAC6抑制剂治疗CRC的新机制。