BIG1介导STING囊泡转运在抗肺癌免疫反应中的作用及分子机制

The exit of stimulator of interferon genes (STING) vesicles from Golgi plays an essential role in the regulation of STING-dependent downstream signaling activity, however, the molecular entities and key mechanisms involved in controlling STING vesicles exiting from Golgi still remain poorly understood. Our latest work suggests that Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is likely to regulate the exit of STING vesicles from Golgi by mediating ARF3 activity, thereby affecting the degree of activation of STING-dependent downstream signaling, and playing an important role in anti-lung cancer immune response (Scientific Hypothesis). In this project, we will verify the above scientific hypothesis by collecting and analyzing human lung cancer specimens, and taking advantage of myeloid cell lineage BIG1 conditional knockout mice to study the effects of BIG1 deficiency on anti-lung cancer immune response and anti-lung cancer immunotherapy of STING agonist, and taking advantage of mouse primary bone marrow-derived dendritic cells to study the effects of BIG1 or ARF3 deficiency on STING levels, STING-dependent downstream signaling , STING intracellular distribution and trafficking. The implementation of this project will provide an experimental basis for targeting and regulating BIG1, a key factor in mediating the exit of STING vesicles from Golgi, to enhance STING-dependent downstream signaling and anti-lung cancer immune response.

高尔基体上干扰素基因刺激蛋白(STING)囊泡的排出在STING下游信号通路活性调控中发挥重要作用，然而参与控制STING囊泡从高尔基体上排出的分子实体及关键机制至今仍是未知的。我们前期研究工作提示布雷菲德菌A抑制型鸟苷酸交换蛋白1（BIG1）很可能通过介导ARF3活化调控高尔基体上STING囊泡排出，从而影响STING下游信号激活程度，并在抗肺癌免疫反应及治疗中发挥重要作用（科学假说）。本项目拟对肺癌患者的临床标本进行收集与分析，并利用髓系细胞BIG1条件敲除小鼠探索BIG1缺失对肺癌免疫反应及STING激动剂抗肺癌免疫治疗效果的影响，以及利用小鼠原代树突状细胞探索BIG1或ARF3缺失对STING水平、下游信号活性、胞内分布及转运等方面的影响对上述假说进行验证。本项目的实施将为靶向调控高尔基体上STING囊泡排出的关键因子BIG1来增强STING信号从而增强抗肺癌免疫反应提供实验依据。

Brefeldin A 抑制型鸟苷酸交换蛋白1（Brefeldin A-inhibited guanine nucleotide-exchange protein 1, BIG1）是ARF（ADP-ribosylation factor）鸟苷酸交换因子家族成员，其主要分布于反面高尔基体网状结构上，在蛋白质修饰、分拣与包装，然后分门别类地运送到细胞特定的部位或分泌到细胞外等过程中发挥重要作用，但是关于BIG1的抗肿瘤免疫作用还未有报道。本项目通过构建髓系细胞BIG1条件敲除小鼠来探索BIG1缺失对肿瘤免疫反应及STING激动剂抗肿瘤免疫治疗效果的影响，以及利用小鼠骨髓来源巨噬细胞（BMDMs）探索BIG1缺失对STING蛋白水平以及下游信号通路活性等方面的影响。研究结果显示：（1）髓系细胞BIG1敲除可以显著增强STING激动剂抑制小鼠皮下结直肠癌移植瘤的增殖以及黑色素瘤肺转移灶的形成，同时可以显著延长肺转移瘤小鼠的生存期。流式数据显示髓系细胞BIG1敲除可以显著诱导肿瘤组织相关巨噬细胞向M1型极化，同时可以显著增强STING激动剂诱导的巨噬细胞向M1型极化。（2）在小鼠骨髓来源巨噬细胞（BMDMs）中，BIG1缺失可显著抑制STING激动剂引起的STING蛋白下调，但是STING的mRNA水平没有显著差异；并且BIG1缺失进一步激活了STING下游关键蛋白TBK1和IRF3，以及进一步促进了IFN-β、CXCL10、IL-6的表达。（3）免疫共沉淀结果显示，BIG1和STING的N端跨膜区域相互作用，STING激活后BIG1与STING在核周部位存在显著的共定位现象。本项目的实施不仅可以揭示BIG1缺失在调控小鼠皮下移植瘤及转移瘤发生发展中的表型和机制；更重要的是，通过靶向调控STING通路活性的关键因子BIG1深入探索其在抗肿瘤免疫治疗中的潜力，为肿瘤免疫治疗提供新靶点。