ERBB3-V104L通过上调PD-L1表达促进胆囊癌免疫逃逸的作用及其调控机制研究

The treatment targeting PD-1/PD-L1 showes obvious curative effect in a variety of carcinoma, nevertheless, PD-L1 expression and regulatory mechanism in gallbladder carcinoma is still elusive. Our previous study analyzed exome sequence from GBC tumors and identified that non-silent ERBB3 mutations are frequent events in gallbladder cancer patients. Additionally, we found that V104L mutation is the hottest spot among these non-silent mutations. Furthermore, the mutant of ERBB3-V104L can significantly promote the ability of GBC cells to proliferate and metastasis. Most importantly，V104L mutant could upregulate the PD-L1 expression level in GBC cells. These indicate ERBB3-V104L mutant play a key role in immune escape in the process of GBC progression. Thus we hypothesize that ERBB3-V104L mutant upregulates PD-L1 expression in GBC cells and promotes T cell apoptosis to inhibit CD4, CD8 T cell activation and proliferation, which negatively regulate the immune response and result in immune escape of GBC. This study aims to clarify the role of ERBB3-V104L in the immune escape of GBC and to explore the regulatory mechanism of PD-L1 expression using various systems including molecular, cellular, tissue and animal models.. This study will also provide theoretical basis for the potential using of ERBB3-V104L and PD-L1 as a new target for gallbladder cancer treatment which combining immunotherapy and targeted therapy and will shed light on the future treatment of gallbladder cancer.

靶向PD-1/PD-L1免疫检查点治疗在多种肿瘤中显示出明显的疗效，但是其介导的免疫逃逸在胆囊癌中的作用及调控机制尚属空白。我们前期发现ERBB3在胆囊癌病人中发生高频突变，其中V104L是突变的热点区域；ERBB3-V104L可促进胆囊癌的增殖能力，并可上调PD-L1的表达，提示ERBB3-V104L在胆囊癌发生发展中发挥重要功能，并与胆囊癌免疫逃逸相关。据此我们提出假说，ERBB3-V104L通过上调PD-L1的表达，促进微环境中杀伤性T细胞耗竭，从而负性调控机体的免疫应答，导致胆囊癌免疫逃逸的发生。本课题拟从分子、细胞、组织以及动物水平等多层次揭示ERBB3-V104L通过上调PD-L1影响胆囊癌T细胞免疫逃逸的分子机制；进一步研究联合靶向ERBB3及PD-L1对胆囊癌的治疗价值。本课题将为胆囊癌发生、发展及免疫逃逸机制提供新的研究基础，为胆囊癌靶向治疗联合免疫治疗提供理论依据。

胆囊癌是胆道系统最常见的恶性肿瘤，中国属高发地区，其恶性程度高，5年总体生存率仅为5％，预后极差。靶向PD-1/PD-L1免疫检查点治疗在多种肿瘤中显示出明显的疗效，但是其介导的免疫逃逸在胆囊癌中的作用及调控机制尚属空白。我们前期发现ERBB3在胆囊癌病人中发生高频突变，其中V104L是突变的热点区域；ERBB3-V104L可促进胆囊癌的增殖能力，并可上调PD-L1的表达，提示ERBB3-V104L在胆囊癌发生发展中发挥重要功能，并与胆囊癌免疫逃逸相关。在此基础上，本课题从分子、细胞、组织以及动物水平等多层次揭示ERBB3-V104L通过上调PD-L1影响胆囊癌T细胞免疫逃逸的分子机制；进一步研究联合靶向ERBB3及PD-L1对胆囊癌的治疗价值。首先，我们扩大样本量（157对）通过全基因组外显子检测验证了前期结果，发现ERBB3在胆囊癌中发生高频突变（8%），V104L位点（5/12）仍是ERBB3突变的热点区域。进一步点突变过表达ERBB3-V104L后，结果显示ERBB3-V104L明显促进GBC-SD 细胞的增殖及转移能力。随后我们对ERBB3-V104L突变型和ERBB3野生型细胞进行芯片检测，结果显示突变株PD-L1表达明显上调，并通过流式细胞技术、蛋白印迹实验等证实。我们进一步通过T细胞细胞毒试验明确PBMCs明显抑制ERBB3-WT胆囊癌细胞株的增殖并促进了肿瘤细胞的凋亡，然而突变组T细胞活性受到明显的抑制，在组织样本中也有类似的结果，这些结果进一步明确了ERBB3-V104L通过上调PD-L1促进胆囊癌的免疫逃逸。我们继续通过体外、体内实验发现Sapitinib（ERBB阻断剂）及Atezolizumab（抗PD-L1抗体）具有明显抑制肿瘤增殖的效果，联合治疗效果更为明显。最后，我们对ERBB3-V104L通过上调PD-L1影响胆囊癌T细胞免疫逃逸的分子机制进行探讨，结果显示ERBB3-V104L能够通过对HMGB1-HMGB2-PDL1轴中HMGB1进行剥夺，解除HMGB1对HMGB2介导的PD-L1蛋白质再折叠及胞膜呈递过程的抑制作用，通过增加中间丝至胞膜重排及PD-L1胞内非降解作用再循环，从而促进PD-L1介导的胆囊癌T细胞免疫逃逸。综上所述，本课题为胆囊癌发生、发展及免疫逃逸机制提供新的研究基础，为胆囊癌靶向治疗联合免疫治疗提供理论依据。