肺腺癌EGFR突变通过miRNA-33b及miRNA-4711调控PD-L1表达的分子机制研究

Epidermal growth factor receptor (EGFR) mutation is one of the most important oncogenic driver in lung adenocarcinoma. Programmed cell death 1 ligand 1 (PD-L1), which is known as an immune checkpoint, plays a critical role in mediating lung cancer immune escape. We found that the expression of PD-L1 in lung adenocarcinoma or lung cancer cells with EGFR mutation was up-regulated. In addition, transfecting the EGFR mutation into human cells can also up-regulate the expression of PD-L1. Based on gene chip analysis, bioinformatics analysis, and the in vitro verification, we found that the expression of miR-33b and miR-4711 was inhibited in cells with EGFR mutation, accompanied with overexpression of PD-L1. According to these preliminary data, we hypothesize that EGFR mutation may up-regulate PD-L1 expression through inhibiting the expression of miR-33b and miR-4711 in lung adenocarcinoma. In order to test this hypothesis, we plan to conduct both the in vitro and in vivo studies. For the in vitro study, lung cancer cell lines with EGFR mutation will be treated with the mimics of miR-33b, miR-4711, or EGFR siRNA to confirm the role of these two microRNAs in EGFR mutation mediated PD-L1 overexpression, as well as the target genes of these two microRNAs. Furthermore, the in vivo study will be carried out with a mouse model of primary lung adenocarcinoma. The lentivirus-mediated gene transfection of mutated EGFR cDNA will be done through intra-tracheal injection. The mice will be treated with mimics of miR-33b and miR-4711, or Gefitinib to study the relationship among the expression of miR-33b, miR-4711 and PD-L1 in lung adenocarcinoma with EGFR mutation. The findings from the in vitro and in vivo studies will be further verified in human lung adenocarcinomas from the patients. This project is expected to reveal the mechanisms involved in EGFR mutation mediated PD-L1 overexpression in lung adenocarcinoma, thus providing novel targets for lung cancer therapy.

EGFR突变是肺腺癌最重要的驱动基因之一，免疫检查点蛋白PD-L1则介导肺癌免疫逃逸。我们前期发现EGFR突变肺腺癌及相应细胞株高表达PD-L1，转染EGFR突变基因也上调PD-L1表达。结合基因芯片、生物信息学分析及体外验证，我们发现EGFR突变可抑制miR-33b及miR-4711的表达，miR-33b及miR-4711的拟合物则下调PD-L1的表达。我们据此推测肺腺癌EGFR突变可能通过抑制miR-33b及miR-4711上调PD-L1的表达。本项目围绕该假设，分别在不同EGFR突变肺癌细胞株、小鼠EGFR突变肺癌模型中通过上调miR-33b及miR-4711表达、或抑制EGFR，证实miR-33b及miR-4711在EGFR突变调控PD-L1表达中的作用，并确定其靶基因，进而在人肺腺癌组织标本中验证其临床意义，阐明肺腺癌EGFR突变调控PD-L1表达的分子机制，寻找肺癌治疗新靶点。

肺癌是我国发病率及死亡率最高的恶性肿瘤，临床上以腺癌最常见。EGFR突变是肺腺癌最重要的驱动基因之一，免疫检查点蛋白PD-L1介导肺癌免疫逃逸。针对EGFR突变的靶向治疗药物EGFR受体酪氨酸激酶抑制剂（EGFR-TKIs）是近年来肺癌治疗领域的重大进展，但耐药是其治疗失败的重要原因。本研究观察了多种EGFR突变肺癌细胞株，发现其PD-L1表达均高于EGFR野生型细胞株，且EGFR-TKI处理可降低PD-L1表达，其糖基化修饰的蛋白B3GNT3表达也下调，基于TCGA数据库分析提示B3GNT3高表达与肺癌预后不良相关；临床标本的研究显示PD-L1与EGFR突变无关，但B3GNT3阳性表达更常见于EGFR突变患者，而B3GNT3与PD-L1双阳性患者预后较差。对耐吉非替尼（PC-9GR）及耐奥西替尼（PC-9OR）的PC-9细胞进行转录组测序发现其PD-L1表达均较PC-9细胞明显增高，伴EGFR旁路激活。基因富集分析显示EGFR-TKI耐药后多条激活通路与AXL相关，且AXL高表达与EGFR相关。qPCR及Western Blot均证实PC-9OR细胞AXL及PD-L1表达增高，AXL高表达可增强PC-9细胞对奥西替尼的耐受性，沉默AXL可下调PC-9OR细胞PD-L1的表达，并提升其对奥西替尼的敏感性。观察EGFR-TKI治疗前后组织标本发现EGFR-TKI耐药肺癌AXL及PD-L1表达均显著增强。分别以PC-9、PC-9OR、沉默AXL的PC-9（PC-9OR-shRNA1/2）和过表达AXL的PC-9（PC-9OE）建立小鼠皮下移植瘤，给予奥西替尼-/+AXL抑制剂，发现抑制AXL可增强奥西替尼对耐药肿瘤的治疗作用，由此认为AXL可能通过上调PD-L1表达参与奥西替尼耐药的发生。综上，肺腺癌PD-L1表达与EGFR突变状态无关，但EGFR突变患者高表达PD-L1糖基化基因B3GNT3，与PD-L1阳性患者不良预后有关；AXL可能通过上调PD-L1表达参与EGFR-TKI耐药的发生，EGFR-TKI联合AXL抑制剂有望增强EGFR-TKI的疗效。