司美替尼下调KRAS G12V突变型非小细胞肺癌细胞PD-L1水平的探索性研究  

Exploratory study on downregulation of PD-L1 in KRAS G12V-mutant non-small cell lung cancer cells by selumetinib

作　　者：马韵芳 潘丽娜[1] 李圳 高蓓莉[2] 胡家安[1] 徐志红[1] MA Yun-fang;PAN Li-na;LI Zhen;GAO Bei-li;HU Jia-an;XU Zhi-hong(Department of Geriatrics,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of Respiratory and Critical Care,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)

机构地区：[1]上海交通大学医学院附属瑞金医院老年病科,上海200025 [2]上海交通大学医学院附属瑞金医院呼吸与危重症科,上海200025

出　　处：《上海交通大学学报（医学版）》2021年第6期741-748,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市卫生和计划生育委员会科研课题(201840083);上海市卫生和计划生育委员会重要薄弱学科建设计划(2015ZB0503);上海市高校产学研课题(RC20190079)。

摘　　要：目的·探究不同Kirsten大鼠肉瘤病毒癌基因(Kirsten rat sarcoma viral oncogene,KRAS)突变型非小细胞肺癌(non-small cell lung cancer,NSCLC)患者肿瘤组织及细胞系与程序性死亡蛋白配体1(programmed cell death ligand 1,PD-L1)表达的关系及其可能的调控机制。方法·通过实时荧光定量PCR和荧光活化细胞分选法,在NSCLC细胞系中验证PD-L1 mRNA和蛋白的表达与KRAS突变状态的关系。通过免疫组织化学法检测77例早期(Ⅰa~Ⅱb期)NSCLC患者肿瘤组织中PD-L1的表达情况。通过Western blotting研究KRAS突变对RAS下游信号通路分子蛋白激酶B(protein kinase B,PKB,又称为AKT)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、核糖体S6蛋白激酶(ribosomal S6 protein kinase,p70S6K)、细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)的影响。用RAF抑制剂达拉非尼、有丝分裂原活化蛋白激酶(mitogen activated-protein kinase,MEK)抑制剂司美替尼、磷脂酰肌醇3-激酶抑制剂GDC0941、AKT抑制剂MK2206和mTOR抑制剂雷帕霉素分别处理6个KRAS突变NSCLC细胞株,检测这5种酪氨酸激酶抑制剂对PD-L1表达的调控作用。结果·在NSCLC细胞系中,KRAS突变细胞系的PD-L1 mRNA和蛋白的表达水平均显著高于KRAS野生型细胞系(均P<0.05),且KRAS G12V细胞系PD-L1的mRNA表达水平最高,KRAS G12C细胞系PD-L1的蛋白表达水平最高。对于早期NSCLC患者,PD-L1在KRAS G12V和G12D突变型患者肿瘤组织中高表达的比例显著高于KRAS野生型。Western blotting结果显示,KRAS突变NSCLC细胞系中,p70S6K被激活,而ERK、AKT、mTOR未被激活。进一步研究发现,1μmol/L GDC0941、0.5μmol/L MK2206和10 nmol/L雷帕霉素均未对6个KRAS突变NSCLC细胞株的PD-L1表达产生影响,5 nmol/L达拉非尼仅在1个KRAS G12V突变株和1个KRAS L19F突变株引起PD-L1表达上调(均P<0.05),而0.1μmol/L司美替尼在3个KRAS G12V突变株和1个KRAS L19F突变株引起PD-L1表达下调(均P<0.0Objective·To investigate the correlation between Kirsten rat sarcoma viral oncogene(KRAS)-mutant subtypes and programmed cell death ligand 1(PD-L1)expression in the non-small cell lung cancer(NSCLC)tissues and cell lines,and the possible underlying mechanism.Methods·Realtime quantitative PCR and fluorescence-activated cell sorting were used to verify the correlation between PD-L1 mRNA or protein and KRAS mutation status in the NSCLC cell lines.Immunohistochemistry was used to detect the expression of PD-L1 in the tumor tissues of 77 NSCLC patients with early stages(Ⅰa-Ⅱb).The effect of KRAS mutation on the cytokines in the RAS downstream signaling pathway,i.e.,protein kinase B(PKB or AKT),mammalian target of rapamycin(mTOR),ribosomal S6 protein kinase(p70 S6 K),and extracellular regulated protein kinase(ERK),was studied by Western blotting.Dabrafenib(RAF inhibitor),selumetinib[mitogen activated-protein kinase(MEK)inhibitor],GDC0941(phosphoinositide 3-kinase inhibitor),MK2206(AKT inhibitor),and rapamycin(mTOR inhibitor)were used to treat 6 KRAS-mutant cell lines,respectively,to detect the regulation of these five tyrosine kinase inhibitors on PD-L1 expression.Results·In the NSCLC cell lines,the expressions of PD-L1 mRNA and protein in the KRAS mutant cell lines were significantly higher than those in the KARS wild-type cell lines(P<0.05).KRAS G12 V-and G12 C-mutant cell lines showed the highest expression of PD-L1 mRNA and protein,respectively.For the NSCLC patients with early stages,the proportions of PD-L1 overexpression in the tumor tissues of the patients with KRAS G12 V and G12 D mutants were significantly higher than those of the patients with KRAS wild-type.Western blotting showed that in the KRAS-mutated NSCLC cell lines,p70 S6 K was activated,while ERK,AKT and mTOR were not activated.Moreover,1μmol/L GDC0941,0.5μmol/L MK2206,and 10 nmol/L rapamycin could not affect the expression of PD-L1 in the 6 KRAS-mutant cell lines.Darafenib(5 nmol/L)up-regulated PD-L1 in only one KRAS G12 V-mutant cell line

关 键 词：KRAS突变 非小细胞肺癌 免疫治疗 程序性死亡蛋白配体1 司美替尼 

分 类 号：R734.2[医药卫生—肿瘤]