ARAF突变在肺癌细胞RAF抑制剂耐药中的作用  

Role of ARAF mutation in RAF inhibitor resistance in lung cancer cells

作　　者：王耀元 陈娟 侯玮 Wang Yaoyuan;Chen Juan;Hou Wei(Department of Respiratory,No.215 Hospital of Shaanxi Nuclear Industry,Xianyang 712000,China)

机构地区：[1]陕西省核工业二一五医院呼吸科,咸阳712000

出　　处：《肿瘤研究与临床》2024年第10期728-733,共6页Cancer Research and Clinic

摘　　要：目的探讨ARAF突变在肺癌细胞RAF抑制剂耐药中的作用。方法通过CRISPR-Cas9方法构建敲除ARAF基因的A549和NCI-H1688肺癌细胞株。通过慢病毒包装感染方法,在敲除ARAF基因的肺癌细胞中过表达野生型ARAF(ARAF WT)和突变型ARAF(ARAF V145L、S214F、S214C或D429A),对照组为未经过处理的敲除ARAF基因的A549或NCI-H1688细胞。使用不同浓度RAF抑制剂belvarafenib或AZ-628处理以上各组A549或NCI-H1688细胞。CCK-8法检测细胞活力,计算各组细胞对belvarafenib或AZ-628的半数抑制浓度(IC_(50))。以ARAF WT组A549细胞IC_(50)(263 nmol/L)belvarafenib处理各组A549或NCI-H1688细胞,蛋白质印迹法检测ARAF及MEK/ERK/RSK信号通路中关键蛋白表达水平。选择6~8周龄雄性BALB/c裸鼠,腋窝皮下注射稳定过表达ARAF WT或突变型ARAF的敲除ARAF基因的A549或NCI-H1688细胞悬液,构建异种移植瘤模型,以每天30 mg/kg belvarafenib或AZ-628溶液连续灌胃,比较第16天各组肿瘤体积。结果与ARAF WT组比较,ARAF D429A组A549或NCI-H1688细胞belvarafenib或AZ-628的IC_(50)均低,ARAF S214F和ARAF S214C组IC_(50)均高,差异均有统计学意义(均P<0.05)。与ARAF WT组比较,经belvarafenib处理的ARAF WT组、ARAF D429A组A549细胞或NCI-H1688细胞p-MEK、p-ERK、p-RSK的相对表达水平均低,差异均有统计学意义(均P<0.05),ARAF、MEK、ERK、RSK的相对表达水平差异均无统计学意义(均P>0.05);与ARAF WT组比较,经belvarafenib处理的ARAF S214F组、ARAF S214C组p-MEK、p-ERK、p-RSK相对表达水平均高,差异均有统计学意义(均P<0.05),ARAF、MEK、ERK、RSK的相对表达水平差异均无统计学意义(均P>0.05)。ARAF WT组、ARAF D429A组A549细胞或NCI-H1688细胞移植瘤裸鼠经belvarafenib或AZ-628灌胃后,第16天肿瘤体积均低于对应的未经belvarafenib或AZ-628干预的ARAF WT组、ARAF D429A组细胞移植瘤裸鼠,且经belvarafenib或AZ-628灌胃的ARAF D429A组A549细胞或NCI-H1688细胞移植瘤裸鼠第16天肿瘤体积均Objective To explore the role of ARAF mutation in RAF inhibitor resistance in lung cancer cells.Methods The lung cancer cell lines A549 and NCI-H1688 with ARAF gene knockout were constructed using CRISPR-Cas9 method.Overexpression of wild-type ARAF(ARAF WT)and mutant ARAF(ARAF V145L,S214F,S214C,or D429A)in lung cancer cells with ARAF gene knockout was achieved through lentiviral packaging infection method,while the control group consisted of untreated A549 or NCI-H1688 cells with ARAF gene knockout.A549 or NCI-H1688 cells in each group were treated with different concentrations of RAF inhibitor belvarafenib or AZ-628.The CCK-8 method was used to detect cell viability,and the half maximal inhibitory concentration(IC_(50))of cells in each group against belvarafenib or AZ-628 was calculated.A549 or NCI-H1688 cells in each group were treated with IC_(50)(263 nmol/L)of belvarafenib in the ARAF WT group,and the expression levels of key proteins in the ARAF and MEK/ERK/RSK signaling pathways were detected by Western blotting.The 6-8 week old male BALB/c nude mice were selected and subcutaneously injected stable overexpression of ARAF WT or mutant ARAF A549 or NCI-H1688 cell suspension with ARAF gene knockout into the axilla to construct a xenograft tumor model,and 30 mg/kg belvarafenib or AZ-628 solution was administered continuously by gavage per day;tumor volume was compared among the groups at the 16th day.Results Compared with the ARAF WT group,the IC_(50) of belvarafenib or AZ-628 in A549 or NCI-H1688 cells of the ARAF D429A group was lower,while the IC_(50) of the ARAF S214F and ARAF S214C groups was higher,and the differences were statistically significant(all P<0.05).Compared with the ARAF WT group,the relative expression levels of p-MEK,p-ERK and p-RSK in A549 or NCI-H1688 cells treated with belvarafenib in the ARAF WT and ARAF D429A groups were all lower,and the differences were statistically significant(all P<0.05),but there were no statistically significant differences in the relative expression levels of AR

关 键 词：肺肿瘤 原癌基因蛋白质A-raf 突变 抗药性 肿瘤 RAF抑制剂 

分 类 号：R734.2[医药卫生—肿瘤]