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

doi:10.3969/j.issn.1674-8115.2021.06.006

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (6): 741-748.doi: 10.3969/j.issn.1674-8115.2021.06.006

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

马韵芳¹(), 潘丽娜¹, 李圳¹, 高蓓莉², 胡家安¹(), 徐志红¹()

^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

出版日期:2021-06-28 发布日期:2021-06-29
通讯作者: 胡家安,徐志红 E-mail:mayunfang@126.com;jahu_rj@aliyun.com;zhihxu@163.com
作者简介:马韵芳（1987—），女，硕士生；电子信箱：mayunfang@126.com。
基金资助:
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)

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

Yun-fang MA¹(), Li-na PAN¹, Zhen LI¹, Bei-li GAO², Jia-an HU¹(), Zhi-hong XU¹()

^1.Department of Geriatrics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Department of Respiratory and Critical Care, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Online:2021-06-28 Published:2021-06-29
Contact: Jia-an HU,Zhi-hong XU E-mail:mayunfang@126.com;jahu_rj@aliyun.com;zhihxu@163.com
Supported by:
Scientific Research Project of Shanghai Municipal Commission of Health and Family Planning(201840083);Weak Discipline Construction Project of Shanghai Municipal Commission of Health and Family Planning(2015ZB0503);Production, Study and Research Project of Shanghai Colleges and Universities(RC20190079)

摘要/Abstract

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，1 μmol/L GDC0941、0.5 μmol/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0.1 μmol/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<0.05），且呈一定剂量依赖性。结论·KRAS突变，尤其是G12V突变型的NSCLC细胞或组织的PD-L1表达水平明显高于KRAS野生型；司美替尼可下调KRAS G12V突变型NSCLC细胞的PD-L1水平，推测KRAS G12V突变的NSCLC细胞可能通过上调MEK上调PD-L1的表达。

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

Abstract:

Objective·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·Real-time 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 (p70S6K), 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 G12V- and G12C-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 G12V and G12D mutants were significantly higher than those of the patients with KRAS wild-type. Western blotting showed that in the KRAS-mutated NSCLC cell lines, p70S6K was activated, while ERK, AKT and mTOR were not activated. Moreover, 1 μmol/L GDC0941, 0.5 μmol/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 G12V-mutant cell line and one L19F-mutant cell line (P<0.05), while selumetinib (0.1 μmol/L ) inhibited the expressions of PD-L1 in three KRAS G12V-mutant cell lines and one L19F-mutant cell line (P<0.05) in a dose-dependent manner.

Conclusion·The expressions of PD-L1 in the tumor tissues and cell lines of NSCLC with KRAS mutation, especially G12V mutation subtype, were higher than those in the NSCLC tissues and cell lines with KRAS wild-type. Selumetinib can downregulate the expression of PD-L1 in KRAS G12V-mutant NSCLC cells, which suggests that KRAS G12V-mutant NSCLC cells may up-regulate the expression of PD-L1 by upregulating MEK.

Key words: KRAS mutation, non-small cell lung cancer (NSCLC), immunotherapy, programmed cell death ligand 1 (PD-L1), selumetinib

中图分类号:

R734.2

马韵芳, 潘丽娜, 李圳, 高蓓莉, 胡家安, 徐志红. 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[J]. 上海交通大学学报(医学版), 2021, 41(6): 741-748.

Yun-fang MA, Li-na PAN, Zhen LI, Bei-li GAO, Jia-an HU, Zhi-hong XU. Exploratory study on downregulation of PD-L1 in KRAS G12V-mutant non-small cell lung cancer cells by selumetinib[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(6): 741-748.

图/表 7

图1 NSCLC细胞株PD-L1 mRNA表达与KRAS、EGFR、TP53突变状态及肺癌组织学类型间的关系Note:A. KRAS mutation status. B. EGFR mutation status. C. TP53 mutation status. D. Different histological types. Mut—mutation; ADC—adenocarcinoma; SCC—squamous-cell carcinoma. ①P=0.003, ②P=0.000, compared with KRAS WT.

Fig 1 Correlations between PD-L1 mRNA expression and KRAS, EGFR, TP53 mutation status and histological types in the NSCLC cell lines

图2 FACS检测PD-L1在NSCLC细胞系中的表达情况Note:A. Expressions of PD-L1 in the representative NSCLC cell lines detected by FACS. NCIH520 and RERFLCAI cell lines are KRAS WT cell lines; NCIH358, IALM and NCIH1373 cell lines are KRAS-mutant cell lines. Blank—incubated with the FACS buffer; Iso—incubated with PD-L1 homologous antibody in the FACS buffer; PD-L1—incubated with PD-L1 antibody in the FACS buffer. B. Expressions of PD-L1 in 20 NSCLC cell lines detected by FACS. C. Comparison of PD-L1 expression between the KRAS-mutant cell lines and WT cell line. ①P=0.000, compared with WT.

Fig 2 Expression of PD-L1 in the NSCLC cell lines detected by FACS

图3 早期NSCLC患者肿瘤组织免疫组化染色

Fig 3 Immunohistochemical staining of tumor tissues in the early NSCLC patientsNote:The TPS in the KRAS WT, G12D and G12V patients were 16.2%, 88.2% and 73.7%, respectively.

表1 不同KRAS状态的早期NSCLC患者肿瘤组织PD-L1表达情况

Tab 1 Expression of PD-L1 in the tumor tissues of early NSCLC patients with different KRAS states

KRAS status	Total/n	TPS<1%/n(%)	1%≤TPS<50%/n(%)	TPS≥50%/n(%)	χ²(P value)^①
WT	10	0 （0）	9 （90）	1 （10）	-
G12A	15	7 （47）	8 （53）	0 （0）	66.57 (0.000)
G12C	12	7 （58）	5 （42）	0 （0）	85.45 (0.000)
G12D	21	5 （24）	10 （48）	6 （28）	45.31 (0.000)
G12V	19	0 （0）	9 （47）	10 （53）	42.85 (0.000)

图4 Western blotting检测RAS下游信号分子的表达Note:A. Lane 1-14 is CaLu3, HARA, KNS62, LC1sq, NCIH460, NCIH2347, CORL23, NCIH441, SW900, RERFLCAD2, IALM, NCIH1373, NCIH358, and RERFLCAD1, respectively. B. Semi-quantitative results of the proteins′ expression in the KRAS WT and mutant cell lines. ①P=0.031, compared with KRAS WT.

Fig 4 Expression of RAS downstream signaling molecules by Western blotting

图5 FACS检测LY2584702对PD-L1表达的影响Note:Each cell line was treated with LY2584702 at different concentrations for 24 h. ①P=0.039, ②P=0.001, compared with the control group. LY—LY2584702.

Fig 5 Influence of LY2584702 on the expression of PD-L1 by FACS

图6 FACS检测TKIs对KRAS突变NSCLC细胞株PD-L1表达的调控Note:A. Changes of PD-L1 expression after GDC0941 (PI3K inhibitor), MK2206 (AKT inhibitor), and rapamycin (mTOR inhibitor) treatment for 24 h. B. Changes of PD-L1 expression after treatment with different concentrations of dabrafenib or selumetinib for 24 h. ①P=0.010, ②P=0.006, ③P=0.033, ④P=0.008, ⑤P=0.001, ⑥P=0.014, ⑦P=0.020, ⑧P=0.025, ⑨P=0.002, ⑩P=0.003, compared with the corresponding control group.

Fig 6 Regulation of TKIs on the expression of PD-L1 in KRAS-mutant NSCLC cell lines by FACS

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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

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