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 PDF全文下载地址:
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