Background: Lung cancer is the malignant tumor with the highest incidence and mortality in China, among which non-small cell lung cancer (NSCLC) accounts for about 80%. Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapy has been playing an important role in treatment of NSCLC. However, unavoidable therapeutic resistance significantly limits the clinical efficacy of EGFR-TKI. As a key member of the forkhead box protein family, FOXC1 is aberrantly expressed in NSCLC and involved in NSCLC progression. The aim of this work is to investigate the effect and potential mechanism of FOXC1 on gefitinib resistance in NSCLC.
Methods: Western blot was performed to assess the expression of FOXC1 protein in HCC827/GR cells. Immunohistochemistry (IHC) assays were performed in human NSCLC tissues with gefitinib resistance. HCC827/GR cells were transfected with shRNA specifically targeting FOXC1 mRNA and stable cell lines were established. The effects of FOXC1 on cell viability and apoptosis were analyzed using a new methyl thiazolyl tetrazolium assay (MTS assay) and flow cytometry. Self-renewal ability was determined by mammosphere-formation analysis. Quantitative real-time PCR (qRT-PCR) and Western blot were employed to detect the expression of SOX2, Nanog, OCT4 and CD133. Flow cytometry analysis were further used to detect the level of CD133. IHC assays were used to detect the levels of SOX2 and CD133 in NSCLC tissues with genfitiinb resistance. Correlations of the expressions of FOXC1, CD133 and SOX2 with each other in lung adenocarcinoma samples were analyzed based on The Cancer Genome Atlas (TCGA) database.
Results: The expression of FOXC1 is significantly increased in HCC827/GR cells compared with HCC827 cells (P<0.05). IHC results showed FOXC1 was highly expressed in NSCLC tissues with gefitinib resisitance. Knockdown of FOXC1 significantly increased the sensitivity of HCC827/GR cells to gefitinib. The cell viability was decreased and the apoptosis was promoted (P<0.05). Moreover, FOXC1 knockdown apparently inhibited the expression of SOX2 and CD133, and decreased the mammosphere-formation capacity in HCC827/GR cells. In NSCLC tissues with gefitinib resistance, the expressions of SOX2 and CD133 were significantly higher compared with gefitinib-sensitive tissues (P<0.01). Meanwhile, the expressions of FOXC1, CD133 and SOX2 with each other were positively correlated (P<0.05).
Conclusions: FOXC1 could increase gefitinib resitance in NSCLC, by which mechanism is related to the regulation of cancer stem cell properties.
【中文题目：干扰FOXC1逆转非小细胞肺癌吉非替尼耐药的作用】 【中文摘要：背景与目的 肺癌是我国发病率和死亡率最高的恶性肿瘤，其中非小细胞肺癌（non-small cell lung cancer, NSCLC）约占肺癌的80%。表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）靶向治疗已成为NSCLC临床治疗的主要手段，然而不可避免的耐药性出现极大限制了EGFR-TKI的治疗效果。叉头框蛋白C1（forkhead box protein C1, FOXC1）是叉头框蛋白家族重要成员，在NSCLC异常表达并参与调控NSCLC进展。本研究旨在探讨干扰FOXC1对NSCLC吉非替尼耐药的影响及可能机制。方法 应用Western blot、免疫组化方法检测FOXC1在NSCLC吉非替尼耐药细胞和组织中的表达情况；应用FOXC1 shRNA转染HCC827/GR细胞，筛选稳定干扰FOXC1的HCC827/GR细胞，采用新型四氮唑盐比色法（Methyl-thiazolyldiphenyl-sulfophenyl-tetrazolium bromide assay, MTS）法、流式细胞术及微球体形成实验检测细胞增殖、细胞凋亡及细胞自我更新能力；采用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）、Western blot检测干性标志物SOX2、Nanog、OCT4和CD133的表达水平；运用流式细胞术检测CD133的表达情况；免疫组化检测耐药组织中SOX2和CD133的表达情况；基于癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库的肺腺癌数据集，分析FOXC1、SOX2和CD133表达的相关性。结果 FOXC1在NSCLC吉非替尼耐药细胞HCC827/GR的表达水平显著高于HCC827敏感细胞（P<0.05），免疫组化结果显示FOXC1在NSCLC吉非替尼耐药组织中高表达。与对照组相比，稳定干扰FOXC1的HCC827/GR细胞对吉非替尼的半数抑制浓度（50% inhibitory concentration, IC50）值显著降低（P<0.01），细胞增殖能力降低、凋亡率升高（P<0.05）。干扰FOXC1能够抑制SOX2、CD133的表达，并减弱HCC827/GR耐药细胞的微球体形成能力。免疫组化结果显示，吉非替尼耐药组织中SOX2和CD133表达显著高于敏感组织（P<0.01）。相关性分析结果显示，FOXC1、SOX2和CD133表达两两呈正相关（P<0.05）。结论 FOXC1参与NSCLC吉非替尼耐药，其机制可能与FOXC1调控肿瘤干细胞（cancer stem cells, CSCs）特性有关。】 【中文关键词：肺肿瘤；FOXC1；吉非替尼耐药；肿瘤干细胞】.
Keywords: Cancer stem cells; FOXC1; Gefitinib resistance; Lung neoplasms.