ROR1激活AKT/FOXO1信号介导非小细胞肺癌吉非替尼耐药

ROR1-mediated Gefitinib Resistance of Non-small Cell Lung Cancer by Activating AKT/FOXO1

EGFR-TKI靶向治疗在非小细胞肺癌(non-small cell lung cancer,NSCLC)综合治疗中显示出重要作用;然而,耐药性却极大限制其临床治疗效果。受体酪氨酸激酶样孤儿受体(receptor tyrosine kinase-like orphan receptor 1,ROR1)是Ⅰ型受体酪氨酸激酶家族中的成员,在肿瘤发生发展中发挥重要作用。本研究拟探讨ROR1介导非小细胞肺癌吉非替尼耐药的作用及机制。采用吉非替尼反复诱导非小细胞肺癌HCC827细胞,建立吉非替尼耐药细胞株HCC827/GR。应用荧光定量PCR和Western印迹检测HCC827/GR内ROR1的表达。采用shRNA的方法体外检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化,采用体外检测ROR1过表达前后HCC827对吉非替尼耐药的变化。体内检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化。Western印迹检测HCC827/GR内ROR1下游信号分子的活化。实时荧光定量PCR及Western印迹结果显示,HCC827/GR耐药细胞中的ROR1 mRNA和蛋白质表达水平显著高于HCC827敏感细胞。体外干扰ROR1表达,可明显增强HCC827/GR耐药细胞对吉非替尼的敏感性(IC_(50) 15.3±3.69 vs.4.2±1.38),增加吉非替尼诱导的细胞凋亡(20.5±2.52 vs.41.8±3.74)。体外过表达ROR1显著增强HCC827敏感细胞对吉非替尼的耐药性(IC_(50) 0.8±0.52 vs.2.2±0.87)。体内裸鼠移植瘤实验同样发现,干扰ROR1能增强HCC827/GR移植瘤对吉非替尼的敏感性。进一步研究发现,AKT/FOXO1信号在HCC827/GR耐药细胞中异常活化,而干扰ROR1能够抑制AKT的磷酸化,并上调FOXO1的表达。上述结果表明,ROR1参与非小细胞肺癌吉非替尼耐药,抑制ROR1能够逆转吉非替尼耐药,其机制与ROR1调控AKT/FOXO1信号有关。

EGFR-TKI targeted therapy has been playing an important role in the treatment of non-small cell lung cancer（ NSCLC）. However,unavoidable therapeutic resistance significantly limits the clinical efficacy of TKI. Receptor tyrosine kinase-like orphan receptor 1（ ROR1） is a member of the type I receptor tyrosine kinase family and plays an important role in cancer development and progression. The aim of this work is to investigate the effect and molecular mechanism of ROR1 on gefitinib resistance in NSCLC. EGFR-TKI-resistant HCC827/GR cells were established from parental HCC827 cells by continuous exposure to gefitinib. ROR1 expression on the mRNA and protein levels was detected by qRT-PCR and Western blot respectively. Influence of ROR1 to gefitinib resistance in HCC827/GR cells was assayed by ROR1 shRNA in vitro. Influence of ROR1 to gefitinib resistance was assayed by ROR1 overexpression in HCC827 cells in vitro or by ROR1 shRNA in HCC827/GR cells in xenograft mouse model. ROR1 downstream signal molecules were detected by Western blot. We found that the mRNA and protein levels of ROR1 are significantly increased in HCC827/GR. Knockdown of ROR1 significantly increased gefitinib sensitivity of HCC827/GR cells and gefitinib-induced cell apoptosis in vitro.Overexpression of ROR1 significantly increased gefitinib resistance of HCC827 cells in vitro. Moreover,in vivo assays also showed that knockdown of ROR1 significantly increased gefitinib sensitivity（ P 〈0. 05）. Furthermore,we found that abnormal activation of AKT/FOXO1 signaling was detected in HCC827/GR cells,and knockdown of ROR1 significantly inhibited the phosphorylation of AKT,but increased the expression of FOXO1. Taken together,these results indicated that ROR1 could increase gefitinib resistance in HCC827 cells,by which mechanism is related to activation of AKT/FOXO1 signaling.