YAP蛋白在HER2阳性乳腺癌拉帕替尼耐药中的作用

Role of yes-associated protein in resistance of HER2^+ breast cancer to lapatinib

目的建立乳腺癌拉帕替尼耐药细胞株,探讨Hippo通路中YAP蛋白在HER2阳性乳腺癌细胞拉帕替尼耐药中的作用及机制。方法采用逐步增加剂量与高浓度反复间歇诱导乳腺癌细胞株Skbr3对拉帕替尼耐药,耐药株命名为Skbr3-LR。用拉帕替尼同时处理亲本细胞和耐药细胞株,CCK-8法检测其耐药指数;结晶紫染色和平板克隆形成实验检测细胞的存活状况;定量PCR检测上皮-间质化改变。定量PCR及Western blot检测YAP、TAZ的表达。以维替泊芬阻断耐药细胞株Skbr3-LR中YAP下游通路的激活,CCK-8法检测其细胞活性的改变并计算IC50值的变化;结晶紫染色检测细胞存活数量的变化。定量PCR及Western blot检测YAP表达的改变。结果与亲本细胞比较,结晶紫染色和平板克隆形成实验结果均显示,耐药株存活细胞数量明显增多。亲本细胞耐药指数为3.61μmol/L,耐药株耐药指数上升约8倍,为29.26μmol/L。PCR结果显示,与亲本细胞比较,耐药细胞株的上皮标志物E-cadherin表达降低,而间质标志物Twist、Vinemtin和N-cadherin表达上升,YAP/TAZ表达上升,差异均有统计学意义(P<0.05)。Western blot结果显示,耐药细胞株中总YAP和磷酸化YAP表达均较亲本细胞显著上升(P<0.05)。以维替泊芬阻断耐药细胞株YAP下游通路的激活后,其耐药指数为2.98μmol/L,较未处理的Skbr3-LR耐药指数下降约4倍;其存活细胞数量明显降低,YAP表达也明显降低(P<0.05)。结论成功建立了拉帕替尼获得性耐药的乳腺癌细胞模型,其耐药机制可能与Hippo信号通路下游的关键效应因子YAP表达异常有关。

Objective To construct a lapatinib-resistant breast cancer cell line,and to study the role and underlying mechanism of yes-associated protein(YAP)in Hippo signaling pathway in the process.Methods The drug-resistant cell model of human breast cancer was established by gradually increasing the dose of drug and repeated intermittent high concentration inducing.After lapatinib treatment,the parent cell line Skbr3 and the drug-resistant cell line Skbr3-LR were detected by CCK-8 assay for drug resistance index,crystal violet staining and plate colony for cell survival,and quantitative PCR for the expression of epithelial mesenchymal transformation(EMT)markers.The expression of YAP/TAZ at mRNA and protein levels were measured by quantitative PCR and Western blotting,respectively.After verteporfin was used to block the downstream pathway of YAP in Skbr3-LR cells,CCK-8 assay was employed to measure the cell viability to obtain IC50,cell crystal violet staining was performed to count the number of survival cells,and the change of YAP expression was be detected by quantitative PCR and Western blotting.Results The results of crystal violet staining and plate colony showed that there were more survival cells in the Skbr3-LR cells when compared with the parent Skbr3 cells.The drug resistance index was 3.61μmol/L in the parent Skbr3 cells,and was increased by 8 times in the drug-resistant Skbr3-LR cells(29.26μmol/L).Compared with the parent cells,the expression level of the marker of epithelial cells,E-cadherin was decreased,while those of mesenchymal markers,Twist,Vinemtin,N-cadherin,and of YAP/TAZ were increased in the drug resistant cells(all P<0.05).And Western blot assay also indicated the enhanced expression of YAP and p-YAP proteins(P<0.05).After the Skbr3-LR cells were treated with verteporfin,the IC50 to Lapatinib was decreased from 11.25 to 2.98μmol/L,by 4 times,and there were less survival cells and the expression of YAP was decreased(P<0.05).Conclusion The lapatinib-resistant model of breast cancer Skbr3-LR cells is successfully established.And the mechanism of cell resistant to lapatinib may be associated with the abnormal expression of YAP protein.