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

Abstract

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 3.61 μmol/L in the parent Skbr3 cells, and was increased by 8 times in the drug-resistant Skbr3-LR cells (29.26 μmol/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 2.98 μmol/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.

Keywords

• lapatinib
• breast cancer
• hippo
• yes-associated protein