YAP promotes erlotinib resistance in human non-small cell lung cancer cells

Abstract

Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P<0.05), as did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05). Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration, invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.

Keywords: Hippo pathway; epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance; erlotinib; non-small cell lung cancer; yes-associated protein.

Figure 1. Forced overexpression of YAP in HCC827 promotes resistance to erlotinib in HCC827 cells

A. Western blotting showed that YAP protein expression increased in YAP plasmid-transfected HCC827 after erlotinib treatment. B. YAP mRNA expression increased more in HCC827 cells with YAP forced overexpression than in pcDNA 3.1 transfected HCC827 cells after erlotinib treatment and DMSO control treatment (***P < 0.001). C. The IC50 of erlotinib was 15.58μM for cells transfected with YAP plasmid, and 2.48 μM for cells transfected with pcDNA 3.1. D. After treatment with erlotinib, cell viability of YAP plasmid-transfected HCC827 cells increased compared to pcDNA3.1-transfected HCC827 cells (***P < 0.001).

Figure 2. Inhibition of YAP by siRNA enhanced the cytotoxicity of erlotinib to H1975 cells

A. Western blotting showed that YAP protein expression level decreased after YAP siRNA transfection and erlotinib treatment in H1975 cells. B. YAP mRNA expression significantly decreased in H1975 cells with YAP siRNA transfection after erlotinib treatment (***P < 0.001). C. The IC50 of erlotinib was 5.60μM for H1975 with YAP silencing by YAP siRNA, and 17.12μM for H1975 transfected by control siRNA. D. After treatment with erlotinib, cell viability of H1975 cells with YAP silencing by YAP siRNA decreased compared to control siRNA-transfected H1975 cells (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3. The YAP inhibitor verteporfin increased sensitivity of H1975 cells to erlotinib

A. Cell viability analysis in H1975 cells after verteporfin treatment. B. Cell viability analysis of H1975 cells after erlotinib combined with 1μM verteporfin versus erlotinib alone. C. Cell viability of H1975 cells significantly decreased after combination treatment (***P < 0.001). D. Western blotting shows YAP protein expression level decreased after combination treatment in H1975 cells.

Figure 4. Hippo reporter activity and downstream gene expression in HCC827 and H1975 cells

A. GTIIC reporter activity of the hippo pathway significantly decreased in HCC827 cells after erlotinib treatment and increased with YAP forced overexpression (***P < 0.001). B. GTIIC reporter activity significantly decreased in YAP siRNA-transfected H1975 cells after erlotinib treatment (***P < 0.001). C. GTIIC reporter activity significantly decreased in H1975 cells after verteporfin alone and after combined treatment with verteporfin and erlotinib (*P < 0.05, **P < 0.01). D. Hippo downstream gene expression of AREG and CTGF in HCC827 cells with YAP forced overexpression significantly increased even after erlotinib treatment (*P < 0.05, **P < 0.01, ***P < 0.001). E. Hippo downstream gene expression of AREG and CTGF in YAP siRNA-transfected H1975 cells significantly decreased after erlotinib treatment (***P < 0.001). F. Hippo downstream gene expression of AREG and CTGF significantly decreased after verteporfin treatment alone and after combined treatment with erlotinib and verteporfin treatment (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5. Analysis of cell migration, invasion and tumor sphere formation abilities after erlotinib alone, verteporfin alone and combination treatment with verteporfin and erlotinib in H1975 cells

(A) Decrease in cell migration ability in H1975 cells after combination treatment (B) Decrease in cell invasion ability in H1975 cells after combination treatment (C) Quantitative analysis of transwell invasion assay result, indicating combination treatment decreased cell invasion ability in H1975 cells (***P < 0.001). (D) Decrease in tumor sphere formation ability in H1975 cells after verteporfin treatment alone, and after combined treatment with verteporfin and erlotinib. (E) Quantitative analysis shows verteporfin treatment alone or combination treatment decreased tumorsphere formation ability in H1975 cells (*P < 0.05, **P < 0.01).

Figure 6. Western blot analysis of YAP protein expression after time-dependent treatment with erlotinib alone and combined treatment with verteporfin and erlotinib

ERBB3 mRNA expression after YAP forced overexpression, erlotinib treatment and combination treatment with verteporfin and erlotinib. A. YAP protein level in H1975 cells increased after 48 and 72 hours of erlotinib treatment. B. In HCC827 cells, YAP protein level decreased after 8 to 24 hours of erlotinib treatment and rebounded after 48 and 72 hours of treatment. C. YAP protein level decreased in H1975 cells after combined treatment for 8, 16, 24 and 48 hours and rebounded after 72 hours. D. In HCC827 cells, YAP protein level decreased after combined treatment for 8, 16, 24, and 48 hours and then rebounded after 72 hours. E. ERBB3 mRNA expression significantly increased in HCC827 cells with YAP forced overexpression (*P < 0.05). F. ERBB3 mRNA expression significantly increased after erlotinib 1μM treatment and decreased after combination treatment (**P < 0.01,*P < 0.05,). G. A schematic diagram of a hypothetical model of how YAP promotes erlotinib resistance. In HCC827 cells, activation of YAP forms an autocrine loop with the ERBB3 pathway to bypass the EGFR signaling pathway after continuous erlotinib treatment. In H1975 cells, inhibition of YAP by siRNA or the YAP inhibitor verteporfin enhances the sensitivity to erlotinib.