Wnt/β-Catenin Pathway-Mediated PD-L1 Overexpression Facilitates the Resistance of Non-Small Cell Lung Cancer Cells to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

Abstract

Background: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is considered highly effective treatment for advanced non-small cell lung cancer (NSCLC), who often develop drug resistance after 10 months of treatment. Herein, the aim was to unravel the mechanism behind the resistance to icotinib in NSCLC.

Methods: Establishment of icotinib-resistant PC-9 cells (PC-9R) was achieved through repeated exposure to increasing concentrations of icotinib for more than 12 months. PC-9R cells were transfected with programmed cell death ligand 1 (PD-L1) knockdown plasmid (PD-L1-KD)/overexpression plasmid (PD-L1-OE), and treated with Wnt pathway agonist CHIR99021 or β-catenin antagonist ICG-001. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium assay was employed for detecting cell sensitivity to icotinib. The invasion and migration abilities of the cells were evaluated using Transwell and scratch assays. Quantification of PD-L1, matrix metalloproteinase (MMP)-2, MMP-9 and Wnt/β-catenin pathway-related proteins was conducted by means of quantitative real-time polymerase chain reaction or Western blotting.

Results: Half-maximal inhibitory concentrations (IC₅₀) of PC-9 and PC-9R cells to icotinib were 1.73 μM and 25.18 μM, respectively. The expression of PD-L1, Wnt family member 1 (Wnt1) and β-catenin was higher in PC-9R cells than in PC-9 cells (p < 0.05). The transfection of PD-L1-OE resulted in elevated IC₅₀, migration, invasion, and MMP-2 and MMP-9 expression in PC-9R cells (p < 0.05), while transfection with PD-L1-KD had the opposite effect (p < 0.05). The expression of PD-L1, β-catenin, MMP-2 and MMP-9, and IC₅₀, migration and invasion was increased following PC-9R cells treatment with CHIR99021 (p < 0.05). These impacts were observed to be in direct contrast in the case of ICG-001 treatment (p < 0.05).

Conclusion: Activation of the Wnt/β-catenin pathway mediates the high expression of PD-L1 to promote the resistance of NSCLC cells to icotinib. Thus, targeted inhibition of PD-L1 expression is of benefit for the treatment of NSCLC.