Abstract: Objective To investigate the effect of Wnt signaling pathway and epidermal growth factor receptor (EGFR) on gefitinib resistance of non-small cell lung cancer (NSCLC) and its mechanism. Methods EGFR expressions in parental cells and gefitinib-resistant HCC827 / R cells were detected by qRT-PCR and Western blot assay. Immunohistochemical (IHC) staining was used to detect the expressions of EGFR in NSCLC tissues before and after drug resistance. Activation status of Wnt signaling pathway in cells were detected by luciferase assay. TCF/LEF transcription factor binding sites on the EGFR promoter region were predicted in the Jaspar database. The regulation of transcription factors on gene expression was detected by Chip and luciferase assays. Functional blockade assay was used to detect whether Wnt signaling pathway/EGFR pathway mediated gefitinib resistance. Results Compared with the parental cells, the expression of EGFR in HCC827/R cells was significantly increased at mRNA and protein levels (P＜0.05). The results of IHC showed that high expressions of EGFR in NSCLC tissue samples after drug resistance were detected in 2 of 3 paired NSCLC tissue samples before and after gefitinib resistance. Luciferase results showed that Wnt/β-catenin signaling pathway was abnormally activated in resistant HCC827/R cells compared with that of parental cells (P＜0.05). Bioinformatics analysis predicted that there were TCF3/ TCF4 sites, which were downstream transcription factors of the Wnt/β-catenin signaling pathway, the binding site located on the promoter region of EGFR gene (- 1476~ - 1468), and Chip and Luciferase experiments confirmed that EGFR gene expression could be up-regulated by Wnt/β - catenin signaling pathway (P＜0.05). The results of functional blockade experiments showed that when Wnt3a was used to stimulate parental HCC827 cells while knocking down EGFR, the inhibition rate of 3.13 μM gefitinib on cells was restored compared with that of Wnt3a alone (P＜0.05). Conclusion Wnt/ β-catenin signaling pathway up-regulates EGFR expression to promote gefitinib resistance in NSCLC, which provides the new experimental evidence for improving the therapeutic effect of gefitinib targeted therapy on NSCLC.

Key words: carcinoma, non-small-cell lung, Wnt signaling pathway, receptor, epidermal growth factor, gefitinib, drug resistance