Rab25 promotes erlotinib resistance by activating the β1 integrin/AKT/β-catenin pathway in NSCLC

Abstract

Objectives: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has significant therapeutic efficacy in non-small-cell lung cancer (NSCLC) patients. However, acquired resistance is inevitable and limits the long-term efficacy of EGFR-TKI. Our study aimed to investigate the role of ras-associated binding protein 25 (Rab25) in mediating EGFR-TKI resistance in NSCLC.

Materials and methods: Rab25 expression in NSCLC patients was measured by immunohistochemical staining. Western blotting was used to analyse the expression of molecules in the Rab25, EGFR and Wnt signalling pathways. Lentiviral vectors were constructed to knock in and knock out Rab25. The biological function of Rab25 was demonstrated by cell-counting kit-8 and flow cytometry. The interaction between Rab25 and β1 integrin was confirmed by co-immunoprecipitation.

Results: Rab25 overexpression induced erlotinib resistance, whereas Rab25 knockdown reversed this refractoriness in vitro and in vivo. Moreover, Rab25 interacts with β1 integrin and promotes its trafficking to the cytoplasmic membrane. The membrane-β1 integrin induced protein kinase B (AKT) phosphorylation and subsequently activated the Wnt/β-catenin signalling pathway, promoting cell proliferation. Furthermore, high Rab25 expression was associated with poor response to EGFR-TKI treatment in NSCLC patients.

Conclusions: Rab25 mediates erlotinib resistance by activating the β1 integrin/AKT/β-catenin signalling pathway. Rab25 may be a predictive biomarker and has potential therapeutic value in NSCLC patients with acquired resistance to EGFR-TKI.

Keywords: EGFR-TKI resistance; NSCLC; Rab25; β-catenin; β1 integrin.

Figure 1

Rab25 mediates erlotinib resistance in lung cancer cells. (A) Western blotting showing the effect of erlotinib on EGFR signalling pathway molecules in the parental and erlotinib‐resistant lung cancer cell lines. Rab25 protein (B) and mRNA (C) expression were analysed by real‐time PCR and Western blotting, respectively, in the PC9 and HCC827 cells transfected with the Rab25‐overexpressing vector and control vector. Western blotting and real‐time PCR analyses of Rab25 protein (D) and mRNA (E) levels after Rab25 knockdown in the PC9/ER and HCC827/ER cells. **P < 0.01, mean ± SEM, Student's t test. The effect of Rab25 overexpression in the PC9 and HCC827 cells (F) and the effect of Rab25 knockdown in PC9/ER and HCC827/ER cells (G) on the sensitivity of the cells to erlotinib were measured using a CCK‐8 assay. (H, I) Erlotinib‐mediated cell apoptosis in the indicated cells was detected by flow cytometry

Figure 2

The Wnt signalling pathway is involved in Rab25‐mediated erlotinib resistance. (A, B) Western blotting showing key molecules of the Wnt signalling pathway in the indicated cells. (C) Cell proliferation in the indicated cells was measured by an EdU assay. (D) Propidium iodide staining detected cell cycling in the indicated cells, and ModFit was used to analyse the S phase ratio of the cells. **P < 0.01, mean ± SEM, ANOVA. After treatment with LiCl for 24 h, the P‐GSK‐3β and β‐catenin levels were measured in the PC9/ER/shRab25 and HCC827/ER/shRab25 cells by Western blotting (E), and cell proliferation (F) and cell cycling (G) were detected in these cells.

Figure 3

β1 integrin mediates the Rab25‐induced Wnt signalling activation. Western blotting analysis showing the effects of Rab25 overexpression on β1 integrin, p‐GSK‐3β, β‐catenin, PI3K and p‐AKT expression in the PC9 and HCC827 cells (A), and the effects of Rab25 knockdown on these molecules in the PC9/ER and HCC827/ER cells (B). (C) PC9 and HCC827 cells were transfected with the Rab25 overexpression lentiviral vector and β1 integrin interfering RNA, and then, the p‐GSK‐3β, β‐catenin, cyclinD‐1 and p‐AKT levels were measured by Western blotting. Cell lysates from PC9 and HCC827 cells were subjected to immunoprecipitation with anti‐β1 integrin (D) and anti‐Rab25 (E) antibodies, and then, the immunoprecipitates were assessed using the indicated antibodies. (F) Cytoplasmic Rab25 colocalized β1 integrin in the PC9 and HCC827 cells treated with erlotinib. (G) β1 integrin recycling to the plasma membrane in the PC9/ER and HCC827/ER cells treated with erlotinib. Protein localization was detected by immunofluorescent staining and observed by confocal microscopy. Scale bars, 10 μm

Figure 4

Rab25 mediates erlotinib resistance in lung cancer in vivo. (A) Mice were implanted subcutaneously with the indicated cell lines (5 × 10⁶), and tumours were grown to ~100 mm³ and then treated with erlotinib for 14 d. Subsequently, the mice were euthanized, and the tumours were dissected. (B) Tumour growth curve representing the mean ± SEM of the tumour volumes of five mice in the indicated xenograft groups. **P < 0.01, one‐way ANOVA. (C‐F) Immunohistochemical staining assay to confirm the expression of Rab25, β1 integrin and β‐catenin in the indicated group of tumour samples. Tumour cell proliferation was measured by Ki‐67 staining. Scale bars, 50 μm

Figure 5

Rab25 expression is associated with the therapeutic response to EGFR‐TKI treatment in NSCLC. (A) IHC staining analysis of Rab25 expression in NSCLC patient tissue samples. A score of 0‐1 indicates low expression of Rab25, and a score of 2‐3 indicates high expression. (B) Patients with high expression (grey bar) and low expression (black bar) were assigned based on their therapeutic responses to EGFR‐TKI. **P < 0.01, Pearson's chi‐square test. (C) IHC staining analysis of Rab25 expression in tissue samples from patients who had acquired resistance to erlotinib or gefitinib. Scale bars, 100 μm. (D‐E) Kaplan‐Meier analysis of progression‐free survival and overall survival in NSCLC patients. (F) Graphical model for the mechanism by which Rab25 regulates β1 integrin/AKT/β‐catenin signalling pathway