Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3β/snail signalling pathway

Abstract

Background: Epithelial mesenchymal transition (EMT) is known to be associated with chemoresistance as well as increased invasion/metastasis. However, the relationship between EMT and resistance to an epidermal growth factor receptor (EGFR) -targeting drug in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we investigated the acquisition of EMT by gefitinib in HNSCC cell line (UMSCC81B).

Methods: We isolated fibroblastoid variant (81B-Fb) from gefitinib-resistant UMSCC81B-GR3 cells obtained after increasing the doses of gefitinib treatment in vitro and examined EMT and its underlying mechanism.

Result: 81B-Fb cells exhibited fibroblast-like morphology, increased motility, loss of E-cadherin, acquisition of vimentin and snail expression. In 81B-Fb cells, downregulation of EGFR, which is mediated by increased ubiquitination, and activation of downstream protein kinase B (Akt), glycogen synthase kinase-beta (GSK-3β) signalling and upregulation of snail expression were observed compared with UMSCC81B cells. LY294002, but not U0126, suppressed foetal bovine serum or heregulin-β1-induced phosphorylation of Akt/GSK-3β and snail expression together with the inhibition of 81B-Fb cell motility. Furthermore, forced expression of EGFR resulted in partial restoration of gefitinib sensitivity and reversal of EMT.

Conclusion: These results suggest that EMT in the gefitinib-resistant cells is mediated by the downregulation of EGFR and compensatory activation of Akt/GSK-3β/snail pathway.

Figure 1

Isolation of fibroblastoid subline with EMT phenotype (81B-Fb) from gefitinib-resistant UMSCC81B-GR3 cell line obtained after long-term gefitinib treatment in vitro. (A) Immunohistochemistry of UMSCC81B-GR3 subcutaneous tumour in nude mouse. E-cadherin(−)/vimentin(+) tumour cells are seen at the invasion front of UMSCC81B-GR3 tumour (arrows). Bars=100 μm. (B) Phase-contrast photomicrographs of cultured UMSCC81B, UMSCC81B-GR3 and fibroblastoid 81B-Fb cells. Arrow=fibroblastoid tumour cells. Bars=30 μm. (C) Comparison of gefitinib sensitivity between UMSCC81B cells () and 81B-Fb cells (). Bars= s.d., ^*P<0.05.

Figure 2

EMT phenotypic expression of 81B-Fb cells compared with parental UMSCC81B cells. (A) Western blot analysis of EMT-associated proteins. (B) mRNA expression of EMT-associated genes of UMSCC81B cells () and 81B-Fb cells (). Loss of E-cadherin and acquisition of vimentin and snail expression are apparent. (C) Motility of UMSCC81B cells () and 81B-Fb cells () as measured by wound-closure assay. (D) In vitro growth rate of UMSCC81B cells () and 81B-Fb cells (). Bars=s.d., ^*P<0.05.

Figure 3

Downregulation of EGFR expression in 81B-Fb cells compared with UMSCC81B cells. (A) Western blot and immunofluorescence analysis of EGFR protein expression of 81B-Fb cells. Change of subcellular localisation of EGFR from membrane to the cytoplasm is apparent in 81B-Fb cells in the presence of FBS. (B) Internalisation of EGFR in UMSCC81B and 81B-Fb cells in response to EGF. Cells were serum-starved for 24 h, and stimulated with EGF (20 ng ml⁻¹). Accumulation of EGFR in the endosome (arrows) is seen in both cells at 15 min after EGF stimulation. (C) Immunoprecipitation analysis of ubiquitination of EGFR in response to FBS or EGF. Cells were serum-starved for 24 h, pretreated with 10 mM MG132 for 2 h and then stimulated with EGF for 15 min or FBS for several hours. (D) mRNA expression of various EGFR ligands of UMSCC81B cells () and 81B-Fb cells (). Bars=s.d., ^*P<0.05.

Figure 4

Western blot analysis of the effects of gefitinib on the phosphorylation of EGFR and downstream signalling pathways in 81B-Fb cells compared with UMSCC81B cells. Cells were starved in serum-free medium for 24 h, exposed to gefitinib at increasing concentrations for 2 h and were then stimulated with EGF (10 ng ml⁻¹) for 10 min.

Figure 5

Effects of PI3K inhibitor and MEK1/2 inhibitor on the signalling pathway and EMT phenotypes in 81B-Fb cells. (A) Activation of Akt/GSK-3β and upregulation of snail expression in response to FBS in 81B-Fb cells compared with UMSCC81B cells. (B) Inactivation of Akt and downregulation of snail by LY294002 in 81B-Fb cells. Cells were cultured in DMEM with 10% FBS and exposed each inhibitors for 12 h. LY: LY294002 (50 μM), U: U0126 (20 μM). (C and D) Effects of signal inhibitors on the morphology (C) and motility (D) of 81B-Fb cells. LY294002 (25 μM) but not U0126 (10 μM) disturbs fibroblastoid morphology and inhibits migration of 81B-Fb cells. Bars=s.d., ^*P<0.05. Abbreviation: NS=not significant.

Figure 6

Effects of EGFR transfection on the growth, differentiation and gefitinib sensitivity of 81B-Fb cells. (A) Subcellular localisation of EGFR in the UMSCC81B cells, 81B-Fb cells, EGFR transfectant of 81B-Fb cells (Tf-1) and vector control. Note, restoring membrane expression of EGFR in Tf-1 cells compared with 81B-Fb cells and vector control cells. (B) Quantitative RT–PCR analysis. E-cadherin mRNA expression is significantly increased in EGFR transfectants in accordance with increased EGFR expression. (C) In vitro growth of 81B-Fb cells (□), EGFR transfectant (Tf-1; ) and vector control (). (D) Comparison of gefitinib sensitivity among various tumour cells with () and without gefitinib (). Bars=s.d., ^*P<0.05. Abbreviation: NS=not significant.