Glycogen synthase kinase-3 inhibition overcomes epithelial-mesenchymal transition-associated resistance to osimertinib in EGFR-mutant lung cancer

Abstract

A novel epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, osimertinib, has marked efficacy in patients with EGFR-mutant lung cancer. While epithelial-mesenchymal transition (EMT) plays a role in the resistance to various targeted drugs, its involvement in EGFR-inhibitor resistance remains largely unknown. Preclinical experiments with osimertinib-resistant lung cancer cells showed that EMT was associated with decreased microRNA-200c and increased ZEB1 expression. In several resistant clone cells, pretreatment with the histone deacetylase inhibitor quisinostat helped overcome the resistance by reverting EMT. Furthermore, drug screening from a library of 100 kinase inhibitors indicated that Glycogen synthase kinase-3 (GSK-3) inhibitors, such as LY2090314, markedly inhibited the growth and induced apoptosis of resistant cells, specifically those with a mesenchymal phenotype. These results suggest that GSK-3 inhibition could be useful to circumvent EMT-associated resistance to osimertinib in EGFR-mutant lung cancer.

Keywords: epidermal growth factor receptor; epithelial-mesenchymal transition; glycogen synthase kinase-3; osimertinib; resistance.

FIGURE 1

Osimertinib‐resistant H1975 clones were generated in vitro. A, H1975 parental cells and the resistant clones (OR#1, OR#2, OR#3, and OR#4) were established through stepwise dose escalation. B, MTT assay was used to assess the growth inhibition of H1975 cells and the resistant clones treated with osimertinib or rociletinib for 72 h (n = 3). Results are expressed as the mean ± SEM. IC₅₀ values were calculated and demonstrated. C, H1975 parental cells and resistant clones were treated with 1 µmol/L osimertinib for 2 h. The changes in phosphorylation (p‐) of indicated proteins were analyzed by western blotting. D, Control and epidermal growth factor receptor (EGFR) siRNAs were transfected into parental and resistant cells, and EGFR knockdown was confirmed by western blot. Cell viability was analyzed after 72 h using the MTT assay (n = 3). *P < .001

FIGURE 2

Epithelial‐mesenchymal transition (EMT) is induced in osimertinib‐resistant H1975 clones. A, H1975 parental cells and the resistant clones were evaluated to determine any morphologic changes consistent with EMT, using a light microscope. Scale bar = 200 μm. B, Expression of EMT markers was analyzed using western blotting with the indicated Abs. C, Immunofluorescence assay of EMT markers. Scale bar = 50 μm

FIGURE 3

Quisinostat reversed epithelial‐mesenchymal transition by upregulating microRNA (miR)‐200c expression through inhibition of ZEB1. A, Heatmap representation of miRNA array data showing the expression levels of miRNAs in decreasing order of fold change more than 2 between parental H1975 and OR#3 cells (n = 2). B, Relative expression of miR‐200c was measured in H1975 parental cells and resistant clones (n = 3). Error bars represent SEM. *P < .0001. C, H1975‐resistant clone cells were treated with a miR‐200c mimic. Change in ZEB1 and E‐cadherin expression was analyzed by western blotting. D, ZEB1 or Slug siRNA was introduced into H1975‐resistant clone cells, and cell lysates were analyzed by western blotting with the indicated Abs 48 h later. E, Change of miR‐200c promoter activity in OR#3 cells treated with 5 HDAC inhibitors (n = 3). Error bars represent SEM. *P < .01. F, Change in ZEB1 and E‐cadherin expression in resistant clones treated with 0.03 µmol/L quisinostat was analyzed by western blotting. G, Resistant clones were pretreated with 0.03 µmol/L quisinostat for 48 h, followed by treatment with osimertinib for 72 h. Cell viability was measured using MTT assay (n = 3). Error bars represent SEM. *P < .0001

FIGURE 4

Glycogen synthase kinase (GSK)‐3 inhibitor suppressed the growth of epithelial‐mesenchymal transition‐associated resistant cells. A, Drug screening by treatment with 1 μmol/L each drug to identify compounds that suppress the growth of OR#1 cells (n = 3). Pink columns represent GSK‐3 inhibitors. B, C, OR#1 cells were treated with 1 μM GSK inhibitors for 72 h. Cell viability was measured using MTT assay (n = 3) D, H1975 parental cells and resistant clones were treated with LY2090314 for 72 h. Cell viability was measured using MTT assay (n = 3). E, H1975 parental cells and the resistant clones were treated with 0.1 μmol/L LY2090314 in combination with 1 μmol/L osimertinib (n = 3). Error bars represent SEM. *P < .001

FIGURE 5

Glycogen synthase kinase (GSK)‐3 inhibition induced apoptosis of epithelial‐mesenchymal transition‐associated resistant cells. A, Control and GSK‐3 siRNAs were transfected into H1975 parental cells and resistant clone cells. At 24 h posttransfection, cells were treated with 1 μmol/L osimertinib and cell viability was measured after 72 h using MTT assay (n = 3). Error bars represent SEM. *P < .0001. B, Control and GSK‐3 siRNAs were transfected into H1975 parental and resistant clone cells. Cell lysates were analyzed using western blotting with the indicated Abs at 48 h posttransfection. C, Control and GSK‐3 siRNAs were transfected into resistant clone cells. At 24 h post‐transfection, cells were treated with 1 μmol/L osimertinib and cell lysates were analyzed using western blotting with the indicated Abs after 72 h (n = 4). D, OR#1, OR#3, and OR#4 cells were pretreated with 0.03 μmol/L quisinostat for 48 h, followed by GSK‐3 siRNA for 72 h. Cell viability was measured using MTT assay (n = 3). Error bars represent SEM. *P < .0001. E, Control, GSK‐3α, and GSK‐3β siRNAs were transfected into OR#3 cells and cell viability was measured after 72 h using MTT assay (n = 3). Error bars represent SEM. *P < .0001

FIGURE 6

Summary scheme of the role of microRNA (miR)‐200c in the activation of the glycogen synthase kinase (GSK)‐3/AKT axis in osimertinib‐sensitive and osimertinib‐resistant cells. EMT, epithelial‐mesenchymal transition