Cyclooxygenase-2 mediates gefitinib resistance in non-small cell lung cancer through the EGFR/PI3K/AKT axis

Abstract

Gefitinib is a potent inhibitor of EGFR and represents the front-line treatment for non-small cell lung cancer (NSCLC) therapeutics. However, NSCLC patients are prone to develop acquired resistance through as yet, undefined mechanisms of resistance. Here, we investigated the role of COX-2 during gefitinib resistance in NSCLC cells and revealed its underlying mechanism(s) of action. We report the upregulation of COX-2 in gefitinib-resistant NSCLC tissues and cells, which is associated with poor prognosis. In vitro assays in NSCLC cells (PC9/GR) showed that COX-2 facilitates gefitinib resistance in NSCLC cells through its effects on P-gp, MRP1, and BCRP, and cancer cell migration and invasion. In vivo, COX-2 silencing could repress tumor growth. We found that the overexpression of COX-2 enhances the transcription of MMP-2, MMP-7, and MMP-9 which mediates PI3K-AKT activation. In summary, we demonstrate that COX-2 mediates the gefitinib resistance of NSCLC cells through its interaction with EGFR and the PI3K-AKT axis. This highlights COX-2 as a novel molecular target for NSCLC.

Keywords: COX-2; EGFR; PI3K-AKT; gefitinib resistance; non-small cell lung cancer.

Figure 1

COX-2 is overexpressed in NSCLC tissue and correlates with poor patient prognosis. (A) COX-2 mRNA assessed by qRT-PCR analysis in 32 paired NSCLC tissues. (B) COX-2 mRNA levels in NSCLC patients showing gefitinib resistance vs. gefitinib sensitive patients. (C) COX-2 mRNA expression in the NSCLC cells vs control cells analyzed by qRT-PCR. (Mean ± SEM, p ≤0.05). (D) COX-2 protein expression in NSCLC cells vs. normal cells. (E) COX-2 mRNA and protein expression in PC9/GR (gefitinib resistant) and naive PC9 cells. (F) OS according to COX-2 expression assessed through the GEPIA. Two-sided log-rank tests were used to determine p-values.

Figure 2

COX-2 promotes gefitinib chemo resistance and the metastatic phenotypes of NSCLC cells. (A) RT-qPCR showing COX-2 expression in PC9 cells treated with gefitinib. (B-C) PC9 cells overexpressing FLAG-COX-2 or silenced for COX-2 expression (KD, shCOX-2#1, #2) in PC9 cells (PC9/GR). Data are the mean ± SEM. (D) Cell viability assessments and IC50 of gefitinib in PC9 vs. PC9/GR cells. Data were compared through ANOVA analysis. (E) Transwell assays to show PC9 and PC9/GR cell invasion. Data were compared via a Student's t test. (F) P-gp, MRP1, and BCRP expression assessed via RT-qPCR. Data were compared via a Student's t test. (G-H) Xenograft assays for the assessment of tumor volume and weight in mice subcutaneously injected with PC9 cells. Data are the mean ± SEM. Data were compared via ANOVA assessments.

Figure 3

COX-2 influences the expression of tumor cell MMPs through PI3K-AKT signaling. (A) Vimentin, E‐cadherin and N‐cadherin expression assessed by WB. (B) WB analysis of MMP expression in the indicated cell lines. GAPDH was probed as a control. (C) WB assessment of p‐AKT, p-STAT3, p-JAK2, and p‐p38 levels in the indicated cells. GAPDH was probed as a loading control. (D) Relative mRNA expression of the indicated MMPs. Data are the mean ± SEM, compared through a Student's t test. (E) PI3K activity and (F) PTEN expression on the indicated cell lines. GAPDH was probed as a loading control. (G) IC50 of gefitinib in PC9/GR cells co-transfected with COX-2 siRNA and/or AKT overexpression plasmids. Data were compared via a one-way ANOVA. (H) Migration and invasion assays. Data are the mean ± SEM compared through a Student's t‐test.

Figure 4

COX-2-EGFR binding leads to PI3K/AKT activation. (A-B) Cells were treated with EGF or HGF (B) for the indicated time points and WB for the indicated proteins was performed. (C) Co-Ip assays of PC9 cells transfected with HA-EGFR, FLAG-COX-2 or empty vector controls. (D) Cells were stimulated with EGF exposure for 0 or 15 min and WB analysis was performed as described.