Global evaluation of Eph receptors and ephrins in lung adenocarcinomas identifies EphA4 as an inhibitor of cell migration and invasion

Abstract

The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. We aimed to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma compared with normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were carried out in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer compared with non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, possibly by affecting cancer cell migration and invasion.

Figure 1. The expression of EphA4 is associated with an improved outcome

(A) Kaplan-Meier curves for (A) overall survival (N=422) and (B) relapse-free survival (N=327) as a function of EphA4 expression in patients who underwent surgical resection of lung adenocarcinoma (19). EphA4 expression was stratified using tertiles: high, moderate, or low expression. (C) EphA4 gene expression and tumor cell differentiation in lung adenocarcinoma (well versus moderately differentiated, p=0.0053; moderately versus poorly differentiated, p=0.0022). (D) EphA4 gene expression and smoking status (p>0.05). (E) EphA4 gene expression and EGFR mutation status (p=0.0277). Abbreviations: Mod.: Moderately; MT: mutant; WT: wild-type.

Figure 2. Distribution of Eph receptor and ephrin gene expression in NSCLC versus bronchial epithelial cell lines or normal-appearing lung

Gene expression profiles were processed from four publicly available studies including a large panel of NSCLC cell lines (A) (17, 18) and 3 independent studies comparing lung adenocarcinomas and normal lung (B-D) (19, 21, 22). (E-H) EphA4 gene expression in NSCLC versus normal bronchial epithelial cells (E) (17, 18) and in lung adenocarcinoma versus normal lung in 3 independent studies (F-H) (19, 21, 22). Log₂ fold-change of Eph receptors and ephrins in cancer versus normal cells/tissues are displayed in the same order to allow comparison. Striped bars represent genes downregulated in cancer versus normal. Bars striped in the other direction represent genes upregulated in cancer versus normal. Black bars represent no significant change. Statistical significance was determined by a paired (D, H) or non-paired (A-C and E-G) two-sided Student’s t-test.

Figure 3. EphA4 gene expression correlates well with protein expression

(A) EphA4 protein expression in 7 NSCLC cell lines by Western blotting. The bottom panel is a quantitation of the blot. (B) EphA4 gene expression on the Affymetrix platform, by qPCR, and by Western blotting was compared, with the noted correlation coefficients. (C) Comparison of EphA4 protein and gene expression.

Figure 4. EphA4 decreases NSCLC cell migration and invasion

(A) Effect of EphA4 knockdown (H460, H1792) and overexpression (A549, H661) on cell migration. (B) Effect of EphA4 knockdown (H460, H1792) and overexpression (A549, H661) on cell invasion. Data come from three independent assays; *p<0.05, **p<0.01.

Figure 5. EphA4 increases occludin expression and decreases ERK activation

Following EphA4 knockdown (H460, H1792) or overexpression (A549, H661), the expressions of the noted proteins and phospho-proteins were measured using Western blotting. Quantitation of noted proteins is shown for panel A. Abbreviations: IP: immunoprecipitation; IB: immunoblot.