KPNA2 promotes migration and invasion in epithelial ovarian cancer cells by inducing epithelial-mesenchymal transition via Akt/GSK-3β/Snail activation

Abstract

Background: Increased karyopherin alpha 2 (KPNA2) expression has been demonstrated in epithelial ovarian carcinoma (EOC) tissue. However, its role in the disease is not clear. Here, we investigate the mechanism of involvement of KPNA2 in EOC. Methods: Stable cell lines expressing KPNA2, or KPNA2 shRNAs, were constructed. The effects of KPNA2 overexpression and knockdown on EOC cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) were evaluated using relevant assays and western blot analysis. Key components of the Akt/GSK-3β/Snail signaling pathway were detected using western blotting and immunofluorescence. Results: KPNA2 overexpression increased the migration and invasion of EOC cells (EFO-21 and SK-OV3); these cells also exhibited characteristics of EMT. Key proteins in the Akt/GSK-3β/Snail signaling pathway were also upregulated in cells overexpressing KPNA2. In contrast, knockdown of KPNA2 effectively suppressed migration and invasion of these EOC cells. Conclusions: KPNA2 may reduce the migration and invasion of EOC by inhibiting the Akt/GSK-3β/Snail signaling pathway and suppressing EMT.

Keywords: EOC; KPNA2.

Figure 1

Effects of KPNA2 on epithelial ovarian carcinoma (EOC) cell motility. A, Motility as measured by testing the wound closure rate at 0 hours (red dotted line) and 36 hours (black dotted line) (×200; *P < 0.001). B, Representative micrographs, and quantification of wound closure rate in EFO-21 and SK-OV3 cell lines stably overexpressing KPNA2 or infected with KPNA2 shRNA(s) relative to the control. Data were obtained from three independent experiments and described similar results.

Figure 2

Effect of KPNA2 on EOC cell invasion. A, Invasion ability induced by fetal bovine serum as analyzed by the Transwell migration assay (×200; *P < 0.001). B, quantification of invasive properties induced by fetal bovine serum in EFO-21 and SK-OV3 cell lines stably overexpressing KPNA2 or infected with KPNA2 shRNA(s) relative to the control. Data were obtained from three independent experiments and described similar results.

Figure 3

Effect of KPNA2 on epithelial-to-mesenchymal transition (EMT) and the Akt/GSK-3β/Snail pathway. A, Western blot analysis of KPNA2, Akt, p-Akt, GSK-3β, p-GSK-3β, snail, twist, vimentin, E-cadherin, fibronectin, and α-catenin protein expression in the indicated EOC cells. β-actin was used as a loading control. B-K, Expression levels were quantified using ImageJ software. Error bars represent the standard deviation (SD) of three independent experiments. Expression levels were normalized to β-actin (*P < 0.001, **P < 0.0001).

Figure 3

Effect of KPNA2 on epithelial-to-mesenchymal transition (EMT) and the Akt/GSK-3β/Snail pathway. A, Western blot analysis of KPNA2, Akt, p-Akt, GSK-3β, p-GSK-3β, snail, twist, vimentin, E-cadherin, fibronectin, and α-catenin protein expression in the indicated EOC cells. β-actin was used as a loading control. B-K, Expression levels were quantified using ImageJ software. Error bars represent the standard deviation (SD) of three independent experiments. Expression levels were normalized to β-actin (*P < 0.001, **P < 0.0001).

Figure 4

Effect of KPNA2 gene expression on epithelial-to-mesenchymal transition in EOC cells. KPNA2-overexpressing and KPNA2 shRNA(s)-infected SK-OV3 cells were placed on coverslips pre-coated with 10% fetal bovine serum/RPMI-1640. After an additional 24 hours, cells were stained for E-cadherin, α-catenin, N-cadherin, vimentin, and 4',6-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy. The red signal represents staining for the corresponding protein, while the blue signal signifies nuclear DNA staining with rhodamine.

Figure 5

Scheme of the c-Myc-mediated KPNA2-induced epithelial-to-mesenchymal transition (EMT) in EOC cells. KPNA2 induces EMT, which subsequently contributes to invasion by EOC cells. In addition, KPNA2 also transports c-Myc into the nucleus and triggers the Akt/GSK-3β/Snail pathway, which is required for KPNA2-induced EMT.