KPNA2 promotes cell proliferation and tumorigenicity in epithelial ovarian carcinoma through upregulation of c-Myc and downregulation of FOXO3a

Abstract

Karyopherin alpha 2 (KPNA2), a member of the karyopherin family, has a central role in nucleocytoplasmic transport and is overexpressed in many cancers. Our previous study identified KPNA2 as significantly upregulated in epithelial ovarian carcinoma (EOC), correlating with poor survival of patients. However, the precise mechanism of this effect remains unclear. The aim of the present study was to examine the role of KPNA2 in the proliferation and tumorigenicity of EOC cells, and its clinical significance in tumor progression. Real-time quantitative RT-PCR analysis revealed high expression levels of KPNA2 in 162 out of 191 (84.8%) fresh EOC tissues, which was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation, histological type, recurrence, and prognosis of EOC patients. Our results showed that upregulation of KPNA2 expression significantly increased the proliferation and tumorigenicity of EOC cells (EFO-21 and SK-OV3) in vitro and in vivo, by promoting cell growth rate, foci formation, soft agar colony formation, and tumor formation in nude mice. By contrast, knockdown of KPNA2 effectively suppressed the proliferation and tumorigenicity of these EOC cells in vitro and in vivo. Our results also indicated that the molecular mechanisms of the effect of KPNA2 in EOC included promotion of G1/S cell cycle transition through upregulation of c-Myc, enhanced transcriptional activity of c-Myc, activation of Akt activity, suppression of FOXO3a activity, downregulation of cyclin-dependent kinase (CDK) inhibitor p21Cip1 and p27Kip1, and upregulation of CDK regulator cyclin D1. Our results show that KPNA2 has an important role in promoting proliferation and tumorigenicity of EOC, and may represent a novel prognostic biomarker and therapeutic target for this disease.

Figure 1

KPNA2 is overexpressed in EOC cells and tissue samples; and KPNA2 upregulation is associated with poor prognosis. (a) Gene expression microarray analysis showing that KPNA2 was upregulated (10-fold) compared with HOSE samples. (b) Real-time qRT-PCR analysis showing the average expression levels of KPNA2 in EOC (n=191) and HOSE (n=10) tissue samples. Expression levels are normalized to β-actin mRNA. Error bars represent S.E. (c) Western blot analysis of KPNA2 protein expression in two HOSE samples and seven EOC cell lines; β-actin was used as a loading control. (d) KPNA2 protein expression in two HOSE samples and seven EOC cell lines was quantitated using ImageJ software (Wayne Rashband). (e) Kaplan–Meier analysis showing that the expression of KPNA2 was significantly associated with poor overall survival in 191 EOC cases (P=0.012, log-rank test). (f) Kaplan–Meier analysis showing that expression of KPNA2 was significantly associated with poor relapse-free survival in 191 EOC cases (P<0.001, log-rank test)

Figure 2

KPNA2 is essential for EOC cell proliferation. Western blotting analysis of KPNA2 overexpression and KPNA2 knockdown in (a) EFO-21 and (b) SK-OV3 cell lines with two specific shRNAs; β-actin was used as a loading control. MTT assays of KPNA2 overexpression and KPNA2 knockdown in (c) EFO-21 and (d) SK-OV3 cell lines. Each bar represents the mean (± S.D.) of three independent experiments. (e–h) Representative micrographs (left panel) and quantifications (right panel) of the following crystal violet-stained cell lines: EFO-21-vector, EFO-21-KPNA2, EFO-21-si-scramble, EFO-21-siKPNA2 #1, EFO-21-siKPNA28 #2; and SK-OV3-vector, SK-OV3-KPNA2, SK-OV3-si-vector, SK-OV3-siKPNA2 #1, SK-OV3-siKPNA2 #2, *P<0.05

Figure 3

KPNA2 is essential for EOC cell tumorigenicity in vitro and in vivo. Representative micrographs (left panel) and quantifications (right panel) of soft agar colony formation assays for the following cell lines, relative to the control: (a and b) EFO-21-vector, EFO-21-KPNA2, EFO-21-si-scramble, EFO-21-siKPNA2 #1, EFO-21-siKPNA2 #2; and (c and d) SK-OV3-vector, SK-OV3-KPNA2, SK-OV3-si-vector, SK-OV3-siKPNA2 #1, SK-OV3-siKPNA2 #2. Each bar represents the mean (± S.D.) of three independent experiments. Xenograft model in nude mice. (e) EFO-21-vector, EFO-21/KPNA2 (KPNA2), EFO-21-si-scramble, and EFO-21/siKPNA2 #1 (siKPNA2) cells were injected subcutaneously into the left and right flanks of the mice. (f) Representative pictures of tumor growth 30 days after inoculation. (g) Tumor volumes were measured on the indicated days. All data are shown as mean±S.D. (h) Western blotting analysis of KPNA2 expression in EFO-21-vector, EFO-21/KPNA2 (KPNA2), EFO-21-si-scramble, and EFO-21/siKPNA2 #1 (siKPNA2) generated tumors 30 days after injection. (i) Immunohistochemical analysis of EFO-21-vector, EFO-21/KPNA2 (KPNA2), EFO-21-si-scramble, and EFO-21/siKPNA2 #1 (siKPNA2) generated tumors 30 days after injection. Sections obtained from tumors were incubated with anti-ki67 antibody. Representative fields are shown ( × 200 magnification) *P<0.05; **P<0.01

Figure 4

Depletion of KPNA2 induces G1/S arrest of EOC cells (BrdU incorporation assay). (a and b) Representative micrographs (top panel), and (c and d) quantification (middle panel) of BrdU-incorporating cells in KPNA2-overexpressing and two KPNA2 shRNA(s)-infected cell lines, relative to the control. The cells were fixed, subjected to BrdU staining, and visualized under a fluorescence microscope. Data were obtained from three independent experiments and showed similar results. Red, BrdU; blue, DAPI. (e and f) Flow cytometry analysis of the indicated EOC cells transfected with the KPNA2 overexpression construct or KPNA2 shRNA(s). The proportion of S-phase cells as significantly reduced in KPNA2 shRNA(s)-transfected cell lines (P<0.05) compared with the control group; in contrast, the proportion of S-phase cells in the line transfected with the KPNA2 construct clearly increased (P<0.05) *P<0.05

Figure 5

Depletion of KPNA2 induces G1/S arrest of EOC cells (by real-time qRT-PCR analysis). (a and b) Relative mRNA expression of KPNA2, cyclin D1, ki-67, p21Cip1, and p27Kip1 in the indicated EOC cells was determined by real-time qRT-PCR. Expression levels were normalized to β-actin. (c and d) Western blotting analysis of KPNA2, cyclin D1, ki-67, p21Cip1, and p27Kip1 proteins (top) in the indicated EOC cells. (e and f) Expression levels were quantitated using ImageJ software (Wayne Rashband; bottom); β-actin was used as a loading control. Error bars represent the S.D. of three independent experiments

Figure 6

Downregulation of KPNA2 decreases transcriptional activity of c-Myc and the activity of Akt, and increases the activity of FOXO3a. (a and b) c-Myc reporter activity in EOC cells transduced with vector KPNA2; si-scramble; siKPNA2 #1; and siKPNA2 #2, relative to the control. (c and d) Western blotting analysis of phosphorylated Akt (p-Akt), total Akt, phosphorylated FOXO3a (p- FOXO3a), total FOXO3a, and c-Myc proteins in the indicated EOC cells. (e and f) Expression levels were quantitated using ImageJ software (Wayne Rashband; bottom); β-actin was used as a loading control. Error bars represent the S.D. from three independent experiments. *P<0.05 compared with the control

Figure 7

Knockdown of KPNA2 causes subcellular redistribution of E2F1 in EOC cells. KPNA2-knockdown (a) and -overexpressing (b) SK-OV3 cells transfected with pMSCV-c-Myc plasmid. At 48 h after transfection, cells were lysed and fractionated into nuclear and cytoplasmic fractions, followed by western blotting. GAPDH was used as the cytosolic control and lamin B as the nuclear control. (c) Quantification analysis of subcellular distribution acquired from western blotting using ImageJ software (Wayne Rashband). (d) c-Myc reporter activity in KPNA2-knockdown and -overexpressing SK-OV3 cells transfected with pMSCV-c-Myc plasmid, relative to the control. Error bars indicate the S.D. from three independent experiments. *P<0.05 compared with the control