Overexpression of P21-activated kinase 4 is associated with poor prognosis in non-small cell lung cancer and promotes migration and invasion

Abstract

Background: P21-activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is an important oncogene whose expression is increased in many human cancers and is generally positively correlated with advanced disease and decreased survival. However, little is known about the expression and biological function of PAK4 in human non-small cell lung cancer (NSCLC).

Methods: PAK4 expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR, and western blotting. Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression. siRNA-mediated gene silencing and protein kinase assay was applied to demonstrate the role and the mechanism of PAK4 in lung cancer cell migration, invasion.

Results: The results showed that PAK4 was overexpressed in NSCLC cell lines and human NSCLC tissues. PAK4 expression was detected both in the membranes and cytoplasm of NSCLC cancer cells in vivo. Moreover, increased expression of PAK4 was associated with metastasis, shorter overall survival, advanced stage of NSCLC. Furthermore, PAK4 expression was positively correlated with phosphorylation of LIMK1 expression levels. Knockdown of PAK4 in NSCLC cell lines led to reduce the phosphorylation of LIMK1, which resulted in decrease of the cell migration and invasion. In addition, PAK4 bound to LIMK1 directly and activated it via phosphorylation.

Conclusions: These data demonstrate that PAK4 mediated LIMK1 phosphorylation regulates the migration and invasion in NSCLC. Therefore, PAK4 might be a significant prognostic marker and potential therapeutic molecular target in NSCLC.

Fig. 1

PAK4 expression in NSCLC cell lines. a: Western blots of PAK4 expression in HBE cell line and A549, NCI-520, NCI-460, and NCI-H596 NSCLC cell lines (n = 3 replicate experiments; p < 0.05 vs. HBE cells). b: Real-time PCR analysis of PAK4 expression in HBE, A549, NCI-520, NCI-460, and NCI-H596 cells (n = 3 replicate experiments; p < 0.001 vs. HBE cells)

Fig. 2

Expression of PAK4 and the association with progression in NSCLC. a Representative western blots of PAK4 expression (left) in NSCLC tissues (T) and matched adjacent non-tumor tissues (N); the quantitative intensities′ data of PAK4 protein bands of 20 NSCLC tissues and matched adjacent non-tumor tissues. b: The mRNA expression of PAK4 in 20 NSCLC tissues and matched adjacent non-tumor tissues by RT-PCR. c: PAK4 expression in primary NSCLC cancer tissues of 10 patients without metastasis and 10 patients with metastasis. d: Representative immunohistochemical staining (top panels) and statistical analysis (bottom panels) of squamous cell cancer (left) and adenocarcinoma (right) of human NSCLC tissues (T) and matched adjacent non-tumor tissues (N) under at × 40 original magnification. Positive cells are stained brown. P, patient

Fig. 3

Kaplan-Meier survival curve illustrating the prognostic significance of PAK4 expression in NSCLC. a: The patients (n = 210) were divided into high or low PAK4 expression groups according to the proportion of positively stained tumor cells and staining intensity. 5 year survival was calculated using the Kaplan-Meier method and analyzed using the log-rank test. b: 5 year survival was calculated only in the stage I + II cancer patients. c: 5 year survival was calculated only in the stage III + IV cancer patients

Fig. 4

PAK4 knockdown suppressed NSCLC cell migration and invasion. The protein (a) and mRNA (b) expression of PAK4 in A549 and NCI-H520 cells transfected with siRNA(si-PAK4) or control siRNA(control) . Transwell migration assay (c) and Matrigel invasion assay (d) of A549 and NCI-H520 cells transfected with si-PAK4 or control under at × 40 original magnification

Fig. 5

LIMK1 was required for PAK4-mediated NSCLC cell migration and invasion. a: The protein expression of LIMK1, p-LIMK1, cofilin, and p-cofilin in A549 and NCI-H520 cells transfected with si-PAK4 or control by western blotting. b: Immunofluorescent staining for PAK4 (green) and LIMK1 (red) in A549 cells (upper panels) or in NCI-H520 cells (lower panels). Nuclei were stained with Hoechst33258 (blue). c: A549 and NCI-H520 cell lysates were immunoprecipitated with PAK4 antibody (top panels) or LIMK1 antibody (bottom panels) and subjected to western blotting to ascertain LIMK1 and PAK4 interaction. d: In vitro kinase assay using purified activated PAK4 (S445N), kinase-defective PAK4 (K350M), PAK4 (WT), and LIMK1 protein. The amount of p-LIMK1, PAK4, and LIMK1 were measured using western blotting. e: Correlations between the protein level of PAK4 and the p-LIMK1 in human NSCLC tissues (n = 10). NC, negative control; IgG, immunoglobulin G

Fig. 6

LIMK1 overexpression rescued the effects of si-PAK4 on A549 and NCI-H520 cell migration and invasion. A549 or NCI-H520 cells were transiently transfected with Si-PAK4 or LIMK1 or both, and then seeded for migration (a) and invasion assay (b) under at × 40 original magnification. NC, negative control