PAK5-mediated phosphorylation and nuclear translocation of NF-κB-p65 promotes breast cancer cell proliferation in vitro and in vivo

Abstract

Background: Abnormal proliferation is significantly associated with the promotion of malignant tumor. Growing evidence suggest that the signal pathways of p21^cdc42/rac1-activated kinase 5 (PAK5) have been found in various tumor progression, however, the role of PAK5 in breast cancer remains largely unclear.

Methods: We evaluated PAK5 and p65 staining in breast cancer tissues (BCTs) and paired non-cancerous tissues (NTs) using tissue microarray (TMA) technology. The functions of PAK5 were studied in vitro and in vivo. Cell Counting Kit-8 (CCK-8) and flow cytometry were performed to determine proliferation of breast cancer cells. Phosphorylation assay and co-immunoprecipitation (co-IP) were employed to identify the regulation mechanism of p65 by PAK5. The activation of Cyclin D1 promoter was measured with luciferase reporter assay. Xenograft models in nude mice were established to explore the roles of PAK5 in breast cancer growth.

Results: In this study, we show that PAK5 is highly expressed in breast cancer tissues and the increased PAK5 is significantly associated with breast cancer progression. Overexpression of PAK5 promotes the proliferation and cell-cycle progression by increasing the expression of Cyclin D1 in vitro and in vivo. Mechanistic studies demonstrated that PAK5 can promote the phosphorylation and the nuclear translocation of p65 subunit of nuclear factor-kappaB (NF-κB). Furthermore, p65 can directly bind to the promoter of Cyclin D1 and mediate an increase in its protein expression.

Conclusions: Taken together, our findings suggest that PAK5 may serve as a potential prognosis marker and therapeutic target for human breast cancer.

Keywords: Cell proliferation; Cyclin D1; PAK5; p65.

Fig. 1

PAK5 correlates with a worse prognosis in breast cancer patients. a Representative images of PAK5 and p65 immunohistochemical staining in human breast cancer. Magnification × 400 for a. b PAK5 expression staining was higher in BCTs than in paired adjacent NTs. c p65 expression staining was higher in BCTs than in paired adjacent NTs. d High PAK5 expression correlated with a poorer 5-year overall cumulative survival for 129 breast cancer patients (P = 0.0192, log-rank test). e High p65 expression did not correlate with 5-year overall cumulative survival for 119 breast cancer patients (P = 0.1933, log-rank test)

Fig. 2

The effect of PAK5 on breast cancer cells proliferation. a CCK-8 cell proliferation assays after PAK5 overexpression in BT549 and MDA-MB-231 cells. b CCK-8 cell proliferation assay after PAK5 knockdown in BT549 and MDA-MB-231 cells. c Effects of PAK5 overexpression on the cell cycle of breast cancer cells. The percentage of G1 population cells was measured by flow cytometry after PAK5 overexpression in BT549 and MDA-MB-231 cells. d Effects of PAK5 silencing on cell cycle analysis of BT549 and MDA-MB-231 cells. The percentage of cells at G1 stage was calculated using ModFit LT 3.0 software. Data are presented as mean ± SD for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 3

Impact of PAK5 on the expression of cell cycle regulatory proteins. a, b Western blot analysis of the PAK5 gene overexpression and silencing on Myc, PAK5, Cyclin D1, Cyclin D3, Cyclin A, Cyclin A1, Cyclin B1 and Cyclin B3 in BT549 and MDA-MB-231 cells. c, d Western blot analysis of the PAK5 gene overexpression and silencing on p21 and p27 in BT549 and MDA-MB-231 cells. Data are shown as mean ± SD for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 4

PAK5 activates the nuclear translocation of the p65. a Western blot of intracellular p65 from BT549 and MDA-MB-231 cells transfected with the pcDNA3.1-Myc-PAK5 plasmid or vector control. b Western blot of intracellular p65 from BC cells transfected with the PAK5 siRNA or control siRNA. c, d Western blot determined cellular distribution of p65 in BT549 and MDA-MB-231 cells transfected with the pcDNA3.1-Myc-PAK5 plasmid or vector control. e, f Western blot determined cellular distribution of p65 in BC cells transfected with PAK5 siRNA or control siRNA. g, h Immunofluorescent staining of MDA-MB-231 and BT549 cells analyzed by confocal microscopy (magnification, × 400). The antigenic sites of p65 were detected using Alexa Fluor 488 conjugated goat anti-mouse IgG (green). Data are shown as mean ± SD for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Fig. 5

PAK5 interacts and phosphorylates p65. a MDA-MB-231 cells were transfected with pcDNA3.1-Myc-control plasmids and pcDNA3.1-Myc-PAK5 plasmids. Lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Myc and anti-p65 antibody. b MDA-MB-231 cells were transfected with pcDNA3.1-Myc-control plasmids, pcDNA3.1-p65 plasmids and pcDNA3.1-Myc-PAK5 plasmids. Lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Myc, and anti-p65 antibody. c MDA-MB-231 cells transfected with pcDNA3.1-Myc-control plasmids and pcDNA3.1-Myc-PAK5 plasmids. Then concentrated protein was used for WB. The top p-p65 represents the mobility shift of phosphorylated p65 proteins in SDS-PAGE with polyacrylamide-bound Mn²⁺-Phos-tag. The bottom p65 represents non-phosphorylated counterpart in SDS-PAGE with polyacrylamide-bound Mn²⁺-Phos-tag. d, e Luciferase reporter assay was carried out in BT549 and MDA-MB-231 cells. The relative luciferase activities of pcDNA3.1 + pGL3-basic, pcDNA3.1-p65 + pGL3-basic and pcDNA3.1-p65 + pGL3-Cyclin D1 groups normalized against pcDNA3.1 + pGL3-Cyclin D1 group. Data are shown as mean ± SD for three independent experiments. **, P < 0.01

Fig. 6

PAK5 promotes the tumor development of mice. a Xenograft tumor formation of MDA-MB-231 cells in nude mice. b, c The volume and weight of tumor cancer models were analyzed and calculated. d Western blotting analyzed the protein levels of PAK5, p65 and Cyclin D1 in harvested tumor tissues. e Representative images PAK5 and p65 immunohistochemical staining with PAK5 antibody in control and PAK5^OE groups tumor models. *, P < 0.05