Cross-talk between p21-activated kinase 4 and ERα signaling triggers endometrial cancer cell proliferation

Abstract

Cross-talk between estrogen receptor alpha (ERα) and signal transduction pathways plays an important role in the progression of endometrial cancer (EC). Here, we show that 17β-estradiol (E₂) stimulation increases p21-activated kinase 4 (Pak4) expression and activation in ER-positive EC cells. The estrogen-induced Pak4 activation is mediated via the PI3K/AKT pathway. Estrogen increases Pak4 and phosphorylated-Pak4 (p-Pak4) nuclear accumulation, and Pak4 in turn enhances ERα trans-activation. Depletion or functional inhibition of Pak4 abrogates EC cell proliferation induced by E₂, whereas overexpression of Pak4 rescues cell proliferation decreased by inhibiting the estrogen pathway. Pak4 knockdown decreases cyclin D1 expression and induces G1-S arrest. Importantly, Pak4 suppression inhibits E₂ induced EC tumor growth in vivo, in a mouse xenograft model. These data demonstrate that estrogen stimulation increases Pak4 expression and activation, which in turn enhances ERα transcriptional activity and ERα-dependent gene expression, resulting in increased proliferation of EC cells. Thus inhibition of Pak4-ERα signaling may represent a novel therapeutic strategy against endometrial carcinoma.

Keywords: cross-talk; endometrial carcinoma; estrogen receptor alpha (ERα); p21-activated kinase 4 (Pak4); proliferation.

Figure 1. Estrogen increases Pak4 expression and activation

(A-B) E₂ induces Pak4 mRNA and protein levels. Serum-starved Ishikawa, RL95-2 and MCF-7 cells were treated with 10 nM E₂, and cells were harvested at the indicated time. (A) The levels of Pak4 mRNA were determined by qRT-PCR, using β-actin as an internal control. Values represent mean ± s.d. (n = 3). ***P<0.001 compared with control. (B) The protein levels of Pak4 were assessed by Western blot. (C) Ishikawa and RL95-2 cells were serum-starved for 24 h, and then treated with 10 nM E₂ for indicated times. The levels of p-Pak4ser⁴⁷⁴ were measured by Western blot, using β-actin as a loading control. All experiments were carried out in triplicates.

Figure 2. E₂ activates Pak4 via PI3K/AKT pathway

(A) Ishikawa and RL95-2 cells were treated with 10 nM E₂ for up to 90 min, and Western blot was used to detect p-AKT Ser⁴⁷³ and total AKT levels. (B-C) RL95-2 cells were treated with E₂ for 60 or 90 min in the presence or absence of 20 μM LY 294002. Upper panel (B) p-AKT Ser⁴⁷³ and (C) p-Pak4ser⁴⁷⁴ levels were determined by western blotting, using β-actin as a loading control. Lower panel: Densitometric analysis of (B) p-AKT and (C) p-Pak4 in the immunoblots. Values represent mean ± s.d. (n = 3). *P< 0.05, ***P<0.001 compared with control, according to t-test. All experiments were carried out in triplicates.

Figure 3. E₂ promotes Pak4 and p-Pak4 nuclear accumulation

Immunofluorescence staining of (A) Pak4 and (B) p-Pak4 in RL95-2 cells treated with or without E₂. Original magnification×400, bar=25μm. (C) Immunoblot analyses of Pak4 and p-Pak4 in subcellular protein fractions extracted from RL95-2 cells (T, total celllysate; C, cytoplasmic fraction; N, nuclear fraction). Cells were serum-starved for 24 h, and then treated with 10 nM E₂, for indicated times.

Figure 4. Pak4 enhances ERα transcription and ERα target gene expression

(A) Left: Protein and mRNA levels of Pak4 were measured in wt Pak4-overexpressing Ishikawa cells by Western blot and qRT-PCR analysis, respectively; Right: ERα mRNA levels detected by qRT-PCR. Values are the mean ± SD from at least three independent experiments. (B) Left: Western blot and qRT-PCR of Pak4 levels in two different shPak4-transfected RL95-2 cells. Right: ERα mRNA levels detected by qRT-PCR. Values are the mean ± SD from at least three independent experiments. (C) Ishikawa cells were stably transfected with wt Pak4, ca Pak4, kinase-dead Pak4, or the control vector. (D) RL95-2 cells were stably transfected with two different shPak4 or the control vector. The ERE-Luc reporter plasmids were transfected into Ishikawa and RL95-2 cells 24 h before E₂ treatment, and luciferase assay was performed 48 h after E₂ addition. The mRNA levels of ERα target genes were determined by qRT-PCR. Cells were treated with 10 nM E₂ or vehicle for 48 h before RNA extraction. Values represent mean ± s.d. (n = 3), from three independent experiments. *P< 0.05, **P<0.01, ***P<0.001 compared with control, according to t-test. (E) Schematic representation of the estrogen response element and the primers used for ChIP–qPCR. ERE: estrogen response element. TSS: transcription start sites. (F-G) A summary of ChIP-qPCR results for ERα binding in RL95-2 cells with the primer pairs shown in (E). (F) shPak4 and control vector transfected RL95-2 cellswere treated with 10 nM E₂ for 48 h before DNA extraction. (G) RL95-2 cells were treated with 10 nM E₂ in the presence or absence of 1μM PF 3758309.

Figure 5. Pak4 inhibition suppresses E₂-induced cell proliferation and cell cycle progression

(A) Soft agar colony assays of Pak4 knockdown, Pak4 inhibitor PF 3758309 treated RL95-2 cells and control cells. Cells were cultured in the medium with or without E₂ for 2 weeks. (B) RL95-2 cells were transfected with wt Pak4, ca Pak4, kinase-dead Pak4, or the control vector. Cells were cultured in the medium with 10 nM E₂ and 100 nM ICI 182,780 for 2 weeks. Representative images (left) were captured with an inverted phase contrast microscope (magnification, ×200). Columns (right), represent the number of colonies from three independent experiments, each in triplicates; values represent mean ± s.d.; *P< 0.05, **P<0.01, ***P<0.001. (C) RL95-2 cells were stably transfected with shPak4 or control vector with GFP. The fluorescence images showing the transfection efficiency, as well as the decreased size of colonies in Pak4 knockdown cells compared with control cells. Original magnification, ×400. (D) MTT assay of Pak4 knockdown, Pak4 inhibitor PF 3758309 treated RL95-2 cells and control cells. Cells were either treated with E₂, vehicle or left untreated as indicated. (E) RL95-2 cells were transfected with wt Pak4, ca Pak4, kinase-dead Pak4, or the control vector. Cells were treated with 10 nM E₂, 10 nM E₂+ 100 nM ICI 182,780, or vehicle as indicated. All experiments were carried out in triplicates. (F) Cell-cycle profiles of shPak4 RL95-2 cells were assessed by FACS using DNA content profiles (left). Cells were either treated with E₂, vehicle, or left untreated for 96 h before measurement. The percentages of cells in each compartment were calculated (right). Values represent mean ± s.d. (n = 3). *P< 0.05, **P<0.01 compared with control, according to t-test. All experiments were carried out in triplicates.

Figure 6. Pak4 depletion inhibits tumor growth in vivo

(A) Growth rates of tumors in nude mice inoculated with shPak4 RL95-2 cells or control cells. Values represent mean ± s.d. (n = 5). **P<0.01 compared with control, according to t-test. (B) Immunohistochemical staining of Pak4 in control and shPak4 tumors.

Figure 7. Illustration of a positive feedback loop between Pak4 and ERα signaling in endometrial cancer

Estrogen increases Pak4 expression and activation via PI3K/AKT pathway, the increased and activated Pak4 in turn enhances ERα transcriptional activity and cyclin D1 expression, which facilitates EC cell proliferation.