Functional interaction of fibroblast growth factor 8b and androgen in prostate cancer cell proliferation

Abstract

Fibroblast growth factor 8b and androgen play important roles in cell proliferation of prostate cancer. We investigated the effects of fibroblast growth factor 8b and androgen on the proliferation of prostate cell lines and the corresponding intracellular mechanisms. It is found that dihydrotestosterone and fibroblast growth factor 8b stimulated Lncap cell mitosis in a concentration-responsive manner, with 30 ng/mL as the most suitable concentration, respectively. Dihydrotestosterone treatment alone did not enhance the expression and phosphorylation level of fibroblast growth factor receptor but significantly enhanced the level of fibroblast growth factor receptor phosphorylation elicited by fibroblast growth factor 8b. Phosphorylations of extracellular signal–regulated kinase, p38, and c-Jun NH2-terminal kinase were stimulated by dihydrotestosterone or fibroblast growth factor 8b. Among these major downstream pathways for mitogen-activated protein kinase, c-Jun NH2-terminal kinase signaling was most significantly enhanced. Protein kinase C phosphorylation was higher than AKT by the combined stimulation of dihydrotestosterone and fibroblast growth factor 8b. The phosphorylation of CDC2 was significantly induced by dihydrotestosterone and fibroblast growth factor 8b synergetically, and Smad underwent the same induction as CDC2. So the promoting effect of fibroblast growth factor 8b on cell cycle might contribute to the G2/M transition. This study indicated that the functional interaction between fibroblast growth factor 8b and androgen was essential for the prostate cancer cell proliferation.

Introduction

Prostate cancer is a kind of tumor depending on steroid hormones. This means that the proliferation of prostate cancer cells is affected by androgen receptor (AR) signaling pathway. As we know, cell proliferation is also mediated by growth factors, so the growth factor receptor pathway may have crosstalk with AR pathway in prostate cancer. Fibroblast growth factor 8 (FGF-8), a member of the FGF family, has been shown to play a central role in cell proliferation in both tumorigenesis and embryogenesis.^1,2 The FGF-8 gene contains a functional androgen-response element responsible for its transcriptional activation by AR signaling.³ It is reasonable to further detect the functional interactions between FGF-8 and androgen in prostate cancer. Meanwhile, FGF-8-activated intracellular signaling pathways in prostate cancer cells also need to be investigated.

In humans, there are four protein isoforms of FGF-8.⁴ Among them, the FGF-8b isoform is endowed with the strongest tumorigenic and angiogenic potential and is highly expressed in breast, prostate, and ovarian cancers.^1,5 In this study, we investigated the effects of FGF-8b and androgen on the proliferation of prostate cell lines and the corresponding intracellular mechanisms.

Materials and methods

Cell lines

The prostate cancer cell lines PC-3 (CLR-1435) and Lncap (CRL-1740) were obtained from American Type Culture Collection (ATCC) and cultured as follows: Lncap in RPMI 1640 (L0495; Bio-West, Logan, UT, USA), PC-3 in F-12K (21127-022; Life Technologies, Carlsbad, CA, USA) plus 10% fetal bovine serum (FBS; S181P; Bio-West) in a humidified incubator with a 5% CO₂ atmosphere at 37°C. Then, the indicated concentrations of dihydrotestosterone (DHT), FGF-8b, and the inhibitors were added, respectively, to the culture medium which has been replaced with serum-free fresh medium.

Western blot analyses

Total proteins were extracted using the CelLytic extraction kit containing protease inhibitors (Roche, Basel, Switzerland). The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membranes. After blocking in 5% nonfat milk, the primary antibodies and anti-rabbit horseradish peroxidase second antibody (1:5000) were used to probe the target proteins. The bands were visualized using the ECL Plus Western Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin were used as loading controls. The primary antibodies included anti–fibroblast growth factor receptor (FGFR)-1 (Flg), anti-FGFR-2 (Bek), anti-FGFR-3, and anti-FGFR-4 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); FGF-8b polyclonal antibody; anti-phospho-FGFR antibody; anti-phospho- and anti-total-extracellular-signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) antibody; anti-phospho- and anti-total-p38 MAPK antibody; anti-phospho- and anti-total-stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) MAPK antibody; anti-phospho and anti-total-AKT antibody; anti-phospho-PKC (protein kinase C) (pan) antibody; anti-total-Smad1/5/8 (pSmad1/5/8) antibody (Cell Signaling Technology, Inc., Beverly, MA, USA); anti-total-retinoblastoma protein (Rb), total-cell division cycle 2 (cdc2), and total-p53 antibodies (Cell Signaling Technology, Inc.); and anti-actin antibody (Sigma-Aldrich Co., Ltd, Shanghai).

Cell counting kit-8 assays

Cell counting kit (CCK)-8 (Dojindo, Shanghai, China) assays were carried out to measure cell proliferation according to the manufacturer’s instructions. After attaching to the bottom of the wells, cells were cultured in the medium mixed with CCK-8 (10:1) for 2 h. Absorbance was measured by a microplate reader at 450 nm.

Statistical analysis

All statistical analysis was completed using SPSS version 17.0 (IBM Corporation, Somers, NY, USA). Continuous variables were analyzed using independent t tests. Categorical variables were analyzed with Pearson’s chi-square tests. Values of p < 0.05 were considered statistically significant.

Results

Effects of DHT and FGF-8b on prostate cancer cells were firstly examined. Figure 1 demonstrated the results of the cell viability assay. DHT and FGF-8b stimulated Lncap mitosis in a concentration-responsive manner, with 30 ng/mL as the most suitable concentration, respectively (Figure 1(a) and (b)). FGF-8b also stimulated PC-3 cell lines proliferation, which could be suppressed by SU5402, an FGFR-dependent protein kinase inhibitor (Figure 1(c)). The combined effects of DHT and FGF-8b on Lncap cell proliferation were higher than the individual effects of both drugs (Figure 1(d)), and these combined effects could be abolished by SU5402 alone, or by bicalutamide (AR antagonist) alone (Figure 1(e)). This suggested that both signaling of AR and signaling of FGFR were important during the proliferation of prostate cancer cells.

Figure 1.

Effects of androgen and FGF-8b on prostate cancer cell proliferation. (a) Dihydrotestosterone (DHT) and (b) FGF-8b stimulated Lncap cell proliferation with 30 ng/mL as the most suitable concentration, respectively. (c) FGF-8b induced PC-3 proliferation with 30 ng/mL as the most suitable concentration, and SU5402 suppressed this effect. (d) The combined effects of FGF-8b and androgen on Lncap cell proliferation. (e) SU5402 or bicalutamide suppresses the combined effects of FGF-8b and androgen on Lncap cell proliferation.

[Figure omitted. See PDF]

The activities of FGFR in Lncap cells were then examined as well as the effects of FGF-8b and DHT (Figure 2). FGFR expressed in Lncap cells and could be phosphorylated by treatment of FGF-8b, while SU5402 could inhibit this effect. DHT treatment alone did not enhance the expression level and phosphorylation level of FGFR but significantly enhanced the level of FGFR phosphorylation elicited by FGF-8b. Moreover, SU5402 alone or bicalutamide alone could reverse the enhanced levels of phosphorylated FGFR induced by FGF-8b and DHT. These results indicated that FGFR signaling pathway might have crosstalk with AR signaling pathway in prostate cancer cells, and through this crosstalk, androgen affected the FGF-8b-induced proliferation of cancer cells.

Figure 2.

The activities of FGFR and the effect of FGF-8b and androgen on FGFR in Lncap cells. (a) Dihydrotestosterone (DHT) treatment alone did not enhance the expression level and phosphorylation level of FGFR but significantly enhanced the level of FGFR phosphorylation elicited by FGF-8b. (b) SU5402 alone or bicalutamide alone could reverse the enhanced levels of phosphorylated FGFR induced by FGF-8b and DHT.

[Figure omitted. See PDF]

Intracellular signaling pathway was then examined by immunoblots. As shown in Figure 3, phosphorylations of ERK, p38, and SAPK/JNK were stimulated by DHT or FGF-8b. Among these major downstream pathways for MAPK, JNK signaling was most significantly enhanced by the combined treatments with FGF-8b and androgen. Besides, PKC phosphorylation was higher than AKT by the combined stimulation of androgen and FGF-8b.

Figure 3.

Effects of FGF-8b and androgen on intracellular signaling pathway in Lncap cells. Among the major downstream pathways for MAPK, JNK signaling was most significantly enhanced, and PKC phosphorylation was higher than AKT.

[Figure omitted. See PDF]

The key cell cycle regulators were also tested, including CDC2 (a G2/M regulator), Rb (a G1/S regulator), and p53 (a regulator for G2/M and G1/S transition). Among them, the phosphorylation of CDC2 was most significantly induced by androgen and FGF-8b synergetically, and Smad underwent the same induction as CDC2 (Figure 4).

Figure 4.

Cell cycle checkpoints affected by androgen or FGF-8b. The phosphorylations of CDC2 and Smad were significantly induced by androgen and FGF-8b synergetically.

[Figure omitted. See PDF]

Discussion

FGF-8 is an important growth factor, which mainly expresses in the steroid-responsive tissues, such as kidney, breast, prostate, and testis of adult human, and functions autocrinely/paracrinely in the growth of epithelial/stromal cells.¹ FGF-8 also has increased expression in human steroid-responsive tumors, such as breast cancer and prostate cancer.^6,7 It is reasonable that there are possible functional interactions between FGF-8 and steroid-receptor signaling pathway and a possible role of steroids in the tissue-specific expression of FGF-8.⁸ FGF-8 gene expression could be reduced by deprivation of testosterone and be induced again by only 8 h of testosterone stimulation.⁷ Besides, the inappropriate activation of FGF-8b might overcome steroid hormone sensitivity of these tumors, resulting in the progression toward a refractory neoplasm insensitive to hormone deprivation therapy.^3,9,10 In this study, we can see that the combined effects of DHT and FGF-8b on Lncap cell proliferation were higher than the individual effects of both drugs. So FGF-8b and the corresponding signaling pathway may serve as a therapeutic target of prostate cancer in the future.

The FGF ligand and FGFR complex are essential for the downstream signaling pathway. The concentration of FGF ligand could determine the intracellular pathways and the outcome in response to this stimulation, especially during embryonic development.¹¹ Gradients of FGF played a key role in tissue modeling process such as limb development.¹² In this study, the concentration of 30 ng/mL represented the most suitable one for FGF-8b to stimulate the proliferation of prostate cancer cells. The expression pattern of FGFR-splicing variants also affects the intracellular responses of FGFs.¹³ It is reported that FGF-8b binds FGFR4 and the c isoforms of FGFR1–3 instead of b isoforms.¹⁴ In this study, we just concluded that FGFR mediated the effect of FGF-8b in Lncap cell lines, while more details such as the specific type and isoform still need to be investigated.

The basic principles and mechanisms of FGFR signaling pathways have been established.^13,15 But FGF-8-activated intracellular signaling pathways and target genes in prostate cancer cells still need to be investigated. Among intracellular signal transduction cascades mediated by FGFR, the major pathways are MAPK, phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC). Of MAPKs, activation of MEK and ERK was important in the FGFR-signaling axis,¹⁶ while p38 was involved in FGF-induced cytoskeletal changes in prostate cancer cells.¹⁷ In this study, we found that ERK and p38 was moderately activated and JNK phosphorylation was relatively higher by the combined stimulation of androgen and FGF-8b. Besides, PKC phosphorylation was also significantly enhanced by the combined treatments with FGF-8b and androgen. This meant that PLC pathway was also highly activated. However, the PI3K pathway has shown a high basal activity in Lncap cells and did not change much by the treatment of FGF-8b and DHT synergetically.

Abnormity of cell cycle progression is one of the most common characteristics of human malignancies, including prostate cancer. There are cell cycle checkpoints that could reflect the cell cycle progression, such as Rb (G1/S regulator), p58 (regulator for G2/M and G1/S), and CDC2 (G2/M regulator). In this study, the level of Rb or p58 slightly changed in Lncap cells during the period of exposure to FGF-8b or androgen, and CDC2 highly expressed after combined stimulation of FGF-8b and androgen. This indicated that the promoting effect of FGF-8b on cell cycle might contribute to the G2/M transition, which was associated with CDC2 and Smad. However, the detailed mechanisms remained to be elucidated.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was supported by the Research Projects of Shanghai Municipal Commission of Health and Family Planning (grant no. 20164Y0256).