Evaluation of FOXA1 Protein Expression in PC-derived Cell Lines

We next investigated FOXA1 mRNA and protein expression in PC by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis and Western blotting respectively, of four PC-derived cell lines, including androgen independent cell line (PC-3 and DU145), androgen sensitive cell line (LNCaP), androgen insensitive cell line (C4-2) established from castrated host of LNCaP and non-tumorigenic prostate cell line (RWPE-1) [16]–[19] (Figure 2A). The molecular weight of FOXA1 by Western blotting was 49 kDa. Both FOXA1 mRNA and protein expression were significantly higher in the androgen-sensitive LNCaP cells and C4-2 cells than in the other cell lines.

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Figure 2

FOXA1 regulates AR activity in LNCaP cells.

(A) The mRNA expression and the protein expression of FOXA1 in four PC-derived cell lines (PC-3, DU-145, LNCaP, C4-2) and in non-tumorigenic prostate cell line (RWPE-1) were analyzed using qRT-PCR and Western blotting, respectively. (B) LNCaP cells were transfected with a negative control siRNA (nega) or with one of three different FOXA1 siRNAs (siFOXA1#1–3). After 48 h transfection, RNA was extracted and FOXA1 mRNA was analyzed using qRT-PCR analysis. FOXA1 mRNA expression is expressed relative to GAPDH mRNA expression in the same cell. The results shown are the means ± S.E.M. of three independent experiments. Proteins were extracted 72 h after transfection and FOXA1 and GAPDH protein expression was examined by Western blot analyses. (C, D) LNCaP cells were transfected with the pGL3-PSAp 5.8 luciferase reporter plasmid and with one of three different FOXA1 siRNAs. (#1–3) and were then grown in Phenol red–free RPMI-1640 medium containing 5% CS-FBS. (C) After 72 h transfection, luciferase activities were measured using the Dual-luciferase reporter assay system. Luciferase activities are expressed relative to the control, which was assigned a value of 1. Proteins were extracted 72 h after transfection and the expression of AR and GAPDH was examined by western blot analysis. (D) The transfected cells were incubated with the indicated concentrations of dihydrotestosterone for 72 h, and luciferase activities were measured as in (C). (E, F) Non-transfected LNCaP cells were treated with the indicated concentrations of dihydrotestosterone (E) or bicalutamide (F) for 48 h and FOXA1 mRNA expression was then analyzed using qRT-PCR. FOXA1 mRNA expression is expressed relative to GAPDH mRNA expression in the same cell. Values shown are the means ± SD from at least 3 independent experiments, each performed in triplicate. * and ** indicate statistical difference of P<0.05 and P<0.01, respectively.

To obtain cells in which FOXA1 expression was transiently knocked down, we transfected LNCaP cells with one of three different FOXA1 siRNAs (siFOXA1#1–3) plasmids or with a negative control siRNA (nega) plasmid. We then analyzed FOXA1 mRNA and protein expression in these siFOXA1-transfected cells using qRT-PCR and Western blot analyses, respectively. SiFOXA1-transfected cells shows reduction of FOXA1 mRNA level by almost 90% compared to that in nega-transfected cells (nega). FOXA1 protein levels in siFOXA1-transfected cells were also strongly decreased compared with nega-transfected cells (Figure 2B).

The Effect of FOXA1 on Androgen Receptor Activity and the Effect of AR Activation on FOXA1 Expression

To determine the effect of FOXA1 knockdown on AR activity, we transfected the siFOXA1-transfected LNCaP cells with a luciferase expression plasmid driven by the prostate-specific antigen (PSA) promoter, pGL3PSAp-5.8. Although there was no difference in the protein expression of AR in siFOXA1-transfected cells or in nega-transfected cells, siFOXA1-transfected cells showed almost 80% reduction of PSA promoter activity compare to that of nega-transfected cells (Figure 2C). When control LNCaP cells that were only transfected with pGL3PSAp-5.8 were treated with the AR ligand dihydrotestosterone (DHT), PSA promoter activity was increased in a dose-dependent manner. However, co-transfection of these cells with siFOXA1 significantly reduced PSA promoter activity following treatment with various concentrations of DHT (Figure 2D). To look at the influence of FOXA1depletion on endogenous PSA expression, as FOXA1 acts to remodel chromatin, we analyzed PSA mRNA expression in siFOXA1-transfected cells using qRT-PCR. Figure S1A shows that FOXA1 depletion mediated significant reduction of PSA mRNA level compared to that in nega-transfected cells.

To determine whether FOXA1 mRNA expression is mediated by AR activity, we tested the effect of treatment of LNCaP cells with DHT and or with the AR antagonist bicalutamide on FOXA1 mRNA expression. QRT-PCR analysis showed that there was no difference in the mRNA expression of FOXA1 in androgen-sensitive LNCaP cells following growth in medium containing DHT or in medium containing bicalutamide (Figure 2E and F). These results indicated that FOXA1 mediates activation of AR, however, expression of FOXA1 itself is not regulated by androgen.

FOXA1 Regulates Cell Proliferation in PC Cells

To investigate the effect of FOXA1 knockdown on cell proliferation, the cell growth of siFOXA1-transfected LNCaP cells was monitored over 4 days. The siFOXA1-transfected cells showed a significant decrease in cellular growth compared with nega-transfected cells (Figure 3A). On the other hand, FOXA1 overexpressing cells significantly increased cellular growth compared with nega-transfected cells (Figure S1C). To investigate effect of FOXA1 in cell cycle progression, we analyzed the cell cycle stages of siFOXA1-transfected cells using flow cytometric analysis. The percentage of siFOXA1-transfected cells in the G0/G1 phase was higher than that of nega-transfected cells (Figure 3B), suggesting that down-regulation of FOXA1 inhibited cellular proliferation by induction of G0/G1 arrest.

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Figure 3

FOXA1 regulates PC cell proliferation in IGFBP-3 dependent manner.

(A) LNCaP cells were transfected with a negative control siRNA (nega) or with one of three different FOXA1 siRNAs (#1–#3), and were then reseeded into a 24-well culture plate. Cell proliferation was then assayed over 4 days by counting the number of cells for each transfection condition, as indicated. (B) The cell-cycle stage of the cells was analyzed using propidium iodide staining and FACS analysis 24 h after siRNA transfection and the percentage of cells in each transfected population that was in each cycle phase was calculated. (C) LNCaP cells were transfected with a negative control siRNA (nega) or with one of three different FOXA1 siRNAs (siFOXA1#1–3). After 48 h of transfection IGFBP-3 mRNA expression was analyzed by qRT-PCR. IGFBP-3 mRNA expression is expressed relative to GAPDH mRNA expression in the same cell. (D) LNCaP cells were transfected with a negative control siRNA (nega) or with one of three different FOXA1 siRNAs (siFOXA1#1–3). After 72 h of transfection, IGFBP-3 concentration of the media was analyzed by ELISAs. (E) Proteins were extracted 72 h after transfection and the expression of IGFBP3, of the insulin-receptor signaling related proteins (total amount of MAPK, phospho-MAPK, total amount of Akt and phospho-Akt), of G0/G1 arrest-related cell cycle regulatory proteins (p21cip1, p27kip1) and of GAPDH was examined by western blot analysis. Protein molecular weights are indicated at right sides of the panel. These results are the means ± S.E.M. of three independent experiments. **indicates a statistical difference of P<0.01.

Depletion of FOXA1 Regulates IGFBP-3 and IGF Signaling

To gain an insight into genes that are up-regulated by FOXA1 depletion of PC cells, we performed gene expression microarray analysis of cells depleted of FOXA1 (Table 3 and ​and4).4). We identified 421 genes that were more strongly expressed in FOXA1-depleted cells than in the negative control transfectants. Clustering of the up-regulated genes into functional categories indicated that the highest proportion of up-regulated genes were related to cell proliferation, followed by response to wounding, muscle system processes, defense responses, inflammatory responses, negative regulation of cell proliferation, response to hypoxia and cell maturation (Table 3). The up-regulated genes that are related to negative regulation of cell proliferation are listed in Table 4. Of these up-regulated genes, we further studied insulin-like growth factor binding protein 3 (IGFBP-3), since this gene is a well-established tumor-suppressive gene in PC [20]–[23].

To confirm the up-regulation of IGFBP-3 in LNCaP cells by FOXA1 depletion that was indicated by the microarray data, we analyzed IGFBP-3 mRNA and protein expression in LNCaP cells transfected with siFOXA1 plasmids or with the negative control siRNA plasmid, using qRT-PCR and Western blot analyses, respectively. Figure 3C shows that IGFBP-3 mRNA expression in siFOXA1-transfected cells was significantly higher than that in nega-transfected cells. IGFBP-3 protein levels in siFOXA1-transfected cells were also strongly increased compared with the level in nega-transfected cells (Figure 3E). Moreover, to evaluate changes in the levels of secreted IGFBP-3, we performed ELISAs. Figure 3D shows that IGFBP-3 concentration in the media of siFOXA1-transfected cells was also strongly increased (over 30 times) compared with that of nega-transfected cells. Since IGFBP-3 modulates IGF-1 signaling, we next investigated if FOXA1 regulation of IGFBP-3 expression was associated with modulation of the expression level or activation status of IGF-1 downstream signals in these cells (Figure 3E). Western blot analysis indicated marked up-regulation of IGFBP-3, down-regulation of the phosphorylation of the MAPK and Akt, and up-regulation of the protein expression of the cyclin-dependent kinase inhibitors (p21cip1, p27kip1) in siFOXA1-transfected cells compared to nega-transfected cells (Figure 3E). Furthermore, FOXA1 depletion significantly blocked the activation of AKT in response to IGF-1 treatment in LNCaP (Figure S1E). These results showed that FOXA1 regulation of IGFBP-3 is accompanied by regulation of IGF-1 downstream signals.

In order to determine the importance of IGFBP-3 for FOXA1 regulation of PC proliferation, we compared the growth of siFOXA1-transfected LNCaP cells and that of siIGFBP-3-transfected cells. IGFBP-3 was transiently knocked down by transfection of LNCaP cells with the IGFBP-3 siRNA (siIGFBP-3) plasmid and IGFBP-3 mRNA and protein expression in the siIGFBP-3-transfected cells was assessed using qRT-PCR and western blot analysis, respectively. Figure 4A shows that IGFBP-3 mRNA and protein expression in the siIGFBP-3-transfected cells was significantly lower than that in the nega-transfected cells. Although the siFOXA1-transfected cells showed a significant decrease in cellular growth compared with nega-transfected cells (Figure 3A and ​and4B),4B), the anti-proliferative effect of siFOXA1 was reversed by dual transfection of siFOXA1 and siIGFBP-3 (Figure 4B). This result indicated that FOXA1 regulates cell proliferation in an IGFBP-3-dependent manner.

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Figure 4

Regulation of IGFBP-3 expression and AR activity through FOXA1.

(A) LNCaP cells were transfected with a negative control siRNA (nega) or with an IGFBP-3 siRNA (siIGFBP-3) and, 48 h later, IGFBP-3 mRNA and protein levels were analyzed using qRT-PCR and Western blot analysis. IGFBP-3 mRNA expression is expressed relative to GAPDH mRNA expression in the same cell. (B) LNCaP cells were transfected with a negative control siRNA (nega) or with one of three FOXA1 siRNAs (siFOXA1#1–#3) and/or an IGFBP-3 siRNA (siIGFBP-3), and were reseeded into a 24-well culture plate. Cell proliferation was measured over 4 days and the number of cells for each transfected population is indicated. (C, D) LNCaP cells were transfected with a negative control siRNA (nega) or with an AR siRNA (si AR) and/or a FOXA1 siRNA (si FOXA1#1) and, 48 h after transfection, AR mRNA and protein (C) or IGFBP-3 mRNA levels (D) were analyzed using qRT-PCR and/or western blot analysis. The mRNA levels are expressed relative to GAPDH mRNA expression in the same cell. The results shown are the means ± S.E.M. of three independent experiments. ** indicate statistical difference of P<0.01.

Since activation of AR has been reported to down-regulate IGFBP-3 expression [24], we further studied the effect of AR on FOXA1 mediated regulation of IGFBP-3 using siAR-transfected cells (Figure 4C and 4D). Depletion of AR did increase IGFBP-3 mRNA expression (2.8 times). However, the increase in IGFBP-3 expression observed in siFOXA1-transfected cells was much higher (16.4 times) than that in siAR-transfected cells. Moreover, simultaneous depletion of AR and FOXA1 resulted in a further increase in IGFBP-3 expression (24.0 times) compared with depletion of FOXA1 alone, suggesting that FOXA1 and AR cooperatively regulate IGFBP-3 expression (Figure 4D).

Reagents and Antibodies

Lipofectamine 2000 and Lipofectamine™ RNAiMax reagent, siRNAs siFOXA1 (#1–3) (Stealth Select RNAi™ siRNA; HSS104878, HSS104880 and HSS179280, respectively), siIGFBP-3 (Stealth Select RNAi™ siRNA, HSS105267), siAR (Stealth Select RNAi™ siRNA, HSS100619) and negative universal control siRNA (Stealth™ RNAi) were purchased from Invitrogen (Carlsbad, CA, USA). Anti-FOXA1 (C-20) and anti-IGFBP-3 antibodies (H-98) were obtained from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA). Anti-AR [AR441] (ab9474) was obtained from Abcam. Anti-p21cip1 and anti- p27kip1 were from Becton Dickinson Biosciences (San Diego, CA, USA). Anti-Akt, anti-phosphorylated Akt (Ser473), anti-p44/42 MAPK and anti-phosphorylated p42/44 MAPK were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH was purchased from Applied Biosystems (Foster, CA, USA). The luciferase reporter plasmid, pGL3–5.8PSAp, which is driven by a 5.8-kb PSA promoter that includes androgen-response elements, was kindly provided by Dr. A. Mizokami at Kanazawa University, Ishikawa, Japan. DHT, bicalutamide and IGF-1 were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan).

PC Tissue Specimens

PC tissue samples were obtained from 52 patients who underwent radical prostatectomy for PC at Chiba University Hospital between 2006 and 2010 and from whom pretreatment information was available. Pathological evaluation of these tissues was confirmed. Histopathological analysis of the tissues was performed according to the World Health Organization criteria by the Department of Pathology, Chiba University Hospital. Clinicopathological staging was determined by the TNM classification of the International Union Against Cancer. All patients had PC that was histologically confirmed, and tumor samples were checked to ensure that tumor tissue was present in the specimen.

Cell Culture and Transfection

PC-3, DU145, LNCaP and C4-2 cell lines, derived from human PCs, as well as the normal human prostate cell line RWPE-1, were obtained from the American Type Culture Collection (Manassas, VA, USA). The PC cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), and were maintained in an incubator with a humidified atmosphere of 95% air and 5% CO₂ at 37°C. For steroid-free conditions, Phenol red-free RPMI-1640 medium was used with 5% charcoal-stripped FBS (CS-FBS).

PC cells were transfected with DNA and/or siRNA using Lipofectamine 2000 and/or Lipofectamine™ RNAiMax reagent (Invitrogen), according to the manufacturer’s instructions.

Evaluation of FOXA1 mRNA Expression

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized using the ImProm-II™ Reverse Transcription System with random primers (Promega, Tokyo, Japan). QRT-PCR was performed using a LightCycler® 480 Real-Time PCR System (Roche Diagnostics GmbH, Mannheim, Germany). The PCR reactions were carried out in a final volume of 20 µl of a reaction mixture of LightCycler 480 Probe Master (Roche). The initial PCR step consisted of a 5-min hold at 95°C, followed by 45 cycles of 10-sec denaturation at 95°C, 30-sec annealing/extension at 60°C and 1-sec signal acquisition at 72°C. TaqMan® probes and primers for FOXA1 (P/N: Hs00270129_m1), AR (P/N: Hs00171172_m1), IGFBP-3 (P/N: Hs00181211_m1) (Applied Biosystems) and PSA (4410629-T; Roche Diagnostics GmbH, Mannheim, Germany) were used. The transcript amounts for the target genes were estimated from the respective standard curves and were normalized to the amount of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A/N: NM_002046) (Applied Biosystems) transcript that was determined in corresponding samples.

To study the effect of DHT treatment, LNCaP or C4-2 cells were incubated in phenol-red free RPMI-1640 medium containing 5% CS-FBS for 36 h. The cells were then seeded and treated with the indicated concentrations of DHT for 48 h. To study the effect of bicalutamide treatment, LNCaP cells were incubated in RPMI-1640 medium containing 10% FBS with the indicated concentrations of bicalutamide for 48 h. After incubation, total RNA was extracted, cDNA was synthesized and qRT-PCR was performed.

Luciferase Assay

Luciferase Assay were performed according to the protocol described previously [41]. Briefly, LNCaP or C4-2 cells were seeded at a density of 5×10⁴ cells/well into a 24-well culture plate. After 24 h, the medium was changed to phenol red-free RPMI-1640 medium containing 5% FBS and the cells were treated with the indicated concentrations of DHT without antibiotics. The cells were then transfected with 0.4 mg of the luciferase reporter plasmid, pGL3-PSAp 5.8 and siFOXA1(#1–3) using Lipofectamine 2000 (Invitrogen). Cell lysates were prepared 72 h after transfection and luciferase activities were measured using the Dual-luciferase reporter assay system (Promega).

Western Blot Analysis

Protein expression was determined by immunoblotting using the specific antibodies as previously described [42]. Protein expression levels were normalized to GAPDH expression. Cells were harvested 72 h after transfection. Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (150 mmol/L NaCl, 50 mmol/L Tris (pH 8.0), 0.5% deoxycholic acid, 1% NP40 and 1 mmol/L phenyl methyl-sulfonyl fluoride). Protein content was quantified using the bicinchoninic acid protein assay kit (Thermo, Waltham, MA, USA), and protein samples (30 µg) were subjected to SDS-PAGE and transferred to Hybond-C membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked in 5% milk in TBS-T (TBS containing 0.05% Tween 20; 1 h at room temperature), and, following incubation with the respective primary antibody (1100 dilution, overnight at 4°C), membranes were exposed to species-specific horseradish peroxidase-labeled secondary antibodies. Signals were detected using the ECL Plus Western Blotting Detection System (GE Healthcare, Little Chalfont, UK) and were visualized using LAS-4000 mini (Fujifilm, Tokyo, Japan).

IHC

IHC was performed on 4-µm sections of paraffin-embedded specimens using goat anti-FOXA1 polyclonal antibody (C-20, Santa Cruz Biotechnology). Briefly, after deparaffinization and hydration, the slides were treated with endogenous peroxidase in 0.3% hydrogen peroxide solution in 100% methanol for 30 min, after which the sections were blocked for 2 h at room temperature with 1.5% blocking serum (Santa Cruz) in PBS before reacting with anti-FOXA1 antibody (1100 dilution) at 4°C in a moist chamber overnight. Following incubation with the primary antibody, the specimens were washed three times in PBS and were then treated with the Envision reagent followed by color development in 3,3′-diaminobenzidine tetrahydrochloride (Dako, Carpinteria, CA). Finally, the slides were lightly counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted. To avoid non-specific binding, an immunizing peptide blocking experiment was performed. As a negative control, triplicate sections were immunostained without exposure to primary antibodies, thus confirming the staining specificity [42]. To quantify the level of FOXA1 protein expression in those stained slides, we used a previously described IHC scoring system with minor modification [43], [44]. Briefly, the mean percentage of FOXA1-positive tumor cells in each section was determined by analysis of at least five random fields at ×400 magnification. The intensity of the FOXA1 immunoreactive signal was scored as follows: 1+, weak; 2+, moderate; 3+, intense. The percentage of positive tumor cells and the staining intensity of each section were then multiplied to produce the FOXA1 IHC score. Cases with a FOXA1 IHC score >123.0 (the highest score for normal tissue) were defined as positive. These judgments were made by two independent pathologists, neither of whom had any knowledge or information pertaining to the patients’ clinical status.

Growth Assay

Cells were trypsinized and equal numbers (1×10⁴ cells) were seeded in a 24-well culture plate for all transfections. After 24 h, the media was changed to fresh complete media (10% FBS) and cells were transfected with siRNA using Lipofectamine™ RNAiMax reagent (Invitrogen) or FOXA1 expressing vector (Thermo Scientific Cat #MHS1010-7296058 CO USA) using Lipofectamine™2000, according to the manufacturer’s instructions and incubated for 4 days. The cells were trypsinized and counted at each indicated time point. To study the effect of bicalutamide treatment, LNCaP or C4-2 cells were seeded and transfected with siRNA. After 48 h, the media was changed to the indicated concentrations of bicalutamide for 24 h. After incubation, the cells were trypsinized and counted.

Enzyme-linked Immunosorbent Assays (ELISAs)

LNCaP or C4-2 cells were seeded and transfected with siRNA. After 72 h, concentration of IGFBP-3 in the media was measured using Human IGFBP-3 Quantikine ELISA Kit (R & D Systems), according to the manufacturer’s instructions.

Cell Cycle Analysis

Cells were fixed with 70% ethanol (30 min at 4°C), stained with propidium iodide (50 µg/mL) containing 20 µg/mL of RNase (30 min at room temperature), and were subsequently analyzed by flow cytometric analysis using a FACS Canto™ II (Becton Dickinson). The fractions of the cells in the G0–G1, S, and G2–M phases were analyzed using Flow Jo software (Tree Star, Ashland, OR).

Target Gene Search for FOXA1

The human 44 K oligo-microarray (Agilent Technologies, Santa Clara, CA, USA) was used for expression profiling of siFOXA1-transfected cells and of nega-transfected cells. Hybridization and wash steps were performed according to Agilent protocols. The arrays were scanned using a Packard GSI Lumonics ScanArray 4000 (Perkin Elmer, Boston, MA, USA). The microarray data were analyzed using DNASIS array software (Hitachi Software Engineering, Tokyo, Japan), which converted the signal intensity of each spot into text files. The log2 ratios of the median subtracted background intensities were analyzed. Functional categories to which the up-regulated genes belonged were analyzed using gene-analysis software DAVID (http://david.abcc.ncifcrf.gov/). The DAVID functional annotation tool [45], [46] was used to classify gene sets based on Gene Ontology identifiers. Functional categories with a Benjamini-Hochberg statistic less than 0.05 were considered to be statistically significant.

This work was supported by lab technician, Hisayo Karahi, Akemi Otsuka and Noriko Yamamoto.