Research ArticleExperimental Studies

Modulation of PGE2-induced EP4 Expression on Snail Signaling and the Impact on Epithelial-Mesenchymal Transition: Significance of EP4 Antagonism

HYE NA KIM, NARAYANAN K. NARAYANAN, SALAMIA LASANO and BHAGAVATHI NARAYANAN
Anticancer Research December 2011, 31 (12) 4347-4357;

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Abstract

Background: Although significant accumulation of prostaglandin E2 (PGE2) in the human prostate cancer tissues has been reported, there is lack of substantial evidence regarding the key role of PGE2-induced E-prostanoid-4 receptor (EP4) on Snail, a master regulator of epithelial mesenchymal transition (EMT). In this study, we investigated a novel connection between PGE2-induced EP4 and Snail (encodes DNA binding zinc finger protein that acts as transcriptional repressor) signaling in prostate cancer. Materials and Methods: To investigate the key role of serum PGE2, EP4, p-Akt and Snail in prostate cancer progression, we used prostate-specific phosphatase and tensin homolog (PTEN)-knockout (PTEN-KO) mice of different age groups from 4 to 28 weeks. To determine the EP4-specific interaction with Snail in prostate cancer, we used cell-based assays, including siRNA knockdown, and treatment with EP4 antagonist. Results: An interaction between EP4 with Snail was evident in prostate-specific PTEN-KO mice that showed an elevated level of PGE2 in the serum and of EP4, p-Akt and Snail in the tissues. Prostate cancer cells transfected with EP4-siRNA and treatments with EP4 antagonist suggest a link between EP4, and Snail activation, potentially via p-Akt. Cells treated with EP4 antagonist exhibited a significant decrease in Snail, mesenchymal markers and cell migration, and cell cycle arrest with a gain in E-cadherin levels. Conclusion: Our findings provide key evidence that support there being a role of PGE2/EP4/p-Akt in Snail signaling and conferring cell survival advantage. Cancer progression via EMT can be reversed by an EP4 antagonist in this model of prostate cancer.

Among the prostanoids, prostaglandin E2 (PGE2), derived from arachidonic acid, is widely produced in the body and the receptors mediating prostanoid actions have been documented in earlier reports (1-4). Mechanistically, PGE2-induced cell proliferation appears to be mediated by its overall effect on second messenger response, which in turn depends on receptor-ligand concentration and interactions (5). It has been established that PGE2 signaling stimulates the E-prostanoid (EP) receptors, which activate cyclic adenosine monophosphate (cAMP)/phosphatidylinositol 3-kinases (PI3K)/AKT and the extracellular signal-regulated kinase (ERK1/2) pathways, as well as the glycogen synthase kinase (GSK-3β) pathways in some cancer types (6). In previous studies, we found a significant association between EP4, Akt and Nuclear factor kappaB (NF-κB) signaling and their modulation by cyclooxygenase 2 (COX2) inhibitors in preclinical models for prostate cancer (7, 8). Extensive analysis of the data gathered from a PubMed search on EP4 signaling clearly reveals a strong interaction of EP4 with receptor activator of NF-κB ligand (RANKL) through cAMP (9-11), and early growth response protein-1 (Egr-1) regulation via EP4/Extracellular signal-regulated kinase (ERK) and cAMP Responsive Element Binding (CREB) pathway in colon cancer cells (12, 13). Recent studies in prostate cancer models (14, 15) indicate that Snail (encodes DNA binding zinc finger protein that acts as transcriptional repressor), a key member of the epithelial mesenchymal transition (EMT) and also the transcription factor that deregulates E-cadherin expression, promotes tumor invasiveness, tumorigenicity (16) and mediates EMT by binding to the E-box elements of E-cadherin promoter (17-20). Although a significant accumulation of PGE2 in human prostate cancer tissues has been reported (21), there is lack of substantial evidence regarding the influence of PGE2-induced EP4 on Snail activation in prostate cancer. In this study, we provide evidence for a key role of serum PGE2, EP4, p-Akt and Snail in prostate cancer progression, using Phosphatase and tensin homolog (PTEN)-knockout (PTEN-KO) mice of different age groups from 4 to 28 weeks. To dissect the mechanisms of EP4 specific interaction with Snail in prostate cancer, we used cell-based assays, including EP4-siRNA knockdown and migration studies.

Materials and Methods

Serum and prostate tissue samples. Prostate tumor progression associated with an increased expression of EP4, Akt and Snail was determined in genetically modified prostate-specific PTEN-KO mice. Briefly, PTEN-KO mice were developed by deleting the Pten tumor suppressor gene, specifically in the prostatic epithelium that results in the multistep tumorigenesis similar to that of human prostate cancer (22). In this model, conditional homozygous deletion of Pten in mouse prostate epithelium significantly shortens the latency of prostate intraepithelial neoplsia (mPIN) and promotes their progression to metastatic cancer. These conditional prostate-specific PTEN-KO mice were propagated in-house at the departmental animal core facility, by crossing PtenloxP/loxP mice with the PB-Cre4 mice that carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter (22-25), according to the protocol described in our recent publication (26). Briefly, the male ARR2PB-Cre4 transgenic mice obtained from the National Institute of Health (NIH)/National Cancer Institute (NCI) mouse repository, (mouse.ncifcrf.gov) were first crossed with the female B6.129S4-Ptentm1Hwu/J mice purchased from JAX Mice Services (Bar Harbor, ME, USA). Subsequent cross breeding of the F1 generation offsprings resulted in homozygous deletion of Pten in the F2 generation. Pten deletion in these mice has been confirmed by PCR using tail DNA as described earlier (27). Animal were sacrificed at various time points (n=5) between the age 4 and 28 weeks by CO2-asphyxiation and necropsied. At the time of sacrifice, blood was collected by cardiac puncture and serum was obtained by centrifugation at 14,000 rpm for PGE2 analysis. The prostate was micro-dissected and snap-frozen for total RNA and protein extraction. A portion of the prostate tissue was fixed in 10% neutral-buffered formalin for histopathological evaluations and immunohistochemical (IHC) studies.

Histology and IHC detection. Prostate tissues of PTEN-KO mice age 4, 7, 17 and 28 weeks were used for histopathological evaluation of tumor progression. Histological evaluations indicative of tumor progression from mPIN to adenocarcinoma of the dorsolateral prostate were conducted as described in our earlier publication (26). Stromal and tumor regions were differentiated histologically using H&E stained sections in order to identify the regions that specifically stain Snail and/or E-cadherin. Here, prostate cancer tissues from 28-week-old PTEN-KO mice with adenocarcinoma were used to conduct IHC staining for Snail (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and E-cadherin at an antibody dilution of 1:200 in five paraffin-embedded sections each. Positively stained cells were identified using a light microscope under 10 high power fields

PGE2 measurement. To measure PGE2 levels in the serum, Correlate-EIA-prostaglandin (PG) E2 enzyme immunoassay was performed following the manufacturer's protocol (Assay Designs, Ann Arbor, MI, USA), as described by us previously (7). Briefly, at the time of sacrifice, serum samples from 4 and 28-week-old PTEN-KO mice (n=5 each) were collected and stored at −80°C until further analysis. PGE2 standard 50,000 pg/ml was used for subsequent lower dilutions. The assay involves the use of a monoclonal antibody that binds in a competitive manner with PGE2 in the sample or an alkaline phosphatase molecule, which has PGE2 covalently attached. After a short incubation time, the enzyme reaction was stopped, and the yellow color generated was read on a microplate reader at 405 nm. The intensity of bound yellow color is inversely proportional to the concentration of PGE2 in the sample. The results presented here are based on data from three independent assays.

Cell culture. The human prostate cancer cells (PC-3 and LNCaP) obtained from the American Type Culture Collection (Manassas, VA, USA), were grown in RPMI (Gibco Life Technologies, Inc., Gaithersburg, MD, USA) with 10% FBS in addition to adopting the standard cell culture conditions as described earlier (27). The mouse prostate cancer cells (CaP8) derived from the adenocarcinoma of the Pten knockout mice were kindly provided by Dr. Hong Wu, UCLA School of Medicine, CA, USA. The CaP8 cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Atlanta biological, Lawrenceville, GA, USA), as well as 25 μg/ml bovine pituitary extract, 5 μg/ml bovine insulin, and 6 ng/ml recombinant human epidermal growth factor (BioWhittaker, Walkersville, MD, USA), as described earlier (28). Cells were stimulated with 0.5 μM of PGE2 for 1 h or 5 ng transforming growth factor beta (TGF-β) for 24 h before treatment.

Transient transfections. Cells were transfected with two sets of siRNAs designed for EP4 and COX2 gene. Complex of siRNA duplexes and control siRNA along with HiPerFect transfection reagent purchased from Qiagen Inc (Valencia, CA, USA), was prepared as described by us earlier (27). Transfection efficiency with siRNA was assessed by immunofluorescence followed by western blot analysis after 48 h. Snail promoter activity was determined by transfecting PC-3, LNCaP and CaP8 cells plated in 12-well plates at a density of 1×105 cells/well using Snail gene-specific proximal promoter (Snail-1pl) fragment plasmid or control plasmid (Snail-0pl) 4 μg each. Transient transfection assays were followed by treatment with EP2 (AH6809) or EP4 antagonist (AH23848) (Cayman, Ann Arbor, Michigan, USA) at a dose of 5 μM which was appropriately selected based on our dose-dependent assay. Luciferase activities were measured by a dual luciferase assay in which Renilla luciferase activity was used to normalize the activity of firefly luciferase using a Dual-Glo Luciferase Assay System (Promega, Madison WI, USA) and by using LMax-II384 luminomter (Molecular devices, Sunnyvale, CA, USA). All the above experiments were repeated at least three times.

RNA isolation and real-time PCR. Total RNA was extracted from transfected or treated cells using Trizol reagent (GibcoBRL) as described earlier by us (27). cDNA was made from total RNA using the superscript first-strand synthesis system with oligo dT primers (IDT, Coralville, IA, USA) made specifically for Snail, vimentin, fibronectin, E-cadherin, and Gapdh. The following primer sequences were used for amplification: Snail: forward GGG CAG GTA TGG AGA GGA AGA, reverse TTC TTC TGC GCT ACT GCT GCG; E-cadherin: forward CAG CAC GTA CAC AGC CCT AA, reverse GCT GGC TCA AGT CAA AGT CC; Vimentin: forward TCG TTT CGA GGT TTT CGC GTT AGA GAC, reverse CGA CTA AAA CTC GAC CGA CTC GCG. Fibronectin: forward-CCG CCG AAT GTA GGA CAA GA, reverse TGC CAA CAG GAT GAC ATG AAA and GAPDH: forward CCT GGC CAA GGT CAT CCA TGA C, reverse CAT GTA GGC CAT GAG GTC CAC CAC. The amplification conditions were 94°C for 5 m, and 21 cycles for real-time quantitative PCR were performed in triplicate with a Smart Cycler (Cepheid, Sunnyvale, CA) and with SYBR-Green Real time PCR-Supermix (Bio-Rad, Hercules, CA, USA) as described earlier (27). Results were normalized to GAPDH levels. Fold change in the mRNA expression level was calculated as 2ΔΔCt.

Western blot analysis. Relative expression of the molecular targets at the protein level before and after transfection with siRNA or treatment with antagonist for EP4 was determined by western blot analysis using specific antibodies as described earlier (27). Briefly, total protein extracted from prostate cancer tissues or cancer cells were fractionated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We used 50 μg of each sample protein after extraction with a buffer containing 150 mmol/l NaCl, 10 mmol/l Tris (pH 7.2), 5 mmol/l EDTA, 0.1% Triton X-100, 5% glycerol, and 2% SDS in addition to a mixture of protease inhibitors (Boehringer Mannheim, Mannheim, Germany). Fractionated proteins were transferred onto polyvinyl derivative (PVD) membranes. Standard western blot procedure was carried out using specific monoclonal antibody (1:1000 dilution) to detect EP4, Snail, p-Akt (ser-473) p21 and cyclinD1 (Santa Cruz Biotechnology). The level of β-actin expression was used as reference for equal sample loading. Reactive protein bands were detected using the ECL kit (Amersham, Biosciences, Uppsala, Sweden). Densitometric analysis was performed to quantify the reactive protein bands.

Immunofluorescence detection. We performed immunofluorescence detection of Snail in CaP8, and PC-3 and LNCaP cells as described earlier (27). Cells grown in two- or four-well chamber slides were stimulated with 0.5 μM of PGE2 for 1 h or 5 ng TGF-β for 24 h before treatment with AH23848 at a dose of 5 μM for 48 h. Detection of EP4 and Snail was performed by double staining with their specific antibodies (27). Green fluorescence signal (FITC) for Snail and red signal (Rhodamine) for EP4 or E-cadherin over 4’,6-diamidino-2-phenylindole (DAPI) for DNA was viewed at ×400 using an Olympus AX-70 epi-fluorescence microscope (Olympus America, Melville, NY USA).

Cell proliferation assay. Prostate cancer cells were grown in 96-well plates at a density of 1×104 per well (100 μl). Cells were stimulated with 0.5 μM of PGE2 for 1 h, followed by treatment with different concentrations (0-40 μM) of AH23848 (this dose range was chosen based on earlier studies) (2). Cells treated with serum-free medium or DMSO served as the control. Standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay was performed following the manufacturer's protocol (Cayman, Ann Arbor, MI, USA). At the end of the assay, absorbance at 570 nm was detected using a microplate Reader (PerkinElmer, Boston, MA, USA). The assays were performed in triplicate.

Cell migration assay. A 24-well BD FluoroBlok Transwell Insert (BD Biosciences, Bedford, MA, USA) with 8 μM pore size was used to measure cell migration. Briefly, cells pretreated with the EP4 antagonist were seeded (200 μl) into the inserts with RPMI containing 0.25% serum, with the bottom well medium containing only RPMI with 10% FBS. After 48 h of incubation the bottom well was filled with 500 μl of calcein fluorescent dye prepared according to the directions of the manufacturer (BD Biosciences, Bedford, MA, USA). The fluorescence intensity emitted by the migrated cells was measured at 540 nm using a microplate reader. The experiments were repeated three times.

Cell cycle analysis. Prostate cancer cells, PC-3 and CaP8 cells stimulated with 0.5 μM of PGE2 for 1 h followed by treatment with AH23848 for 48 h were used to conduct cell cycle analysis as described earlier (29). Briefly, treated cells were harvested after 48 h by trypsinization and fixed in 10% neutral buffer formalin followed by fixing in 80% ethanol for 24 hours. The cells were then washed in phosphate buffered saline (PBS) and resuspended in 1 ml of 5 mg/ml of propidium iodide with 0.1% RNase A (Sigma, St. Louis, MO, USA) in PBS to measure DNA content. After 30 min incubation at room temperature in the dark, the cells were analyzed by flow cytometry with Coulter Epic Elite ESP (Beckman Coulter, Miami, FL, USA). Cell cycle analysis was performed using Multicycle analysis software (Phoenix Flow System, San Diego, CA, USA). To determine the significant difference in the DNA content between control and treatments, the analysis was repeated with triplicate samples.

Statistical analysis. Data from all the experiments are presented as the mean±SD, from at least three independent experiments. Measures of statistical difference were determined using two-way ANOVA followed by Tukey's multiple comparison procedure (30). Differences among the treatment and control groups were analyzed using Student's t-test. Statistical analyses were performed using GraphPad Prism 4 software (San Diego, CA, USA).

Results

In vivo evidence for an elevated level of serum PGE2, EP4 and Snail expression in a progressive model for prostate cancer deleted for Pten. We measured serum-PGE2, EP4 and Snail expression in samples obtained from prostate-specific PTEN-KO mice of different age groups, known to exhibit progressive changes in the prostate from mPIN to adenocarcinoma stages (22). Findings from ELISA assays showed >3-fold increase in the serum PGE2 in mice with cancer at 28 weeks of age in contrast to the level in the 4-week-old mice (Figure 1A). However in the wild-type mice, no significant change in the PGE2 levels was observed (Figure 1B). Consistent with this, western blot analysis for EP4 and Snail indicated a gradual increase from week 4 to 28 in PTEN-KO mice as compared to the wild-type mice (Figure 1C). Furthermore, the expression of Snail and E-cadherin in the stromal and tumor regions of the prostate were analyzed by IHC. Interestingly, we found detectable levels of Snail and E-cadherin in the non-tumor (stromal) regions of the prostate. However, our findings showed an elevated level of Snail (>4-fold, p<0.001) associated with a significant decrease in staining for E-cadherin (Figure 1D and E) in the prostate tumors of 28 week old PTEN-KO mice, suggesting that the loss of E-cadherin in the tumor is associated with EP4 and Snail expression in the stroma. Based on the endogenous expression level of PGE2, EP4, Snail and E-cadherin, we further investigated the mechanistic aspects of EP4 antagonism in EMT.

Figure 1.
Figure 1.

Serum prostaglandin E2 (PGE2), E-prostanoid-4 receptor (EP4) and Snail (transcriptional repressor) expression in the prostate tumor and stroma of PTEN-KO mice. A, B: PGE2 was analyzed by ELISA assays using serum samples obtained from PTEN-KO and wild-type mice of 4 and 28 weeks of age (n=5). The assays were repeated three times. Box plots represent the mean±SD. **Significant difference at p<0.001. C: Tumor progression is associated with an increase in the protein for EP4 and Snail. Prostate tissues of PTEN-KO mice ages 4, 7, 17 and 28 weeks were used to isolate protein (50 μg/lane) for western blot analysis. The data presented for EP4 and Snail expression are representative of each experiment out of three. Fold increase in the reactive protein band was calculated by densitometric analysis. D, E: Snail and E-cadherin expression in the stroma vs. tumor regions. Prostate cancer tissues from 28-week-old PTEN-KO mice with advanced cancer stage was used to conduct IHC staining using Snail and E-cadherin antibody at a dilution of 1:200 in a minimum of 5 paraffin-embedded sections each. Positively stained cells were identified using a light microscope in 10 high-power fields. Stromal and tumor regions were differentiated histologically using H&E stained sections to identify the regions that specifically express Snail and/or E-cadherin. Box plots represent mean±SD. **Significant difference at p<0.001.

PGE2-induced EP4 and Snail activation in AR-independent prostate cancer cells. It is known from previous studies that overexpression of COX2 is associated with an increase in the accumulation of PGE2 in prostate cancer tissues (21, 31). To determine whether the PGE2-mediated EP4 activation in adenocarcinoma results in a subsequent downstream effect on triggering EMT pathways specifically promoting Snail signaling, we used PC-3 cells that are depleted for COX2 or EP4 by specific siRNAs. Evidence from transient transfection assays followed by western blot analysis showed no change in the expression of either EP2 or EP4 in COX2 knock-down cells (Figure 2A). However, PC-3 cells transfected with EP4-siRNA exhibited a significant decrease in the expression (>5-fold) of both p-Akt and Snail (Figure 2B). These findings suggest a potential interaction between activated EP4, p-Akt, and Snail that may be independent of COX2 but may require EP4 and Akt for Snail expression.

Effect of EP4 antagonist on Snail promoter activity in AR-dependent and -independent prostate cancer cells. We determined the specific effect of EP4 antagonist on Snail promoter activity by transiently transfecting PC-3, LNCaP and CaP8 cells with Snail promoter-driven reporter construct. To determine the pharmacological effect of EP4, the transfected cells were exposed to an established antagonist for EP4 (AH23848) and/or EP2 (AH6809). We found a significant decrease in Snail promoter activity by EP4 antagonist in contrast to a weak effect induced by EP2 antagonist (Figure 3A).

Snail activation mediated by EP4 and/or TGF-β is suppressed by EP4 antagonist. Although the major cellular events of EMT are known to be attributed to the influence of TGF-β on Snail activity (19, 32), we extended our studies to determine whether EP4 agonist may synergistically influence Snail activity in the presence of TGF-β. To determine this, cells were stimulated with TGF-β (5 ng) for 24 h followed by incubation with an EP4 agonist PGE1-OH (1.0 μmol/l) for 48 h and was examined for Snail expression at the mRNA level. While we observed a ~3-fold increase in Snail expression in the presence of TGF-β, this increase was significantly reduced in cells treated with EP4 antagonist. Subsequently, cells transfected with siRNA for EP4 for 48 h followed by incubation with TGF-β showed a similar trend in Snail expression, suggesting the requirement of EP4 for Snail transcription. These findings suggest a synergistic activation of Snail by EP4 under the conditions of an exogenous source of TGF-β (Figure 3B). These results were further confirmed in all three prostate cancer cells by immunofluorescence detection of Snail. As shown in Figure 3C and D, there was a significant decrease in the nuclear signal for Snail in CaP8 cells treated with antagonist for EP4 compared to the control. A similar trend was observed in LNCaP and PC-3 cells by immunofluorescence detection.

Figure 2.
Figure 2.

PGE2-induced EP4, p-Akt and Snail expression. PC-3 cells were pre-treated with PGE2 (0.5 μM) for 1 h to stimulate EP4. Subsequently, cells were transiently transfected with 30 nmol of COX2 siRNA (A), or EP4 siRNA (B) for 24 h. For controls, cells were treated with DMSO (Utreated) and/or control siRNA (Scrambled). Protein lysates (10 μg/lane) from transfected cells were used for Western blot analysis to detect EP2, EP4, p-Akt(ser-473) and Snail protein. Fold changes in the reactive protein bands were calculated based on pixel/densitometric analysis.

Influence of EP4 on the transcription of EMT markers. To investigate the influence of EP4 signaling on the markers of EMT which may facilitate a more aggressive phenotype of prostate cancer, we measured the changes expression of vimentin, and fibronectin in PC-3 cells that are treated with EP4 antagonist but stimulated with PGE2 as described earlier. Results from RT-PCR analysis showed >3-fold decrease in vimentin, fibronectin and Snail in comparison to the untreated control. Our findings, however, also revealed a significant increase in the E-cadherin signal. A similar trend was observed in LNCaP and CaP8 cells (Figure 4A). Interestingly, immunofluorescence detection of E-cadherin in PC-3 showed a significant increase in the signal at the cell-cell contact regions (Figure 4B). The above findings suggest that loss of E-cadherin could be partially abrogated by treatment with EP4 antagonist.

Figure 3.
Figure 3.

A: EP4 antagonist suppresses Snail promoter activity. Prostate cancer cells, PC-3 and CaP8 and LNCaP, seeded in 6-well plates were co-transfected with 1.5 μg of Snail promoter construct ligated to luciferase. After transfection, cells were treated with DMSO or 5 μM each of EP4 antagonist AH23848 or EP2 antagonist AH6809. Reporter activities were measured in the cellular extract after 6 h. The data reported are the relative luciferase activity from three independent experiments. Bar represents mean±SD. **Significant difference at p<0.01. B: Synergy in the activation of Snail by EP4 and TGF-β. Human PC-3, LNCaP and CaP8 cells were stimulated with TGF-β (5 ng) for 24 h followed by incubation with an EP4 agonist PGE1-OH for 48 h for stimulation. To determine the effect of EP4 antagonist AH23848 and/or siEP4 on Snail transcription, total RNA (5 μg) isolated from the treated cells was used for qRT-PCR using specific primers for Snail. Fold change in Snail expression was determined as described in the Materials and Methods. Amplification of GAPDH was used as the internal control. Experiments were repeated three times. Bar represents mean±SD. **Significant difference at p<0.001. C: Effect of EP4 antagonist on Snail expression determined by immunofluorescence. Prostate cancer CaP8 cells of PTEN-KO mice grown in 4-well chamber slides were pre-treated with TGF-β followed by treatment with AH23848 as described. Immunofluorescence detection was performed in CaP8 cells using tetramethyl rhodamine isothiocyanate (TRITC)–conjugated antibody for EP4 (Red) and fluorescein isothiocyanate (FITC)–conjugated antibody for Snail (Green) (1:200 dilution each). Co-staining with DAPI was used for nuclear localization. Specific inhibitory effect of EP4 antagonist on Snail at the cellular level is shown in the merged panel. A similar trend was observed in androgen–dependent LNCaP cells determined by immunofluorescence staining. D: Bar graph represents mean±SD. **Significant difference at p<0.01.

Figure 4.
Figure 4.

A: Effect of EP4 antagonist on epithelial and mesenchymal markers. PC-3 cells pretreated with PGE2 were incubated with DMSO or EP4 antagonist AH23848 (5 μM) for 48 h. Expression levels of vimentin, fibronectin, E-cadherin and Snail were determined by qRT-PCR analysis with specific primers using total RNA isolated from the treated cells. Amplification of Gapdh was used as the internal control. Fold changes in the expression level were calculated as described in the Materials and Methods. B: Gain of E-cadherin and loss of Snail signal in cells treated with EP4 antagonist. PC-3 cells grown in 4-well chamber slides were treated with or without AH23848 for 48 h and processed for immunofluorescence detection. E-cadherin and Snail were analyzed by double staining with TRITC (red) or FITC (Green) conjugated antibodies for Snail and E-cadherin respectively. Fluorescence signaling was captured under an Epi-fluorescence microscope at × 40 magnification. Merged images indicate loss of Snail and an increase in the expression of E-cadherin at the cell–cell contact regions compared to a weak signal in the control (far–right panel; arrow: single cell magnified).

Figure 5.
Figure 5.

EP4 antagonist induces cell growth inhibition and cell-cycle arrest. A: Inhibitory effect of EP4 antagonist on human prostate cancer cell proliferation. Prostate cancer cells were grown in 96-well plates at a density of 1×104 per well. Cells were pre-treated with 0.5 μM of PGE2 for 1 h, followed by treatment with AH23848 for 48 h. The data presented are from MTT assays conducted in triplicate as described in the Materials and Methods. B: Effect of EP4 antagonist on cell migration. This was determined by measuring calcein fluorescence (emission, 530 nm) in a BD Falcon FluorBlok-24-Multiwell Insert system. The rate of cell migration of PC-3 and CaP8 cell was calculated based on the total number of cells migrated under the microporous membrane specifically designed to absorb the fluorescence. The data presented are from three sets of assays including treated, control (DMSO) and blank (calcein) to determine the net fluorescence signal. Bar represents mean±SD. **Significant difference at p<0.01. C: Effect of EP4 antagonist on the cell cycle. PC-3 and CaP8 cells were stimulated with PGE2 followed by treatment with AH23848 for 48 h. Cell-cycle analysis for DNA content was performed as described in the Materials and Methods with PI staining for DNA content. D: Effect of EP4 antagonist on cell-cycle regulatory proteins. Protein lysate isolated from human PC-3 and murine CaP8 cells treated with PGE2 plus 5 μM of EP4-specific antagonist AH23848 for 48 h was used for western blot analysis using specific antibodies for cyclin D1 and p21(Waf1/Cip1) as described in the Materials and Methods. Fold changes in the expression were calculated based on the densitometric analysis of the reactive protein bands.

Effects of EP4 antagonist AH23848 on prostate cancer cell growth and motility. To further investigate the influence of EP4 antagonist on prostate cancer cell proliferation, MTT assay was conducted of PC-3, LNCaP, and CaP8 cells that were stimulated with PGE2 for 1 h before being treated with AH23848. As shown in Figure 5A, there was a dose-dependent inhibitory effect on the rate of cell proliferation by AH23848 treatment compared to the control. The rate of cell migration determined by measuring calcein fluorescence using Falcon FluoroBlok-24-Multiwell Insert system showed AH23848 to have a significant inhibitory effect on cell migration with stimulation by PGE2 (Figure 5B).

EP4 antagonist induces cell cycle arrest in androgen-dependent and -independent prostate cancer cells. Our findings from cell cycle analysis for DNA content in PC-3, and CaP8 cells stimulated with PGE2 followed by treatment with AH23848 for 48 h indicated cell-cycle arrest at the G1/S transition phase (G1: 57.23% and 45.66% respectively). This was associated with a decline in the percentage of cells in the S phase (S: 34.16% and 50.35% respectively (Figure 5C). We also observed a similar trend in LNCaP cells, suggesting a potential impact of EP4 antagonism on DNA synthesis associated with cell-cycle arrest. To further examine the effect of EP4 antagonist on cell-cycle regulatory proteins, we examined the expression of proteins for cyclin D1 and the Cdk inhibitor p21 (Waf1/Cip1) that are associated with G1 arrest. Western blot analysis showed a significant decrease in cyclin D1 expression, but there was no significant change in the expression of Cdk inhibitor p21 (Figure 5D). Protein lysate from LNCaP cells with similar treatments also showed a small increase in p21 expression, suggesting an impact on cell-cycle regulatory mechanisms.

Discussion

The zinc finger family of transcription repressors, including the Snail gene, is a master regulator of EMT in cancer progression and metastasis (17, 18), and is known to be activated by several factors including TGF-β and similar to mothers against decapentaplegic (SMAD) (32, 33). In spite of recent findings that support a key role for Snail in prostate cancer (34, 35), there is a lack of substantial evidence regarding the involvement of an inflammatory axis that could influence Snail through multiple mechanisms. Prostaglandin is one of the eicosanoids that are accumulated in human prostate cancer tissues, which induces a variety of inflammatory cellular responses (21). In this study, we addressed the question of whether PGE2-induced EP4 receptor activation influences Snail activity and whether it could be altered by EP4-specific antagonists. Recently, we have shown there to be an elevated level of p-Akt in prostate cancer tissues of PTEN-KO mice (26), which is consistent with our current observation; in addition we also observed an elevated level of serum PGE2, EP4 and Snail protein. Together, these findings clearly suggest the influence of PGE2/EP4 and p-Akt on Snail activation in prostate cancer partly due to loss of PTEN. Findings from cell-based assays with EP4-siRNAs suggest that Snail activation could occur via PGE2/EP4, and independently of COX2. It is also evident that EP4 antagonist can suppress Snail signaling in prostate cancer cells both at the mRNA and protein level irrespective of their androgen receptor (AR) dependence. Our findings are consistent with a report by Terada et al. (36) in a castration resistant xenograft model for prostate cancer in that EP4 overexpression via AR activation could be one of the mechanisms in castration-resistant prostate cancer progression. Furthermore, our findings on the loss of E-cadherin and gain in Snail in the prostate tissue are consistent with an earlier observation in which Snail acts as survival factor and influences specific aspects of EMT in prostate cancer (34) via stromal - epithelial interactions mediated through PI3k/Akt axis in models that exhibit loss of PTEN. Since Akt is also one of the master regulators of prostate cancer progression and metastasis in PTEN-depleted tumors (37-39), our overall findings further support a close interaction between EP4, p-Akt and Snail activity. Although not much information is yet available on a synergistic activation of Snail by PGE2/EP4 and TGF-β, our data reveal that specific inhibitor of EP4 was able to suppress Snail activated by both PGE2/EP4 and TGF-β in prostate cancer cells. An increased rate of cell proliferation and migration stimulated with PGE2 is consistent with a recent report of Snail-induced EMT conversion with enhanced migratory and proliferative potential (40). It is also evident that prostate cancer cells that express Snail protein are more motile than cells that overexpress E-cadherin, as reported in other studies (41). Thus, our findings on the pharmacological effect of EP4 antagonist on EMT markers, cell migration, cell cycle and its regulatory proteins support a potential role of EP4 as a therapeutic target (42), and thus metastatic prostate cancer progression might be prevented by blocking EMT processes mediated through the inflammatory axis involving PGE2/EP4, p-Akt and Snail.

Acknowledgements

We thank Dr. Hong Wu, Department of Medicine, University of California, Los Angeles, CA, USA for providing CaP8 cells. We acknowledge the services provided by the animal, histopathology and molecular biology core facilities at the New York University School of Medicine, Department of Environmental Medicine, Tuxedo, NY, USA.

  • Received September 5, 2011.
  • Revision received October 25, 2011.
  • Accepted October 27, 2011.

References

  1. ↵
    1. Narumiya S,
    2. Sugimoto Y,
    3. Ushikubi F
    : Prostanoid receptors: structures, properties, and functions. Physiol Rev 79: 1193-1226, 1999.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Jain S,
    2. Chakraborty G,
    3. Raja R,
    4. Kale S,
    5. Kundu GC
    : Prostaglandin E2 regulates tumor angiogenesis in prostate cancer. Cancer Res 68: 7750-7759, 2008.
    OpenUrlAbstract/FREE Full Text
    1. Ma X,
    2. Kundu N,
    3. Rifat S,
    4. Walser T,
    5. Fulton AM
    : Prostaglandin E receptor EP4 antagonism inhibits breast cancer metastasis. Cancer Res 66: 2923-2927, 2006.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Tian M,
    2. Schiemann WP
    : PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-beta signaling during mammary tumorigenesis. FASEB J 24: 1105-1116, 2010.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Hull MA,
    2. Ko SC,
    3. Hawcroft G
    : Prostaglandin EP receptors: targets for treatment and prevention of colorectal cancer? Mol Cancer Ther 3: 1031-1039, 2004.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Fulton AM,
    2. Ma X,
    3. Kundu N
    : Targeting prostaglandin E EP receptors to inhibit metastasis. Cancer Res 66: 9794-9797, 2006.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Narayanan BA,
    2. Reddy BS,
    3. Bosland MC,
    4. Nargi D,
    5. Horton L,
    6. Randolph C,
    7. Narayanan NK
    : Exisulind in combination with celecoxib modulates epidermal growth factor receptor, cyclooxygenase-2, and cyclin D1 against prostate carcinogenesis: in vivo evidence. Clin Cancer Res 13: 5965-5973, 2007.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Sheng H,
    2. Shao J,
    3. DuBois RN
    : Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells. J Biol Chem 276: 14498-1504, 2001.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Chun KS,
    2. Akunda JK,
    3. Langenbach R
    : Cyclooxygenase-2 inhibits UVB-induced apoptosis in mouse skin by activating the prostaglandin E2 receptors, EP2 and EP4. Cancer Res 67: 2015-2021, 2007.
    OpenUrlAbstract/FREE Full Text
    1. Sugimoto Y,
    2. Narumiya S
    : Prostaglandin E receptors. J Biol Chem 282: 11613-11617, 2007.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Takayama K,
    2. Garcia-Cardena G,
    3. Sukhova GK,
    4. Comander J,
    5. Gimbrone MA Jr..,
    6. Libby P
    : Prostaglandin E2 suppresses chemokine production in human macrophages through the EP4 receptor. J Biol Chem 277: 44147-44154, 2002.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Cherukuri DP,
    2. Chen XB,
    3. Goulet AC,
    4. Young RN,
    5. Han Y,
    6. Heimark RL,
    7. Regan JW,
    8. Meuillet E,
    9. Nelson MA
    : The EP4 receptor antagonist, L-161,982, blocks prostaglandin E2-induced signal transduction and cell proliferation in HCA-7 colon cancer cells. Exp Cell Res 313: 2969-2979, 2007.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Fujino H,
    2. Xu W,
    3. Regan JW
    : Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. J Biol Chem 278: 12151-12156, 2003.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Perez-Moreno M,
    2. Jamora C,
    3. Fuchs E
    : Sticky business: orchestrating cellular signals at adherens junctions. Cell 112: 535-548, 2003.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Takeichi M
    : Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251: 1451-1455, 1991.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Hemavathy K,
    2. Ashraf SI,
    3. Ip YT
    : Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene 257: 1-12, 2000.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Batlle E,
    2. Sancho E,
    3. Franci C,
    4. Dominguez D,
    5. Monfar M,
    6. Baulida J,
    7. García De Herreros A
    : The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2: 84-89, 2000.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Cano A,
    2. Perez-Moreno MA,
    3. Rodrigo I,
    4. Locascio A,
    5. Blanco MJ,
    6. del Barrio MG,
    7. Portillo F,
    8. Nieto MA
    : The transcription factor snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76-83, 2000.
    OpenUrlCrossRefPubMed
  19. ↵
    1. De Craene B,
    2. van Roy F,
    3. Berx G
    : Unraveling signalling cascades for the Snail family of transcription factors. Cell Signal 17: 535-547, 2005.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Peinado H,
    2. Ballestar E,
    3. Esteller M,
    4. Cano A
    : Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex. Mol Cell Biol 24: 306-319, 2004.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Chaudry AA,
    2. Wahle KW,
    3. McClinton S,
    4. Moffat LE
    : Arachidonic acid metabolism in benign and malignant prostatic tissue in vitro: effects of fatty acids and cyclooxygenase inhibitors. Int J Cancer 57: 176-180, 1994.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Wang S,
    2. Gao J,
    3. Lei Q,
    4. Rozengurt N,
    5. Pritchard C,
    6. Jiao J,
    7. Thomas GV,
    8. Li G,
    9. Roy-Burman P,
    10. Nelson PS,
    11. Liu X,
    12. Wu H
    : Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4: 209-221, 2003.
    OpenUrlCrossRefPubMed
    1. De Marzo AM,
    2. Platz EA,
    3. Sutcliffe S,
    4. Xu J,
    5. Gronberg H,
    6. Drake CG,
    7. Nakai Y,
    8. Isaacs WB,
    9. Nelson WG
    : Inflammation in prostate carcinogenesis. Nat Rev Cancer 7: 256-269, 2007.
    OpenUrlCrossRefPubMed
    1. Palapattu GS,
    2. Sutcliffe S,
    3. Bastian PJ,
    4. Platz EA,
    5. De Marzo AM,
    6. Isaacs WB,
    7. Nelson WG
    : Prostate carcinogenesis and inflammation: emerging insights. Carcinogenesis 26: 1170-1181, 2005.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Sugar LM
    : Inflammation and prostate cancer. Can J Urol 13: 46-47, 2006.
    OpenUrlPubMed
  26. ↵
    1. Narayanan NK,
    2. Nargi D,
    3. Randolph C,
    4. Narayanan BA
    : Liposome encapsulation of curcumin and resveratrol in combination reduces prostate cancer incidence in Pten knockout mice. Int J Cancer 125: 1-8, 2009.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Narayanan BA,
    2. Narayanan NK,
    3. Davis L,
    4. Nargi D
    : RNA interference-mediated cyclooxygenase-2 inhibition prevents prostate cancer cell growth and induces differentiation: modulation of neuronal protein synaptophysin, cyclin D1, and androgen receptor. Mol Cancer Ther 5: 1117-1125, 2006.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Jiao J,
    2. Wang S,
    3. Qiao R,
    4. Vivanco I,
    5. Watson PA,
    6. Sawyers CL,
    7. Wu H
    : Murine cell lines derived from Pten null prostate cancer show the critical role of PTEN in hormone refractory prostate cancer development. Cancer Res 67: 6083-6091, 2007.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Narayanan BA,
    2. Condon MS,
    3. Bosland MC,
    4. Narayanan NK,
    5. Reddy BS
    : Suppression of N-methyl-N-nitrosourea/testosterone-induced rat prostate cancer growth by celecoxib: effects on cyclooxygenase-2, cell cycle regulation, and apoptosis mechanism(s). Clin Cancer Res 9: 3503-3513, 2003.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Miller RG
    : Simultaneous Statistical Inference 2 nd ed. New York: Springer-Verlag. p. 37, 1981.
  31. ↵
    1. Wallace JM
    : Nutritional and botanical modulation of the inflammatory cascade–eicosanoids, cyclooxygenases, and lipoxygenases–as an adjunct in cancer therapy. Integr Cancer Ther 1: 7-37, 2002.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Smith AP,
    2. Verrecchia A,
    3. Faga G,
    4. Doni M,
    5. Perna D,
    6. Martinato F,
    7. Guccione E,
    8. Amati B
    : A positive role for Myc in TGFbeta-induced Snail transcription and epithelial-to-mesenchymal transition. Oncogene 28: 422-430, 2009.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Massague J
    : TGFbeta in Cancer. Cell 134: 215-230, 2008.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Emadi Baygi M,
    2. Soheili ZS,
    3. Schmitz I,
    4. Sameie S,
    5. Schulz WA
    : Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines. Cell Biol Toxicol 26: 553-567, 2010.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Heeboll S,
    2. Borre M,
    3. Ottosen PD,
    4. Dyrskjot L,
    5. Orntoft TF,
    6. Torring N
    : Snail1 is over-expressed in prostate cancer. APMIS 117: 196-204, 2009.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Terada N,
    2. Shimizu Y,
    3. Kamba T,
    4. Inoue T,
    5. Maeno A,
    6. Kobayashi T,
    7. Nakamura E,
    8. Kamoto T,
    9. Kanaji T,
    10. Maruyama T,
    11. Mikami Y,
    12. Toda Y,
    13. Matsuoka T,
    14. Okuno Y,
    15. Tsujimoto G,
    16. Narumiya S,
    17. Ogawa O
    : Identification of EP4 as a potential target for the treatment of castration-resistant prostate cancer using a novel xenograft model. Cancer Res 70: 1606-1615, 2010.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Ayala G,
    2. Thompson T,
    3. Yang G,
    4. Frolov A,
    5. Li R,
    6. Scardino P,
    7. Ohori M,
    8. Wheeler T,
    9. Harper W
    : High levels of phosphorylated form of Akt-1 in prostate cancer and non-neoplastic prostate tissues are strong predictors of biochemical recurrence. Clin Cancer Res 10: 6572-6578, 2004.
    OpenUrlAbstract/FREE Full Text
    1. Sheng S,
    2. Qiao M,
    3. Pardee AB
    : Metastasis and AKT activation. J Cell Physiol 218: 451-454, 2009.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Stern DF
    : More than a marker… Phosphorylated Akt in prostate carcinoma. Clin Cancer Res 10: 6407-6410, 2004.
    OpenUrlFREE Full Text
  40. ↵
    1. Barrallo-Gimeno A,
    2. Nieto MA
    : The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132: 3151-3161, 2005.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Zhou BP,
    2. Deng J,
    3. Xia W,
    4. Xu J,
    5. Li YM,
    6. Gunduz M,
    7. Hung MC
    : Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol 6: 931-940, 2004.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Lee J,
    2. Banu SK,
    3. Rodriguez R,
    4. Starzinski-Powitz A,
    5. Arosh JA
    : Selective blockade of prostaglandin E2 receptors EP2 and EP4 signaling inhibits proliferation of human endometriotic epithelial cells and stromal cells through distinct cell cycle arrest. Fertil Steril 93: 2498-2506, 2010.
    OpenUrlCrossRefPubMed
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Modulation of PGE2-induced EP4 Expression on Snail Signaling and the Impact on Epithelial-Mesenchymal Transition: Significance of EP4 Antagonism
HYE NA KIM, NARAYANAN K. NARAYANAN, SALAMIA LASANO, BHAGAVATHI NARAYANAN
Anticancer Research Dec 2011, 31 (12) 4347-4357;
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