F-Prostanoid Receptor Regulation of Fibroblast Growth Factor 2

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Endocrinology 148(8):3635–3644 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1517

F-Prostanoid Receptor Regulation of Fibroblast Growth Factor 2 Signaling in Endometrial Adenocarcinoma Cells Kurt J. Sales, Sheila C. Boddy, Alistair R. W. Williams, Richard A. Anderson, and Henry N. Jabbour Medical Research Council Human Reproductive Sciences Unit (K.J.S., S.C.B., R.A.A., H.N.J.), The Queen’s Medical Research Institute, Department of Pathology (A.R.W.W.), and Division of Reproductive and Developmental Sciences (R.A.A.), University of Edinburgh, Edinburgh, Scotland EH16 4TJ, United Kingdom Prostaglandin (PG) F2␣ is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF2␣-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF2␣-FP

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DENOCARCINOMA OF THE endometrium is one of the most frequently diagnosed malignancies of the female genital tract in the Western world, ranking fourth in incidence among invasive tumors in women, after breast, lung, and colon cancers. Incidence rates of the disease vary from 10 –25 women per 100,000, with a clear geographic variation between European (United Kingdom, France, and Spain) and North American (United States of America and Canada) countries, with a greater incidence recorded in North America (1). Adenocarcinoma of the endometrium is typically a disease of postmenopausal women, with approximately 85% of the patients being over 50 yr of age (2). Like many solid tumors, growth and survival of endometrial adenocarcinoma are dependent on the production of growth factors. Of the numerous growth factors reported to date, fibroblast growth factor 2 (FGF2, also called basic FGF) is known to play an important role in neoplastic transformation (3). FGF2 is expressed at increased levels in the endometrium of postmenopausal women (4) and endometrial cancers (5), and recent data have shown that FGF2 overexpression in endometrial adenocarcinoma cells can promote tumor growth when implanted sc in nude mice (6). FGF2 signals via an autocrine/paracrine mechanism involving high-affinity transmembrane receptor tyrosine kinases (FGFR) (7). Although the molecular mechanisms mediating

First Published Online May 3, 2007 Abbreviations: COX, Cyclooxygenase; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; FP, F-series-prostanoid; MEK, mitogen-associated protein kinase; P, PGF2␣-treated; PG, prostaglandin; V, vehicle-treated. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF2␣ on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas. (Endocrinology 148: 3635–3644, 2007)

the role of FGF2 in endometrial adenocarcinomas remain to be fully clarified, a role for prostaglandins (PGs) in the regulation of FGF2 expression in malignant cells has been reported recently (8 –10). In in vitro model systems, overexpression of cyclooxygenase (COX)-1 or COX-2 and subsequent elevation in the biosynthesis of PGs promote the expression and secretion of FGF2 (8, 9). Similarly, a recent study has shown that PGs present in human seminal plasma can also promote the expression of FGF2, via the E-series prostanoid-2 receptor and ERK pathways in endometrial adenocarcinomas (10). PGs are COX metabolites of arachidonic acid, and a role for PGF2␣ via its G protein-coupled receptor [F-series-prostanoid (FP) receptor] has been implicated in numerous benign and neoplastic endometrial pathologies (11–16). In endometrial adenocarcinomas, FP receptor expression is elevated (12, 17) and has been shown recently to mediate the effects of PGF2␣ on neoplastic endometrial epithelial cell function by enhancing cellular proliferation (12) and promotion of tumorigenic and angiogenic factors (17, 18). In this study we investigated an autocrine/paracrine role for FGF2, produced by PGF2␣-FP receptor interaction in endometrial adenocarcinoma cells. We found elevated FP receptor, FGF2, and FGFR1 expression in endometrial adenocarcinomas, colocalized in glandular epithelial cells. Using an endometrial adenocarcinoma cell line overexpressing the FP receptor (Ishikawa FPS cells; European Collection of Cell Culture, Wiltshire, UK) and endometrial adenocarcinoma explants, we found that PGF2␣ rapidly augments the expression and secretion of FGF2 via the FP receptor and ERK signaling pathway. Furthermore, the secreted FGF2 could potentially enhance endometrial tumorigenesis in an autocrine/paracrine manner by inducing mitogenic signaling to

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ERK and elevation of FGF2 and COX-2 expression, and promoting cell proliferation via the FGFR1 receptor, thereby establishing a series of positive feedback loops to sustain tumorigenesis. Materials and Methods Reagents Culture medium was purchased from Life Technologies (Paisley, UK). Penicillin-streptomycin and fetal calf serum were purchased from PAA Laboratories Limited (Middlesex, UK). Anti-phospho-p42/44 ERK (9101) was purchased from Cell Signaling Technologies/New England Biolabs (Hertfordshire, UK). The FP receptor antibody (101802) was purchased from Cayman Chemical Company (Axxora, Nottingham, UK). The FGF2 antibody recognizing the 18-kDa isoform (sc-1360), ERK (sc-93), FGFR1 (sc-121), ␤-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology (Autogen-Bioclear, Wiltshire, UK). PGF2␣ (100 ␮m stock in ethanol, used at a final concentration of 100 nm), AL8810 (10 mm stock in ethanol, used at a final concentration of 50 ␮m), alkaline phosphatase secondary antibodies, indomethacin, PBS, and BSA were purchased from Sigma Chemical Co. (Dorset, UK). The enhanced chemifluorescence system was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). PD98059 (18.7 mm stock in dimethylsulfoxide, used at a final concentration of 50 ␮m) and SU4984 (200 mm stock in dimethylsulfoxide, used at a final concentration of 20 ␮m) were purchased from Calbiochem (Nottingham, UK) and stored at ⫺20 C. Recombinant FGF2 peptide was purchased from PeproTech EC Ltd. (London, UK) and stored as a stock of 200 ng/ml in sterile water at ⫺20 C.

Patients and tissue collection Endometrial adenocarcinoma tissue (n ⫽ 36) was collected from women undergoing hysterectomy who had been diagnosed on endometrial biopsy to have adenocarcinoma of the uterus of endometrioid type. The patients had received no treatment before surgery and were within the age range of 50 –71 yr, with a median age of 60.5 yr. Hysterectomy specimens were collected from the operating theater and placed on ice. With minimal delay, the specimens were opened by a gynecological pathologist. Small samples (⬃5 mm-3 cm) of polypoidal adenocarcinoma tissue were collected from the endometrial lumen. Tissue samples were transferred into neutral buffered formalin and embedded in paraffin wax for immunofluorescence studies. Tissues were snap frozen in dry ice and stored at ⫺70 C (for RNA extraction), and placed in RPMI 1640 culture medium containing 2 mm l-glutamine, 100 U penicillin, and 100 ␮g/ml streptomycin and 8.4 ␮m indomethacin (to inhibit endogenous COX activity) for in vitro culture. The diagnosis of adenocarcinoma was confirmed histologically in all cases, and grading was performed according to the criteria defined by Federation Internationale Obstetrics et Gynaecologie (10). The percentage of tumor cells to stroma was estimated to be approximately 75%:25%. Normal endometrial tissue (n ⫽ 30) at different stages of the menstrual cycle was collected from women undergoing surgery for minor gynecological procedures, with no underlying endometrial pathology, with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) from women with regular menstrual cycles (25–35 d) and processed exactly as described previously. The ages of the control women ranged from 21–39 yr, with a median age of 30.5 yr. None of the control women had received a hormonal preparation in the 3 months preceding biopsy collection. Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to the criteria of Noyes et al. (19). Ethical approval was obtained from Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.

Cell culture Ishikawa endometrial adenocarcinoma cells were obtained from the European Collection of Cell Culture. Stable FP transfectant cells, generated within our laboratory and designated the nomenclature FPS cells, were constructed, characterized, and maintained as described (17), with the addition of a maintenance dose of 200 ␮g/ml G418. The expression

Sales et al. • FP Receptor Signaling Regulates FGF2 Expression

of FP receptor in FPS cells was comparable to that observed in endometrial adenocarcinomas (17).

Immunofluorescent microscopy Tissues. FP receptor and FGF2 or FGFR1 protein expression were colocalized in endometrial adenocarcinomas (n ⫽ 15; five each of poorly, moderately, and well-differentiated endometrioid adenocarcinomas) and normal endometrium (n ⫽ 6; three each of mid-late proliferative phase and secretory phase endometrium) by dual immunofluorescence immunohistochemistry. Tissue sections were prepared as described previously (20) and blocked using 5% normal horse serum diluted in PBS. Subsequently, sections were incubated with goat anti-FGF2/FGFR1 antibody at a dilution of 1:80 for 18 h at 4 C. Control sections were incubated with equivalent concentration of normal IgG from the same host species. Thereafter, sections were washed with PBS and incubated with biotinylated horse antigoat (Dako Corp., High Wycombe, UK), followed by incubation with the fluorochrome streptavidin 488 Alexafluor (Molecular Probes, Inc., Eugene, OR) diluted one in 200 in PBS. Sections were reblocked with 5% normal goat serum diluted in PBS and incubated with rabbit anti-FP receptor antibody at a dilution of 1:100 at 4 C for 18 h. Control sections were incubated with equivalent concentration of rabbit IgG. Thereafter, the sections were washed in PBS and incubated with the fluorochrome streptavidin 546 Alexafluor (Molecular Probes, Inc.) diluted one in 200 in PBS at 25 C for 20 min. Nuclei were counterstained with To-Pro2 (Molecular Probes, Inc.) at a dilution of 1:2000 for 2 min. Sections were mounted in Permafluor (Immunotech-Coulter, Marseille, France) and coverslipped. Fluorescent images were visualized and photographed using a Carl Zeiss (Jena, Germany) laser scanning microscope LM 510. Cells. Approximately 10,000 FPS cells were seeded in chamber slides, allowed to attach and grow overnight, before being fixed in 100% icecold methanol. After fixing, cells were washed in PBS and blocked using 5% normal horse serum diluted in PBS. Immunostaining was performed as outlined for the endometrial tissues previously.

TaqMan quantitative RT-PCR FP receptor, FGF2, and FGFR1 expression. RT-PCR analysis for FP receptor, FGF2, and FGFR1 expression was performed on FPS cells, endometrial adenocarcinomas (n ⫽ 36), and normal tissues (n ⫽ 30) as described previously (12, 21). FGF2 and COX-2 regulation by FGF2. For determination of FGF2 and COX-2 expression in FPS cells, cells were seeded in 5-cm dishes to a density of 5 ⫻ 105 cells in complete medium and thereafter starved by serum withdrawal for at least 12 h in serum-free medium containing 8.4 ␮m indomethacin. For determination of FGF2 and COX-2 expression in tissues, endometrial carcinoma explants (n ⫽ 6) were finely chopped using a sterile scalpel blade and incubated in serum-free medium containing 8.4 ␮m indomethacin for at least 12 h. Thereafter, medium was removed from cells and tissues, and replaced with fresh medium containing indomethacin with either vehicle, AL8810, SU4984, or PD98059 for 1 h before stimulation with serum-free culture medium containing either vehicle, 200 pg recombinant FGF2/ml, or 100 nm PGF2␣, in the absence/presence of AL8810, SU4984, or PD98059 for the time indicated in the figure legends. RNA was extracted using Tri-reagent (Sigma Chemical Co.) following the manufacturer’s guidelines. Once extracted and quantified, RNA samples were reverse transcribed and subjected to RT-PCR analysis using an ABI Prism 7700, as described previously (12). FGF2 and FGFR1 primers and probe for quantitative PCR were designed using the PRIMER express program (PE Applied Biosystems, Warrington, UK). The sequences of the FP receptor and FGF2 primers and probe have been described previously (8, 10, 12). The sequence of the FGFR1 primers and probe are as follows: FGFR1, forward (5⬘-AAA GAA TTC AAA CCT GAC CAC AGA A-3⬘); FGFR1, reverse (5⬘-CAC CAC AGA GTC CAT TAT GAT GCT-3⬘); and FGFR1, TaqMan probe (5⬘-TGG CAT AAC GGA CCT TGT AGC CTC CA-3⬘). Primers were carefully designed to cross exon/intron regions and to avoid the formation of primer-dimers, hairpins, and self-complementarity. Data were analyzed and processed using Sequence Detector v1.6.3 (PE Applied Biosystems). Expression of FP


receptor/FGF2/FGFR1 was normalized to RNA loading for each sample using the 18S ribosomal RNA as an internal standard. Results are expressed as fold increase above vehicle-treated (V) or fold above normal cDNA as mean ⫾ sem.

Protein extraction Tissue. For MAPK studies, carcinoma tissues (n ⫽ 6) were finely chopped using a sterile scalpel blade and incubated in serum-free culture medium containing penicillin/streptomycin (as described previously) and 8.4 ␮m indomethacin (a dual COX-enzyme inhibitor used to inhibit endogenous prostanoid biosynthesis). The next day, tissue was pretreated with vehicle, SU4984, or PD98059 for 1 h before stimulation with vehicle, or 200 pg recombinant FGF2/ml, or conditioned medium from either V or PGF2␣-treated (P) FPS cells for 10 min. After stimulation, tissue was washed with PBS, and protein was harvested by homogenization in protein lysis buffer, clarified by centrifugation, and assayed using protein assay kits (Bio-Rad, Hemel Hempstead, UK) before Western blot analysis. Cells. For FGF2 protein expression, approximately 5 ⫻ 105 cells were seeded in 5-cm dishes, and allowed to attach and grow for at least 16 h. Next, cells were incubated in serum-free culture medium and 8.4 ␮m indomethacin for at least 12 h. Cells were stimulated with vehicle, 100 nm PGF2␣, or 100 nm PGF2␣ and 50 ␮m AL8810, or 100 nm PGF2␣ and 50 ␮m PD98059 for the time indicated in the figure legend. For MAPK studies, cells were pretreated with vehicle, SU4984, or PD98059 for 1 h and then stimulated with vehicle, 200 pg recombinant FGF2/ml, or conditioned medium obtained from FPS cells that had been treated with vehicle, 100 nm PGF2␣, or 100 nm PGF2␣ and 50 ␮m AL8810, or 50 ␮m PD98059 for the time indicated in the figure legend. After stimulation, cells were washed with ice-cold PBS, lysed on ice, and proteins were extracted and quantified as described previously (12). Western blot analysis. Approximately 20 ␮g protein was solubilized in Laemmli buffer [125 mm Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue), then boiled for 5 min. Proteins were resolved and immunoblotted as described previously (12), and incubated with specific primary and alkaline-phosphatase-conjugated secondary antibodies. Immunoreactive proteins were visualized by the enhanced chemifluorescence system according to the manufacturer’s instructions. Proteins were revealed and quantified by PhosphorImager analysis using the Typhoon 9400 system (Molecular Dynamics, Amersham Biosciences). Relative density of immunoblots was calculated by dividing the value obtained from the phosphorylated ERK or FGF2 blots by the value obtained from total ERK or ␤-actin blots and expressed as fold above vehicle controls. All data are presented as mean ⫾ sem. Conditioned medium. Approximately 2 ⫻ 106 cells were seeded in a volume of 20 ml. Cells were allowed to attach and grow for a minimum of 16 h before starvation for at least 12 h with 8.4 ␮m indomethacin in serum-free culture medium. Thereafter, cells were stimulated with either vehicle, 100 nm PGF2␣, or 100 nm PGF2␣ and AL8810, or 100 nm PGF2␣ and PD98059 for 24 h in a final volume of 20 ml. Medium was analyzed for FGF2 content by ELISA. Immunoneutralization of FGF2 from the culture medium was performed by incubation of 2 ml of conditioned medium from each treatment with 1 ␮g of FGF2 antibody, or the equivalent goat IgG at 4 C overnight under constant rotation. Immune complexes were captured with 20 ␮l of a 50% protein A plus protein G slurry (Oncogene, Beeston, Nottingham, UK), and the medium was assayed again for FGF2 content by ELISA.

FGF2 ELISA Tissues. For determination of FGF2 secretion into the culture medium in tissues, endometrial carcinoma explants (n ⫽ 3) were finely chopped using a sterile scalpel blade and incubated in serum-free medium containing 8.4 ␮m indomethacin for at least 12 h. Thereafter, medium was removed and replaced with fresh serum-free medium containing indomethacin and vehicle, 100 nm PGF2␣, or 100 nm PGF2␣ and AL8810, or 100 nm PGF2␣ and PD98059 for 24 h. Culture medium was removed, and FGF2 protein was measured using a Human FGF2 ELISA kit as per the manufacturer’s instruction (Oncogene). The ELISA measures the pre-


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dominant 18-kDa FGF2 isoform present in the cytoplasm and secreted from the cell. Cells. FPS cells were starved for at least 12 h in serum-free medium containing 8.4 ␮m indomethacin. Thereafter, medium was removed and replaced with fresh serum-free medium containing indomethacin and vehicle, 100 nm PGF2␣, or 100 nm PGF2␣ and AL8810, or 100 nm PGF2␣ and PD98059 for 24 h. FGF2 protein secreted into the culture medium was assayed as described previously. The data are presented as mean ⫾ sem.

Proliferation assay Proliferation of FPS cells was determined using a CellTitre 96AQueous One Solution cell proliferation assay (Promega Corp., Madison, WI). Briefly, cells were seeded at 4 ⫻ 103 cells per well in a 96-well plate and allowed to adhere overnight. Cells were next starved for 24 h with 3-␮g/ml indomethacin before addition of FGF2 or vehicle in serum-free medium containing indomethacin. In parallel, wells were treated with serum-free medium containing FGF2 and indomethacin in the presence of SU4984 or PD98059 for 24 h. Control wells received the same concentration of vehicle alone or vehicle and inhibitor. After 24-h treatment, proliferation was measured by addition of the CellTitre 96AQueous One Solution reagent as per the manufacturer’s protocol. Cells were then incubated for 3 h at 37 C and 5% CO2 (vol/vol) to reduce the tetrazolium compound to a 490-nm absorbing formazan compound. Cell proliferation was measured by dividing the absorbance value obtained from cells treated with FGF2 by the absorbance value obtained from controltreated cells and expressed as a percentage over V cells. Data are presented as mean ⫾ sem.

Statistics Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 5.0; Abacus Concepts Inc., Berkeley, CA).

Results FGF2 expression in endometrial adenocarcinoma and normal endometrium

The expression of FP receptor, FGF2, and FGFR1 mRNA in human endometrial adenocarcinoma, normal endometrium, and FPS cells was determined by TaqMan Quantitative RT-PCR analysis (Fig. 1). The expression of FP receptor, FGF2, and FGFR1 mRNA was significantly higher in FPS cells and all cases of endometrial adenocarcinoma investigated compared with normal endometrium (P ⬍ 0.05). In addition, the expression of FGF2 and FGFR1 was greater in endometrial adenocarcinoma compared with FPS cells (P ⬍ 0.05). There was no correlation among the levels of FP receptor, FGF2, or FGFR1 mRNA with grades or stages of adenocarcinoma, however, a large variance was observed within these tissues. Colocalization of FP receptor with FGF2 or FGFR1 in endometrial adenocarcinomas, normal endometrium, and FPS cells

FP receptor and FGF2 or FGFR1 expression was colocalized in endometrial adenocarcinomas by dual immunofluorescence immunohistochemistry and confocal laser microscopy (Fig. 2). Strong FP receptor (FP; red channel) and FGF2/ FGFR1 immunoreactivity (green channel) were localized together (FP/FGF2 or FP/FGFR1; yellow channel) in the glandular epithelial compartment in all poorly, moderately, and well-differentiated endometrial adenocarcinomas, and the well-differentiated endometrial adenocarcinoma cell line

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FIG. 1. FP receptor, FGF2, and FGFR1 expression in endometrial tissue and Ishikawa FPS cells. Relative mRNA expression of FP receptor, FGF2, and FGFR1 in endometrial adenocarcinoma (n ⫽ 36) and normal endometrium (n ⫽ 30) and Ishikawa FPS cells as determined by real-time quantitative RT-PCR analysis (b is significantly different from a, c is significantly different from a and b, and d is significantly different from a– c; P ⬍ 0.05).

stably transfected with the FP receptor (FPS cells). Some diffuse stromal and vascular expression was observed for the FP receptor in endometrial carcinoma tissues, consistent with our previous findings (12). Low FGF2 or FGFR1 immunoreactivity was observed in the stromal compartment of endometrial adenocarcinomas compared with the glandular compartment, and vascular immunoreactivity appeared to be limited to the larger blood vessels. We previously reported a temporal pattern of expression for FP receptor in normal endometrial across the menstrual cycle with expression restricted to the mid-late proliferative phases of the menstrual cycle compared with the secretory phase (20). Consistent with this finding, in the present study, we showed immunoreactivity for FP receptor (red channel) only in proliferative phase endometrium. No FP receptor immunoreactivity was observed in secretory phase endometrium. In contrast, FGF2 and FGFR1 immunoreactivity (green channel) was observed in the glandular epithelium and stroma of all normal endometrial tissues. In proliferative phase endometrium, FP receptor (red channel) expression and FGF2/FGFR1 expression (green channel) colocalized within the epithelial and stromal compartments (yellow channel), as indicated by the arrowheads in the enlarged image. Incubating control sections with nonimmune IgG from the host species (inset) showed no immunoreactivity. PGF2␣ induces FGF2 expression in FPS cells and endometrial adenocarcinoma explants

The role of PGF2␣-FP receptor signaling on the expression of FGF2 was investigated using FPS Ishikawa endometrial adenocarcinoma cells stably expressing the FP receptor to the same levels as observed in endometrial adenocarcinomas (17) and endometrial adenocarcinoma explants. FPS cells were stimulated with 100 nm PGF2␣ or vehicle for 2, 4, 8, 24, 36, and 48 h. PGF2␣ stimulation resulted in a rapid time-dependent increase in the expression of FGF2 mRNA in FPS cells (Fig. 3A), with

FIG. 2. Colocalization of FGF2 and FGFR1 with FP receptor in endometrial tissues and Ishikawa FPS cells. Localization of the site of expression of FP receptor (red) and FGF2/FGFR1 (green), and colocalization of FP receptor with FGF2/FGFR1 (merged; yellow) in poorly (P; n ⫽ 5), moderately (M; n ⫽ 5), and well-differentiated (W; n ⫽ 5) endometrial adenocarcinomas, normal secretory (S) and proliferative phase endometrium (Pr) and Ishikawa FPS cells (FPS), respectively. Enlargement shows FP/FGF2 and FP/FGFR1 colocalization in stromal (arrowhead) and epithelial cells in proliferative phase endometrium. Inset shown for negative control. Scale bar, 100 ␮m.

maximal expression observed by 24 h (P ⬍ 0.01). No such increase in the expression of FGF2 was observed in wild-type cells at either time points investigated (data not shown). PGF2␣-FP receptor signaling to FGF2 in FPS cells and endometrial adenocarcinoma explants was investigated by quantitative RT-PCR analysis (Fig. 3, B and C). FPS cells (Fig. 3B) and adenocarcinoma explants (Fig. 3C) were stimulated with vehicle or 100 nm PGF2␣ in the absence/presence of the specific FP receptor antagonist AL8810 or inhibitor of mitogen-associated protein kinase (MEK) PD98059 for 8 and 24 h (for FPS cells and adenocarcinoma tissues, respectively). PGF2␣ stimulation resulted in a significant increase in the expression of FGF2 mRNA, which was inhibited by cotreatment of FPS cells and tissue explants with the specific FP receptor antagonist AL8810 or MEK inhibitor PD98059 (P ⬍ 0.05). Similarly, a significant elevation in immunoreactivity of the 18-kDa cytoplasmic FGF2 protein was observed in FPS cells treated with 100 nm PGF2␣ after 24 h (Fig. 3D; P ⬍ 0.01). Coincubation of cells with AL8810 or PD98059 abolished the PGF2␣-mediated increase in FGF2 immunoreactivity (P ⬍ 0.01). PGF2␣-FP receptor activation induces FGF2 secretion in Ishikawa FPS cells and endometrial adenocarcinoma explants

We investigated whether FGF2 protein was secreted in the culture medium of FPS cells or endometrial adenocarcinoma



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FIG. 3. FGF2 expression in Ishikawa cells and endometrial cancer explants in response to 100 nM PGF2␣. A, FGF2 expression in Ishikawa FPS cells (n ⫽ 5) as measured by real-time quantitative RT-PCR analysis after treatment of cells for 2, 4, 8, 24, 36, and 48 h with 100 nM PGF2␣ (b is significantly different from a, and c is significantly different from a and b; P ⬍ 0.01). B, FGF2 mRNA expression in FPS cells (n ⫽ 4) treated for 8 h with vehicle, 100 nM PGF2␣, 100 nM PGF2␣ and AL8810, or 100 nM PGF2␣ and PD98059, as determined by quantitative RT-PCR analysis (b is significantly different from a; P ⬍ 0.05). C, FGF2 mRNA expression in endometrial adenocarcinoma explants (n ⫽ 6) as measured by real-time quantitative RT-PCR analysis after treatment of tissue for 24 h with vehicle, 100 nM PGF2␣, 100 nM PGF2␣ and AL8810, or 100 nM PGF2␣ and PD98059 (b is significantly different from a; P ⬍ 0.05). D, FGF2 protein expression in FPS cells (n ⫽ 4) as determined by Western blot analysis after treatment of cells for 24 h with vehicle, 100 nM PGF2␣, 100 nM PGF2␣ and AL8810, or 100 nM PGF2␣ and PD98059. A representative Western blot is shown (b is significantly different from a; P ⬍ 0.01). FPS cells (E; n ⫽ 3) and endometrial adenocarcinoma tissues (F; n ⫽ 3) were treated with vehicle or 100 nM PGF2␣ in the absence/presence of AL8810, PD98059 for 24 h, and FGF2 protein secreted into the culture medium was assayed by ELISA (b is significantly different from a; P ⬍ 0.05). Data are represented as mean ⫾ SEM.

explants treated with 100 nm PGF2␣ by ELISA. PGF2␣ treatment of FPS cells (Fig. 3E) and endometrial adenocarcinoma explants (Fig. 3F) induced a significant elevation in secretion of the 18-kDa isoform of FGF2 protein in the culture medium at 24 h (P ⬍ 0.05). Cotreatment of FPS cells or tissues with PGF2␣ and AL8810 or PD98059 abolished the secretion of FGF2 into the culture medium (P ⬍ 0.05). These data indicate that FGF2 protein expression and secretion are regulated via the PGF2␣-FP receptor activation of the ERK signal transduction pathway.

Autocrine/paracrine regulation of neoplastic epithelial cell function by FGF2 secreted from FPS cells

Because FP receptor, FGF2, and FGFR1 were colocalized within the epithelial compartment of proliferative phase endometrium, endometrial carcinomas, and FPS cells, we next investigated whether the FGF2, secreted into the culture medium of FPS cells determined in Fig. 3E, could induce signaling in FPS cells in an autocrine/paracrine manner via interaction with the FGFR1.

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FIG. 4. Autocrine/paracrine regulation of ERK in FPS cells and endometrial cancer tissues by FGF2. FPS cells (A; n ⫽ 4) and endometrial cancer tissues (B; n ⫽ 6) were pretreated with either the FGFR1 receptor tyrosine kinase inhibitor SU4984 or MEK inhibitor PD98059 or vehicle and then stimulated for 5 min with 200 pg/ml recombinant FGF2, 200 pg/ml recombinant FGF2 and PD98059, or 200 pg/ml recombinant FGF2 and SU4984. ERK phosphorylation was measured by Western blot analysis. C, FPS cells (n ⫽ 4) were pretreated with either vehicle, the FGFR1 receptor tyrosine kinase inhibitor SU4984, or MEK inhibitor PD98059 and then stimulated for 5 min with the conditioned medium assayed for FGF2, as presented in Fig. 3E. ERK phosphorylation was measured by Western blot analysis. D, The conditioned medium (n ⫽ 4) used in Fig. 3E was incubated with either 1 ␮g FGF2 antibody or equivalent concentration of IgG from the same host species for 24 h. Immune complexes were removed by addition of protein A plus G agarose beads and centrifugation and the medium reanalyzed for FGF2 by ELISA. E, FPS cells (n ⫽ 4) were stimulated for 5 min with the immunoneutralized medium preadsorbed with the IgG or FGF2 (from Fig. 3E) and ERK phosphorylation measured by Western blot analysis. F, Western blot analysis of ERK phosphorylation in endometrial adenocarcinoma explants (n ⫽ 6). Adenocarcinoma tissues were pretreated with vehicle, SU4984, or PD98059 and stimulated for 5 min with conditioned medium from either V or P FPS cells, or immunoneutralized P conditioned medium preadsorbed with FGF2 antibody or IgG (b is significantly different from a, and c is significantly different from a and b; P ⬍ 0.05). Data are represented as mean ⫾ SEM.



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FIG. 5. FGF2 elevates the mRNA expression of FGF2 and COX-2 in FPS cells and endometrial adenocarcinoma explants, and enhances cell proliferation via the FGFR1 and ERK pathways. Ishikawa FPS cells (A and B; n ⫽ 4) and endometrial adenocarcinoma biopsy explants (C and D; n ⫽ 4) were treated with vehicle, 200 pg/ml recombinant FGF2, 200 pg/ml recombinant FGF2 and PD98059, or 200 pg/ml recombinant FGF2 and SU4984 for 6 h (A and C) or 8 h (B and D) and FGF2 (A and C) and COX-2 (B and D). mRNA expression was determined by quantitative RT-PCR analysis (b is significantly different from a; P ⬍ 0.01). E, Ishikawa FPS cells (n ⫽ 6) were treated with vehicle, or increasing doses of recombinant FGF2 peptide in the presence/absence of SU4984 or PD98059 for 24 h, and cellular proliferation was determined using the CellTitre 96Aqueous One Solution reagent (b is significantly different from a; P ⬍ 0.05). Data are represented as mean ⫾ SEM.

We treated FPS cells (Fig. 4A) and endometrial adenocarcinoma tissues (Fig. 4B) with 200 pg recombinant FGF2 peptide/ml in the absence or presence of a specific FGFR1 receptor tyrosine kinase inhibitor SU4984 or the MEK inhibitor PD98059 to investigate whether FGF2 could induce ERK signaling via the FGFR1. FGF2 peptide treatment significantly phosphorylated ERK in FPS cells (Fig. 4A) and endometrial adenocarcinoma tissues (Fig. 4B) compared with vehicle treatment. Cotreatment of FPS cells or adenocarcinoma tissues with SU4984 or PD98059 abolished the ability of recombinant FGF2 peptide to phosphorylate ERK (Fig. 4, A and B; P ⬍ 0.05). We investigated whether the FGF2 secreted into the culture medium, after treatment of FPS cells with 100 nm PGF2␣, could similarly phosphorylate ERK. FPS cells were pre-

treated with either vehicle, SU4984, or PD98059 and then stimulated with the undiluted panel of culture medium assayed for FGF2 in Fig 3E. A rapid phosphorylation of ERK was observed in FPS cells (Fig. 4C) in response to the culture medium from PGF2␣-only treated FPS cells (P ⬍ 0.05). This elevation in ERK phosphorylation was abolished by pretreatment of FPS cells with SU4984 or PD98059. Minimal ERK activation was observed in FPS cells treated with the conditioned medium from FPS cells that had been stimulated with PGF2␣ in the presence of the FP receptor antagonist AL8810 or MEK inhibitor PD98059 (Fig. 4C). These data suggest the FGF2 secreted into the culture medium of FPS cells, as observed in Fig. 3E, could activate ERK signaling in a similar manner to recombinant FGF2 peptide via coupling with the FGFR1.

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To confirm that the autocrine/paracrine signaling mediated by the conditioned medium was due to FGF2 signaling via the FGFR1, we immunoneutralized the conditioned medium (assayed in Fig. 3E) by incubating aliquots of medium from each treatment group with either excess FGF2 antibody or equivalent concentration of IgG from the same host species. As shown in Fig. 4D, the FGF2 antibody, but not IgG, significantly depleted the FGF2 present in the conditioned medium collected from cells treated with PGF2␣ (P ⬍ 0.05). We next stimulated FPS cells with the immunoneutralized conditioned medium and investigated ERK phosphorylation (Fig. 4E). We found that the immunoneutralized medium incubated with the FGF2 antibody failed to induce phosphorylation of ERK in the FPS cell line compared with the medium incubated with IgG. In addition, we found that conditioned medium from P FPS cells could similarly phosphorylate ERK in endometrial cancer explants, compared with medium from V FPS cells (Fig. 4F; P ⬍ 0.05). Cotreatment of endometrial biopsy explants with conditioned medium from P FPS cells and SU4984 (P ⫹ SU4984) or PD98059 (P ⫹ PD98059) abolished the ERK phosphorylation induced by treatment of the cells with conditioned medium from P FPS cells alone (P ⬍ 0.05). Similarly, immunoneutralization of conditioned medium from P FPS cells with FGF2 antibody (P ⫹ FGF2-Ab) failed to phosphorylate ERK in cancer biopsy explants, however, the conditioned medium from P FPS cells preadsorbed with IgG (P ⫹ IgG) from the host species promoted a rapid activation of ERK in endometrial cancer tissues (Fig. 4F; P ⬍ 0.05). These data confirm that ERK activation in FPS cells and endometrial adenocarcinoma biopsy explants in response to the conditioned medium from P FPS cells was mediated via FGF2.


Discussion

The expression of E- and F-series-prostanoid receptors, FGF2, and FGFR1 has correlated with proliferation and tumor growth in several in vitro and in vivo model systems (11, 22–29). In addition, a correlation between expression of FGF2 and the COX-PG axis has been recently described (8, 9). In these studies overexpression of COX enzymes (either COX-1 or COX-2) concomitantly up-regulates the expression of growth factors, including FGF2, to promote tumorigenesis (8, 9), however, the specific prostanoids involved in mediating these effects have until now not been explored. Here, we show elevated expression of FGF2 and FP receptor, colocalized within the glandular epithelial compartment of endometrial adenocarcinomas. Previously, we established a role for PGF2␣-FP receptor signaling in endometrial adenocarcinoma explants in enhancing the proliferation of epithelial cells (12). In addition, we have recently mapped out the sequence of signaling events activated by PGF2␣-FP receptor interaction using the FPS cell line and endometrial adenocarcinoma biopsies, and found that PGF2␣-FP receptor signaling can induce the expression of COX-2 via the c-Src and epidermal growth factor receptor-mediated phosphorylation of ERK (17, 18), thereby establishing a positive feedback loop for regulation of the COX-PG axis. We show here that in addition to the autoregulation of the COX-2/PG axis via the FP receptor, PGF2␣-FP receptor signaling can also regulate the expression and secretion of FGF2

FGF2 promotes FGF2 and COX-2 expression in FPS cells and endometrial cancer explants, and enhances cell proliferation via the FGFR1 and ERK pathways

We investigated whether FGF2 could promote the expression of FGF2 and COX-2 in FPS cells and endometrial biopsy explants, thereby establishing a dual positive feedback loop for the regulation of neoplastic cell function. We found that treatment of FPS cells and endometrial biopsy explants with recombinant FGF2 significantly elevated the mRNA expression of FGF2 (Fig. 5, A and C) for FPS cells and endometrial cancer biopsy explants, respectively (P ⬍ 0.01), and COX-2 (Fig. 5, B and D) for FPS cells and endometrial cancer biopsy explants, respectively (P ⬍ 0.01), compared with V cells and tissues. Cotreatment of FPS cells and endometrial cancer biopsies with FGF2 and SU4984 or PD98059 abolished FGF2 and COX-2 mRNA expression in response to FGF2 in FPS cells (Fig. 5, A and B; P ⬍ 0.01) and cancer tissues (Fig. 5, C and D; P ⬍ 0.01). Finally, we investigated the effect of FGF2 on the proliferation of endometrial adenocarcinoma cells. Treatment of FPS cells with FGF2 peptide significantly augmented cell proliferation at all doses compared with V cells (Fig. 5E; P ⬍ 0.05). Cotreatment of cells with FGF2 and SU4984 or PD98059 abolished the FGF2-induced increase in cell proliferation.

FIG. 6. Schematic representation of autocrine/paracrine signaling by FGF2 produced by PGF2␣-FP receptor signaling in endometrial adenocarcinoma cells. PGF2␣, produced via the actions of COX enzymes, interacts with FP receptors in an autocrine/paracrine manner to activate Gq and inositol 1,4,5-trisphosphate, leading to the activation of ERK (17, 18). Activation of ERK signaling can promote the elevated de novo transcription, translation, and secretion of FGF2 protein in FPS cells, as well as induce expression of COX-2 and the release of PGF2␣. In turn, the secreted FGF2 protein from FPS cells can induce ERK phosphorylation via the FGFR1 and increase cellular proliferation. FGF2-FGFR1 interaction also establishes a positive feedback loop that maintains elevated FGF2 and COX-2 expression, thereby enhancing neoplastic cell function.


via the ERK pathway. FGF2, once released from the cell, exerts its function via high-affinity FGF receptors, which are transmembrane receptor tyrosine kinases that belong to the Ig superfamily (7). The data presented also show that FGFR1 is elevated concomitantly with FGF2 in endometrial adenocarcinomas and colocalizes with FP receptor in the glandular epithelial compartment of endometrial adenocarcinomas. In prostate cancer tissue, FGF2 is also significantly elevated compared with normal prostate, and the FGF2 secreted from prostatic stromal cells has mediated a paracrine effect on epithelial cells to promote neoplastic cell growth (7). In endometrial adenocarcinomas, tumorigenesis may be promoted in a similar autocrine/paracrine manner by FGF2, secreted from FP receptor overexpressing epithelial cells in response to circulating PGF2␣. Once released, FGF2 can act on adjacent epithelial cells to promote mitogenic signaling to ERK and transcription of target genes involved in regulating tumorigenesis. To explore whether the FGF2, produced as a consequence of PGF2␣-FP receptor interaction in FPS cells, could signal in an autocrine/paracrine manner in endometrial adenocarcinomas, we treated FPS cells and endometrial adenocarcinoma explants with recombinant FGF2 peptide or conditioned medium from P FPS cells, which we had shown to produce elevated levels of FGF2 in cell culture. Treatment of FPS cells and endometrial adenocarcinoma explants with FGF2 peptide or conditioned medium from P FPS cells rapidly phosphorylated ERK. This phosphorylation was abolished in cells and tissues pretreated with the FGFR1 tyrosine kinase inhibitor SU4984 or inhibitor of MEK (PD98059). We further confirmed that the FGF2 component of the conditioned medium was responsible for the phosphorylation of ERK via FGFR1 by immunoneutralizing the FGF2 from the conditioned medium with an FGF2 polyclonal antibody. In the present study, we show that FGF2 can auto-regulate its own expression in FPS cells and endometrial cancer biopsies, as well as up-regulate the expression of COX-2, which in turn, can promote the further production of PGs to sustain tumorigenesis in a positive feedback manner. Interestingly, we have observed that the expression of FGFR1 in endometrial cancer tissues is significantly greater than expression detected in FPS cells. This elevation in expression of FGFR1 in the cancer tissues compared with our cell line could account for the higher induction of FGF2 and COX-2 mRNA levels that we observe in endometrial cancer biopsies treated with FGF2 compared with FPS cells. In addition to the autocrine/paracrine regulation of tumorigenic genes, our data presented here show that FGF2, once secreted from the cell, can enhance the proliferation of endometrial adenocarcinoma cells. In pancreatic cancer, elevated FGF2 expression promotes tumor progression by enhancing cell proliferation (28). Moreover, Giavazzi et al. (6) have shown that human endometrial adenocarcinoma cells expressing FGF2, under the control of a tetracycline-responsive promoter, can directly modulate tumor growth when implanted sc in nude mice given tetracycline to induce transgene expression. Thus, it is likely that the FGF2 secreted after PGF2␣-FP receptor interaction can promote endometrial tumorigenesis in a similar manner via the FGFR1 and ERK pathways. Antisense cDNA therapy targeted against growth


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factors and their cognate receptors such as FGF2 and FGFR1 have inhibited cancer growth of human melanomas implanted in nude mice (30). Thus, it is feasible that targeted inhibition of potent growth factors and their receptors such as FGF2 and FGFR1, either directly or by targeted disruption of FP receptor signaling in endometrial adenocarcinomas, using specific FP receptor antagonists to prevent the initiation of the FGF2-FGFR1 cascade, may be also of relevance in endometrial tumors as antitumor strategies. In conclusion, we now provide evidence for a second positive feedback loop for the regulation of neoplastic endometrial epithelial cell function via the FGF2-FGFR1 signaling pathway, initiated and driven by the COX-PG axis. As shown in Fig. 6, PGF2␣ via the FP receptor can promote the expression of COX-2, and expression and release of FGF2 protein. In turn, FGF2 can regulate neoplastic cell function in an autocrine/paracrine manner by establishing a positive feedback loop to auto-regulate its own expression via the FGFR1 and ERK pathways, while concomitantly elevating the expression of COX-2, to drive production of PGF2␣. These in turn can enhance tumorigenesis by augmenting cellular proliferation. Acknowledgments We thank Ms. Joan Creiger for patient recruitment and sample collection. Received November 13, 2006. Accepted April 26, 2007. Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh, Scotland EH16 4TJ, United Kingdom. E-mail: [email protected]. Disclosure of potential conflicts of interest relating to this study: The authors have nothing to declare.

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