Suppression of Prostate Tumor Cell Growth by Stromal Cell Prostaglandin D Synthase–Derived Products

Research Article

Jeri Kim,1 Peiying Yang,2 Milind Suraokar,3 Anita L. Sabichi,3 Norma D. Llansa,3 Gabriela Mendoza,3 Vemparalla Subbarayan,3 Christopher J. Logothetis,1 Robert A. Newman,2 Scott M. Lippman,3 and David G. Menter3

Departments of 1Genitourinary Medical Oncology, 2Experimental Therapeutics, and 3Clinical Cancer Prevention, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Abstract seminal fluid (10). Once PGD2 is made, it forms derivative Stromal-epithelial interactions and the bioactive molecules compounds, most of which can transactivate the peroxisome g g produced by these interactions maintain tissue homeostasis proliferator–activated receptor (PPAR ). One PGD2 derivative, 15-deoxy-D12,14-prostaglandin J (15-d-PGJ ), can slow the growth and influence carcinogenesis. Bioactive prostaglandins pro- 2 2 duced by prostaglandin synthases and secreted by the prostate and induce the partial differentiation of selected cancer cells (12). D12,14 into seminal plasma are thought to support reproduction, but Another PGD2 derivative, 15-deoxy- -PGD2 (15-d-PGD2), has g their endogenous effects on cancer formation remain unre- also been shown to stimulate PPAR transactivation in RAW 264.7 solved. No studies to date have examined prostaglandin cell macrophage cultures as effectively as 15-d-PGJ2 (13). L-PGDS enzyme production or prostaglandin metabolism in normal also binds tritiated testosterone and may play a role in androgen prostate stromal cells. Our results show that lipocalin-type transport (14). In castrated rats, testosterone proprionate induces prostaglandin D synthase (L-PGDS) and prostaglandin D L-PGDS synthesis in the epididymis (15). Although multiple studies 2 have shown a strong correlation between elevated L-PGDS (PGD2) metabolites produced by normal prostate stromal cells inhibited tumor cell growth through a peroxisome expression in the male reproductive tract and male fertility proliferator–activated receptor ; (PPAR;)–dependent mech- (14, 16, 17), the mechanistic role L-PGDS plays in normal anism. Enzymatic products of stromal cell L-PGDS included reproductive homeostasis and activity remains unknown. 12,14 In previous studies, we observed that PPARg was highly high levels of PGD2 and 15-deoxy-# -PGD2 but low levels of 12,14 expressed in malignant cells, which promoted selective growth 15-deoxy-# -prostaglandin J . These PGD metabolites 2 2 g activated the PPAR; ligand-binding domain and the peroxi- suppression in tumor cells by PPAR ligands when compared with g some proliferator response element reporter systems. Thus, normal cells that did not express PPAR (18, 19). Because high levels of L-PGDS and PGD2 have been found in normal seminal growth suppression of PPAR;-expressing tumor cells by PGD2 metabolites in the prostate microenvironment is likely to be plasma and reproductive tissue (10), we hypothesized in the g an endogenous mechanism involved in tumor suppression present study that these products stimulate the PPAR expressed that potentially contributes to the indolence and long latency primarily by prostate tumor cells, resulting in specific growth period of this disease. (Cancer Res 2005; 65(14): 6189-98) suppression. As a corollary to this hypothesis, we assumed that normal epithelia not expressing PPARg would remain unaffected by L-PGDS and PGD . To test our hypothesis, we examined the Introduction 2 expression of L-PGDS and its metabolic products in normal Dynamically balanced molecular mechanisms in the prostate prostate cells and their biological effects on normal prostate microenvironment mediate stromal-epithelial function during the epithelial cells and prostate tumor cells. development and homeostatic maintenance of the prostate gland. Perturbation of these molecular dynamics can have a negative or positive influence during prostate carcinogenesis. Materials and Methods Among the many products generated by support tissues in the Cell culture. Normal prostate epithelial cells, prostate stromal cells, and prostate gland, those most likely to profoundly affect the growth of prostate smooth muscle cells isolated from young trauma victims were cancer cells are prostaglandins. Prostaglandins are essential to obtained from Clonetics Corp. (San Diego, CA). The PC-3, LNCaP, and male reproduction (1, 2), and high levels of prostaglandins are DU145 cell lines were obtained from American Type Culture Collection found in semen as products of both prostate and seminal vesicles (Manassas, VA). Control RAW 264.7 cells were provided by Dr. B. Su (3–7). (Department of Immunology, The University of Texas M.D. Anderson Unique among glandular epithelial tissues, the prostate is one Cancer Center, Houston, TX) and HU78 cells by Dr. D. Jones (Department of of the few tissues other than the heart, the brain, and some Hematopathology, The University of Texas M.D. Anderson Cancer Center). adipose tissues that make lipocalin-type prostaglandin D synthase Primary cell cultures were maintained in defined culture medium according to the manufacturer’s instructions as described previously (20). All other cell (L-PGDS), which synthesizes prostaglandin D (PGD ; refs. 8–11). 2 2 lines were maintained in DMEM and F-12 low-glucose medium mixed at a Both L-PGDS protein and PGD2 are prominently found in normal ratio of 1:1 (Life Technologies, Bethesda, MD) supplemented with 10% fetal bovine serum. Reverse transcription-PCR. The RNA STAT-60 reagent (Tel-Test, Inc., Requests for reprints: David G. Menter, Department of Clinical Cancer Friendswood, TX) was used to extract the total RNA, which was treated with Prevention, The University of Texas M.D. Anderson Cancer Center, Box 1360, 1515 DNase I before use in a reverse transcription-PCR (RT-PCR) analysis. RNA Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-0626; Fax: 713-794-4403; E-mail: [email protected]. (1 Ag) was reverse transcribed with mouse mammary tumor virus RT (Life I2005 American Association for Cancer Research. Technologies, Inc., Rockville, MD). L-PGDS (600 bp) was amplified by the www.aacrjournals.org 6189 Cancer Res 2005; 65: (14). July 15, 2005

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Cancer Research primer set 5V-CTGCTCGGCTGCAGGAGAATGGCTACTCATCACAC-3V and Transactivation of the peroxisome proliferator response element. 5V-TGGGGAGTCCTATTGTTCCGTCATGCACTTA-3V, and PGDS (321 bp) PC-3 cells were cotransfected with [acyl-CoA oxidase-peroxisome was amplified by the primer set 5V-CCCAGGTCTCCGTGCAGCCCAACTTC- proliferator response element (PPRE)]-thymidine kinase-luciferase report- CAG-3Vand 5V-TGTACAGCAGGGCGTAGTGGTCGTAGTCA-3Vas described er (250 ng; ref. 27) and hGal cDNA (100 ng); reporter assay analysis previously (21). DP1 receptor (387 bp) was amplified by the primer set 5V- followed as described previously (19). Luciferase activity was normalized GCAACCTCTATGCGATGCAC-3Vand 5V-GAATTGCTGCACCGGCTCCT-3Vas to hGal activity that was cotransfected along with the appropriate described by Sarrazin et al. (22). DP2 receptor (309 bp), otherwise known as reporter. CRTH2 receptor, was amplified by the primer set 5V-CCTCTGT- Peroxisome proliferator–activated receptor ;–specific ligand-bind- GCCCAGAGCCCCACGATGTCGGC-3V and 5V-CACGGCCAAGA- ing domain transactivation. Recombinant pcDNA3 plasmids that AGTAGGTGAAGAAG-3Vas described by Nagata et al. (23). Primer pairs contained cDNA inserts encoding either a fusion protein containing Gal4 (5V-CAGCTCTGGAGAACTGCTG-3Vand 5V-GTGTACTCAGTCTCCACAGA-3V; DNA-binding domain (amino acids 1-147) coupled to a PPARg ligand- ref. 24) were used in RT-PCR analysis to detect 36B4 mRNA (24). The RT- binding domain (LBD; amino acids 174-475) fusion protein or just a Gal4 PCR DNA products were subcloned using a topoisomerase PCR system DNA-binding domain as a control (28) were used to transfect prostate (Invitrogen, Carlsbad, CA) and sequenced by automated sequencing cancer cells in six-well tissue culture plates. Either plasmid (0.5 Ag) plus (SeqWright, Houston, TX) to verify the insert DNA. After sequencing (Gal4UAS)4-thymidine kinase-luciferase reporter plasmid (0.5 Ag; ref. 29) occurred, a 600-bp product was then subcloned into three vectors, a were transfected by using FuGENE 6 (Roche, Indianapolis, IN). Either pIRESNeo2 selectable vector, a pCMVHA-tagged vector, and a pCMVmyc- PPARg ligands (5 Amol/L) dissolved in ethanol or ethanol alone were tagged vector, to yield pLPGDSNeo, pLPGDSHA, and pLPGDSmyc, added 24 hours after the addition of DNA. Luciferase activity was measured respectively. 6 hours after the ligands were added and normalized to the activity of Determination of prostaglandin D2 metabolites in prostate cells. either Renilla luciferase or hGal that was cotransfected with the Various cell lines were plated in 100-mm tissue culture dishes to attain a appropriate normalization reporter at one fifth the concentration of the confluence of 70% to 75%. Cells were then incubated with 10 Amol/L chimeric Gal4/PPARg plasmid. arachidonic acid for 30 minutes and then 1 hour. The culture medium was Construction and transfection with U6-tetO-driven short hairpin collected at each time point, and cells were harvested at 1 hour by RNA plasmids. A tetracycline-inducible version of the human U6 PolIII trypsinization and subjected to PGD2 extraction. promoter construct (U6-tetO) was obtained from Dr. D. Takai (University of Intracellular prostaglandin D2. The intracellular PGD2 was extracted Tokyo, Tokyo, Japan; ref. 30) to use as a base vector. Two DNA oligomers (5V- by using the modified method of Kempen et al. (25). Briefly, cells were GCCCTTCACTACTGTTGACGACGT-3V)and(5V-CGTCAACAGTAGT- resuspended in 500 AL PBS, and 20 AL aliquots were treated with 1 N citric GAAGGGC-3V) that contained a PPARg sequence shown previously to be acid and 10% butylated hydroxytoluene (2.5 AL). PGD2 was extracted thrice an effective small interfering RNA (siRNA) against the PPARg message with 2 mL hexane/ethyl acetate solution (1:1, v/v). The upper organic phases (31) were cloned into the U6-tetO construct. Additional oligomers (5V- were pooled and evaporated under a stream of nitrogen at 25jC. All CGTCAACAGTAGTGAAGGGCCTTTTTGGGCC-3V)and(5V-CAAAAAGG- extraction procedures were done under conditions of minimal light. CCCTTCACTACTGTTGACGACGT-3V) containing a complementary sequence Samples were then reconstituted in a 200 AL methanol to 10 mmol/L to the earlier oligos and a T5 transcriptional termination signal to ensure ammonium acetate buffer (70:30, v/v; pH 8.5) before analysis by liquid termination were subsequently cloned into the modified U6-tetO construct. chromatography tandem mass spectrometry (LC/MS/MS). The resulting plasmid contained a hairpin siRNA against the PPARg

Culture medium prostaglandin D2. PGD2 metabolites in the cell message. The clones were confirmed as being positive for hairpin siRNA culture medium were extracted by using a solid-phase method in which an insertion after restriction digestion, PCR analysis, and direct sequencing. aliquot of 10 AL of 10% butylated hydroxytoluene was added to 1 mL of the A control vector containing a previously reported siRNA against the cell culture medium. The solution was applied to a Sep-Pak C18 cartridge enhanced green fluorescent protein (EGFP; ref. 32) was made in a similar

(Waters Corp., Milford, MA), and PGD2 metabolites were eluted with 1 mL fashion, resulting in a short hairpin (shRNA) that was directed against the methanol. The eluate was evaporated under a stream of nitrogen, and the EGFP. residue was dissolved in a 100 AL methanol to 10 mmol/L ammonium In control experiments, a digital image analysis was done after cells were acetate buffer solution (70:30, v/v; pH 8.5). transfected with a plasmid that expressed EGFP either alone or in Liquid chromatography tandem mass spectrometry. LC/MS/MS combination with an EGFP shRNA-expressing plasmid followed by analysis was done with a Quattro Ultima tandem mass spectrometer treatment with 10 Amol/L PGD2. Phase-contrast images were obtained to (Micromass, Beverly, MA) equipped with a HP1100 binary pump high- observe total cell fluorescence and epifluorescence to determine the level of performance liquid chromatography inlet (Agilent Technologies, Inc., Palo EGFP protein. Similar experiments were done using either EGFP shRNA or Alto, CA). Prostaglandins were separated by using a Luna 3A phenyl-hexyl PPARg shRNA followed by treatment of cells with 10 Amol/L PGD2. Staining analytic column (2 Â 150 mm; Phenomenex, Torrance, CA; ref. 26). The with calcein AM (CAM) and 4V,6-diamidino-2-phenylindole (DAPI) dyes was mobile phase consisted of 10 mmol/L ammonium acetate (pH 8.5) in phase used to evaluate cell viability. A and methanol in phase B. The flow rate was 250 AL/min, with column Cell proliferation assay. Quantification was done by treating cells with temperature maintained at 50jC. The sample injection volume was 25 AL. 40 AL of a PBS solution containing 2.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-

PGD2 was detected by using electrospray-negative ionization and followed 2,5-diphenyltetrazolium bromide followed by formazan solubilization in by multiple reaction monitoring using the transition at m/z 351.2 > 271.2. 100 AL DMSO and reading absorbance at a wavelength of 540 nm on a

PGD2 was fragmented by using argon as the collision gas at a collision cell 96-well plate reader (33). pressure of 2.10 Â 10À3 mm Hg. Results were expressed as nanograms of Statistical analyses. Data were analyzed statistically using the Statview 6 PGD2 per 10 cells. The cell number was measured with an electronic software program (SAS Institute, Inc., Cary, NC). Student’s t tests were used particle counter (Beckman Coulter, Inc., Hialeah, FL). to determine the significance between mean group values. Western blot analysis. Whole cell lysates were prepared as described previously (20). Specifically, protein (100 Ag) was loaded in each lane and run on a 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane Results (Schleicher & Schuell Bioscience, Inc., Keene, NH). After blocking with 3% bovine serum albumin, the blots were exposed to rabbit primary anti-L- Lipocalin-type prostaglandin D synthase is expressed by PGDS antibody (Cayman Chemical Co., Ann Arbor, MI) followed by anti- normal but not by malignant prostate cells. In normal prostate rabbit secondary antibody (Pierce Chemical Co., Rockford, IL). The signals cells, PCR and Western blot analyses indicated that levels of L- were detected by using an enhanced chemiluminescence system (Pierce PGDS RNA and protein, respectively, were greatest in stromal cells, Chemical). less prominent in smooth muscle cells, and lowest in epithelial cells

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Figure 1. L-PGDS expression in prostate cells. A, RT-PCR analysis of L-PGDS expression in normal human epithelial cells (EC), stromal cells (St), and smooth muscle cells (SM) and prostate cancer cell lines PC-3 (PC), LNCaP (LN), and DU145 (DU). L-PGDS mRNA expression was observed in all of the normal cells but none of the tumor cells. Two different sets of primers were used: one set encompassed the entire open reading frame (600 bp) and the other set spanned multiple introns within the open reading frame (321 bp). B, Western blot analysis of 100 Ag total protein was done to determine the expression of L-PGDS protein in normal human epithelial cells, stromal cells, and smooth muscle cells and prostate cancer cell lines PC-3, LNCaP, and DU145. Actin was used as a protein loading control. C, immunofluorescence of L-PGDS (green fluorescence) expression profiles. L-PGDS expression was high in epithelial cells, stromal cells, and smooth muscle primary cell cultures but low in PC-3, LNCaP, and DU145 prostate cancer cell lines. DAPI-counterstained DNA in nuclei was blue, and Alexa 594-phalloidin–counterstained actin was red. L-PGDS was found in the cytoplasm and endoplasmic reticulum of normal cells but not in tumor cells. D, RT-PCR analysis of DP1 and DP2 expression in normal epithelial cells, stromal cells, and smooth muscle cells and PC-3, LNCaP, and DU145 cancer cells. E, RT-PCR control samples (HU78 cell mRNA) were shown to express both DP1 and DP2 receptors.

(Fig. 1A and B). The intracellular distribution of L-PGDS was in the protein–activating transmembrane receptors DP1 and DP2. cytoplasm and endoplasmic reticulum of normal cells (Fig. 1C). Multiple sets of intron-spanning PCR primers were used to analyze In contrast, tumor cells did not exhibit observable intracellular DNase-treated total RNA isolated from these cells. No DP1 or DP2 levels of L-PGDS. cDNA amplimers were observed at 25, 30, or 35 thermocycles in Prostate cells lack G protein–activating transmembrane any of the prostate cells (Fig. 1D). In contrast, HU78 cells in which receptors DP1 and DP2. We used a RT-PCR assay to examine DP1 and DP2 expression had been characterized previously showed normal and tumor-derived prostate cells for the presence of the G both PCR products (Fig. 1E). www.aacrjournals.org 6191 Cancer Res 2005; 65: (14). July 15, 2005

Arachidonic acid–conditioned stromal cell medium express either the PPARg1 or the PPARg2 protein and were not as enhances normal cell growth but suppresses cancer growth. sensitive as PC-3 tumor cells to growth inhibition by the PPARg We reported previously on the expression, phosphorylation ligand 15d-PGJ2 in the absence of the expression of either PPAR patterns, and functions of the human PPARg1 and PPARg2 isoforms isoform. In contrast, PC-3 prostate cancer cells, which express high in prostate cells (18). We found that prostate epithelial cells did not levels of the PPARg1 receptor isoform, were significantly growth inhibited by 15d-PGJ2. In another study, we showed that PPARg was expressed by tumor cells (PC-3 > DU145 > LNCaP), but they did not express 15-lipoxygenase-2, an arachidonic acid–metabolizing enzyme that also produces the PPARg ligand 15-hydroxyeicosatetrae- noic acid (19). In contrast, prostate epithelial cells that did not express PPARg expressed high levels of 15-lipoxygenase-2 (19). In the present study, we determined the effect of normal stromal cell arachidonic acid metabolites on epithelial cell and tumor cell growth (Fig. 2A). The growth of cells containing the highest levels of the PPARg receptor (i.e., PC-3 and DU145 cells) was most inhibited by arachidonic acid metabolites, whereas LNCaP cells, which express lower levels of PPARg, were the least inhibited. In contrast, the growth of epithelial cells, which lack the PPARg receptor, was slightly stimulated. Depletion of prostaglandin D2 by antibodies decreases the effects of arachidonic acid–conditioned stromal cell medium. We next verified that PGD2 production was responsible for the effects produced by stromal cell–conditioned medium (Fig. 2B). In these experiments, stromal cell monolayers were treated with arachidonic acid followed by the lowering of PGD2 levels with a specific antibody. The removal of PGD2 from stromal cell– conditioned medium abrogated the stimulation of epithelial cells and the growth suppression of tumor cells (Fig. 2B). These PGD2 depletion effects were verified by fluorescence analysis of monolayers after staining with the CAM vital dye and the DNA- intercalating dye DAPI. Viable epithelial cells elicited bright green fluorescence and excluded DAPI uptake, whereas dying PC-3 cells lost the green fluorescence and allowed the uptake of DAPI because of a loss of membrane integrity (Fig. 2C). Measurement of prostaglandin D2 metabolites. Total ion chromatography was used to evaluate the retention times of the various PGD2 metabolites and to achieve critical separation profiles

Figure 2. Characterization of prostate stromal cell–conditioned medium. A, effects of stromal cell–conditioned medium on growth of normal prostate epithelial cells and prostate tumor cells (LNCaP, PC-3, and DU145). Stromal cells were established as monolayers and incubated in the absence or presence (St/AA) of arachidonic acid. Controls consisted of stromal cells incubated with medium alone (St/M), medium alone incubated in plastic tissue culture dishes (M), or medium containing arachidonic acid incubated in plastic tissue culture dishes (AA). The conditioned medium was placed into monolayers of PC-3, LNCaP, and DU145 tumor cells and normal epithelial cells, and total propidium iodide was read on a fluorescence plate reader. Significance was determined by performing a Student’s t test comparing arachidonic acid alone with stromal cells plus arachidonic acid, which resulted in Ps from the lowest to the highest significance as follows: *, P < 0.07; **, P < 0.06; ***, P < 0.0007; ****, P < 0.0003. RFU, relative fluorescence units. Representative of two experiments. B, anti-PGD2 antibody depletion of PGD2 from prostate stromal cell–conditioned medium and its effects on both normal epithelial cells and PC-3 tumor cell growth. Stromal cell monolayers were incubated in the presence of arachidonic acid and protein A/G-agarose beads in the presence or absence of anti-PGD2 antibody (anti-PGD2 Ab). Total propidium iodide staining was then measured as in (A). Significance was determined by performing a Student’s t test comparing the conditions with protein A/G alone with those with anti-PGD2 antibody; all results were significant (P < 0.001). Representative of two experiments. C, analysis of cells treated with PGD2-depleted medium conditioned by stromal cell medium alone, stromal cell/arachidonic acid–conditioned medium, protein A/G-agarose beads alone (St/AA Protein A/G), or stromal cell/arachidonic acid agarose beads in the presence of an anti-PGD2 antibody (St/AA Protein A/G anti-PGD2 Ab). Viable cells have green fluorescence, whereas dead cells have lost the green fluorescence and taken up the blue DAPI dye (arrows). Representative of two experiments.

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Figure 3. PGD2 metabolites in prostate cell–conditioned medium. A, total ion chromatography methods were developed to attain critical separation profiles showing PGD2 PGD2 < 15d-PGD2 < 15d-PGJ2. B, mass spectroscopy was done by using deuterated standards as internal controls for separation to distinguish between PGD2 metabolites (PGD2 < 15d-PGD2 < 15d-PGJ2). C, LC/MS/MS determination of PGD2. Monolayers of normal prostate epithelial cells, stromal cells, and smooth muscle cells and of prostate cancer cells (PC-3, LNCaP, and DU145) were placed in fresh serum-free medium containing 0.1% bovine serum albumin before the addition of 10 Amol/L arachidonic acid. Conditioned medium was collected after 30 minutes, subjected to solid-phase extraction, and analyzed for the presence of PGD2 by LC/MS/MS analysis. Columns, mean; bars, SD. D, determination of arachidonic acid conversion to PGD2 by stromal cell monolayers. Monolayers were incubated with 10 Amol/L arachidonic acid, and the cell monolayers or medium was assayed at various time points (15, 30, 60, and 120 minutes) for the presence of PGD2 by LC/MS/MS analysis. Points, mean; bars, SD. E, determination of arachidonic acid conversion to PGD2 and identification of 15d-PGD2 and 15d-PGJ2 metabolites. Stromal cell monolayers and control RAW 264.7 cells were incubated with 10 Amol/L arachidonic acid, and the medium was assayed at 2 hours for the presence of PGD2 metabolites by LC/MS/MS analysis. Columns, mean; bars, SD. F, PGD2 metabolites were examined after treatment of LNCaP, PC-3, and DU145 cells with 1 Amol/L PGD2; the hydrolytic and metabolic profile of PGD2 was analyzed by LC/MS/MS. Representative of experiments that were repeated twice.

(PGD2 < 15d-PGD2 < 15d-PGJ2; Fig. 3A). Mass spectroscopy methods was examined in culture medium and in whole cells at 15, 30, 60, and were then developed using deuterium-labeled standards to 120 minutes. Similar amounts of PGD2 were produced in both cells distinguish between each of the PGD2 metabolic products (PGD2 < and medium, reaching a plateau between 1 and 2 hours (Fig. 3D). The 15d-PGD2 < 15d-PGJ2; Fig. 3B). The high expression levels of L-PGDS conversion of arachidonic acid to PGD2 metabolites has been shown in normal stromal cells, smooth muscle cells, and epithelial cells previously in cultured RAW 264.7 macrophages (13). Using cultured corresponded to high levels of PGD2 and were uniformly restricted RAW 264.7 macrophages as controls, we determined the level of to normal cells as determined by LC/MS/MS analysis (Fig. 3C). arachidonic acid conversion by stromal cells to PGD2, 15-d-PGD2, Arachidonic acid is converted to prostaglandin D2 metabo- and 15-d-PGJ2 in the LC/MS/MS analysis. The production of 15-d- lites by stromal cells. The conversion of arachidonic acid to PGD2 PGD2 and 15-d-PGJ2 at 2 hours by both RAW 264.7 macrophages and www.aacrjournals.org 6193 Cancer Res 2005; 65: (14). July 15, 2005

Figure 4. PGD2 compounds affect prostate cancer cell growth. PC-3 cell monolayers were treated with multiple concentrations of PGD2 compounds and stained at various times with CAM and DAPI. A, digital image analysis was done after 72-hour treatments using epifluorescence microscopy; viable cells have green fluorescence, whereas dead cells have lost the green fluorescence and taken up the blue DAPI dye (arrows). B, quantification using a fluorescence microplate reader showed the suppression of prostate cancer cell growth by these compounds with a relative effectiveness of 15-d-PGJ2 > 15-d-PGD2 > PGD2. C, epifluorescence microscopy of CAM- and DAPI-stained cells that included diluent (control) cells or cells treated with 11Me15KetoD2 or 15KetoD2. D, no growth suppression occurred in cells treated with 11Me15KetoD2 or 15KetoD2. Concentrations used were as follows: open black squares, control samples; open red diamonds, 1 Amol/L; open green circles, 2.5 Amol/L; open blue diamonds, 5 Amol/L; cross in cyan squares, 7.5 Amol/L; cross in magenta diamonds, 10 Amol/L. Representative of duplicate experiments.

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stromal cells was <50% of the production of PGD2 (Fig. 3E). To cancer cell growth was determined (15-d-PGJ2 > 15-d-PGD2 > PGD2). evaluate how effectively PGD2 metabolites were converted in tumor In contrast, control samples treated for 72 hours with diluent or the cells, we treated these cells with various concentrations of PGD2. DP1 or DP2 receptor-activating metabolite 11Me15KetoD2 or Nearly 40% of the PGD2 were converted to 15-d-PGD2 in all cell lines, 15KetoD2 showed high numbers of viable cells and little or no whereas only f1% of the PGD2 was converted to 15-d-PGJ2 (Fig. 3F). DAPI incorporation into cell nuclei (Fig. 4C and D). Similar conversion rates of PGD2 to 15d-PGD2 and 15d-PGJ2 were Transcriptional activation of the peroxisome proliferator– observed when 5 and 10 Amol/L PGD2 were used. activated receptor ; response element. The first enzyme of the Prostaglandin D2 compounds suppress prostate cancer cell peroxisomal h-oxidation pathway, acyl-CoA oxidase, contains growth. We compared the effects of PGD2, 15-d-PGD2, and 15-d- upstream cis-acting regulatory regions called PPREs (34). A PGJ2 on PC-3 prostate cancer cells that express the highest level of reporter construct (acyl-CoA oxidase-PPRE)3-thymidine kinase- PPARg for their effects on growth and death by adding the vital luciferase was highly induced by PGD2, 15-d-PGD2, and 15-d-PGJ2 dyes CAM and DAPI, the latter of which was excluded by intact in PC-3 cells that expressed high levels of PPARg (Fig. 5A). plasma membranes. Fluorescence microscopy of PC-3 prostate Peroxisome proliferator–activated receptor ; ligand- cancer cells grown in 96-well plates treated with PGD2, 15-d-PGD2, binding domain–specific luciferase reporter activation. The or 15-d-PGJ2 metabolites for 72 hours showed a concentration- various PGD2 metabolites were used to activate a chimeric Gal4- dependent reduction in cell number and a corresponding increase PPARg LBD luciferase reporter system in PC-3 cells, which in DAPI incorporation into cell nuclei (Fig. 4). Quantification of provided a measure of their relative effectiveness for such CAM conversion by viable cells in 96-well plates was examined activation: PGD2 was less effective than 15-d-PGD2, which was after treating prostate cancer cells with PGD2, 15-d-PGD2, or 15-d- less effective than 15-d-PGJ2 (Fig. 5B). In contrast, DP1 and DP2 PGJ2 metabolites for 6, 24, or 72 hours on a multiwell fluorescence receptor ligands had no effect on Gal4-PPARg-LBD activation. plate reader (Fig. 4A and B). Treatment of prostate cancer cells with Knockdown of peroxisome proliferator–activated receptor PGD2 or 15-d-PGJ2 caused a concentration- and time-dependent ; expression abrogates the suppression of PC-3 cell growth by decrease in the level of CAM fluorescence over 72 hours. The relative prostaglandin D2. Transfection of PC-3 cells with an EGFP treatment effectiveness of these ligands in suppressing prostate shRNA-containing plasmid had no effect on the inhibition of

Figure 5. Activation of PPAR by PGD2 metabolites. A, activation of PPARg by PGD2 metabolites was examined in PC-3 prostate cancer cells by using an (acyl-CoA oxidase-PPRE)3-thymidine kinase-luciferase reporter plasmid followed by treatment with various prostaglandins (5 Amol/L) or ethanol alone (control). The luciferase (LUC) reporter activity induced by the metabolites was increased over that induced by the control. B, transactivation of the PPARg LBD by PGD2 metabolites was measured in PC-3 prostate cancer cells that were transfected with 0.5 Agofa luciferase reporter gene containing four GAL4-binding sites linked to the thymidine kinase minimal promoter and 0.5 Agof an expression vector for the Gal4 DNA-binding domain fused to the PPARg LBD. Transfected cells were treated with various prostaglandins (5 Amol/L) or with ethanol alone (control). After 6 hours, cells were harvested and luciferase activity was determined. Student’s t test showed significant differences between results for the ethanol control and results for some of the PGD2 metabolites; each PGD2 metabolite with a statistically significant difference (P < 0.01) is marked with an asterisk in (A and B). Representative of nine experiments. Columns, mean; bars, SD.

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growth by PGD2 (Fig. 6A-C). In contrast, the same plasmid that contains a PPARg shRNA interfered with the ability of PGD2 to inhibit PC-3 cell growth (Fig. 6B and C). In parallel experiments, the transfection of the EGFP shRNA plasmid had no effect on PPARg protein expression in PC-3 cells, whereas PPARg shRNA plasmid caused a decrease in PPARg protein levels (Fig. 6D). These data indicated that growth suppression of PC-3 cells by PGD2 was partly dependent on the presence of the PPARg receptor.

Discussion In the present study, we showed that normal prostate cells taken from young trauma victims expressed high levels of L-PGDS and synthesized PGD2. In contrast, prostate tumor cells lost the ability to make L-PGDS but up-regulated the PPARc gene. PGD2 and its metabolites activated the PPARg LBD in the tumor cells. Our data suggest that suppression of prostate cancer growth can involve stromally derived prostaglandin metabolism that is unique to the prostate, which may help explain the indolent nature of prostate cancer development. Besides binding to nuclear receptors, prostaglandins can also bind to membrane receptors. Membranous PGD2 receptors are coupled to G protein and occur as two isoforms, DP1 (35) and DP2 (36). G protein receptors can activate adenylate cyclase, leading to cyclic AMP synthesis, or can antagonize this process (37). They can also increase the phosphatidylinositol turnover that elevates the free intracellular calcium level. In the present study, we did not observe either DP1 or DP2 expression in any prostate cells (Fig. 1D and E), suggesting that the receptor-dependent responses in these cells primarily involve PPARg. Various PPARg-specific ligands that arise from PGD2 can mediate their effects through PPARg transactivation and the suppression of cell growth (13, 28, 38, 39). Other PPARg ligands also suppress xenograft tumor development in the prostates of immunocompromised mice (40) and the growth of prostate cancer cell lines in vitro (41, 42). To our knowledge, we are first to show that, in addition to 15-d-PGJ2, PGD2 and 15-d-PGD2 can also transactivate transcription through the PPARg LBD in prostate cancer cells and suppress their growth (Fig. 5). L-PGDS may also lead to growth inhibition of PPARg-expressing cells through the production of 15d-PGJ2. In the present study, we observed that very little PGD2 was converted to 15-d-PGJ2 by tumor cells; the bulk of the PGD2 was converted to 15-d-PGD2 (Fig. 3F). When tumor cells gain PPARg expression while losing L-PGDS expression, they are likely to become susceptible to influences from Figure 6. PC-3 cell response to PGD2 relies in part on PPARg receptor expression. A, PC-3 cells were transfected with an EGFP expression construct the microenvironment. Stromal-epithelial interactions are dynam- alone or in combination with an EGFP shRNA construct and then treated with ically balanced and rely on a sensitive equilibrium among cell PGD2. EGFP epifluorescence (green) decreased in the presence of EGFP proliferation, differentiation, and apoptosis through interactions shRNA but not PGD2. In contrast, cell growth was reduced by PGD2 but not by EGFP siRNA as shown by the phase-contrast microscopy (bottom). B, PC-3 between normal cells or cancer cells and their microenvironments cells were transfected with either EGFP shRNA or PPAR shRNA and then (43). This feedback between stroma and developing prostate tumor treated with PGD2. Vital staining of samples after 24 hours revealed that epithelial cells or metastases in other microenvironments may viable cells retained CAM green fluorescence, whereas dead cells had lost the green fluorescence and incorporated blue DAPI dye into their nuclei (arrows). present opportunities for targeting both the tumor cells and the C, PC-3 cells were transfected with either EGFP shRNA or PPARg shRNA stromal cells (43, 44). Most studies have examined the growth- and then treated with PGD2 followed by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl promoting effects of stromal factors on prostate cancer, which tetrazolium bromide analysis. According to a Student’s t test, PGD2 significantly inhibited the growth of untransfected (*, P < 0.002) and EGFP involve the bidirectional exchange of support factors, including shRNA-transfected cells (**, P < 0.0001) but was not as effective in inhibiting the androgen and tumor growth factor-h1 (43–47). Changes in the growth of PPARg shRNA-transfected cells (***, P < 0.06). D, no effect on support tissue and generation of reactive stroma can also occur as PPARg protein expression was observed when cells were transfected with EGFP siRNA (lanes 1 and 2). In contrast, PPARg protein expression was part of tumor formation (45, 48). Clinical studies have shown that decreased in PC-3 cells transfected with PPARg shRNA (lanes 3 and 4). stromal factors can be used as predictors of cancer recurrence (49). COS-1 cells transfected with the human PPARg plasmid served as a In contrast, we know very little about potential growth-suppressive specific protein control (lane 5). h-Actin served as a protein gel-loading control and remained unchanged. Representative of a series of experiments factors present in stroma that affect normal homeostasis and repeated twice. tumor development.

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The present study is the first to show that growth-suppressive proteins, crosses between ARR2PB composite promoter-driving factors that inhibit tumor cell growth and induce differentiation Cre-recombinase (54) and a floxed PPARc (55) generated knockout properties in tumor cells are present in stroma (Figs. 2 and 4). In mice characterized by a high incidence of prostatic intraepithelial general, the stromal cells examined in previous growth promotion neoplasia (PIN). PIN involvement that favored the development of studies were isolated from samples taken from patients with invasive prostate cancer was consistent with the normal prostate cancer, benign prostatic hyperplasia, or bladder cancer progression of prostate cancer (56). Because the ARR2PB is a before undergoing cystoprostatectomy. These stromal cell sources second-generation promoter that is efficiently expressed in all are likely to have different biological properties compared with the lobes of the mouse prostate, it is probably more representative of stromal cells used in our study, which were obtained from young how tissue-specific loss of PPARc contributes to cancer progres- trauma victims. These potential differences may be age or disease sion in these animals (54). These prostate-specific gene-targeting state related, possibilities that are currently under investigation in findings are consistent with our present studies that show how our laboratory. normal stromal cell–derived prostaglandins from L-PGDS con- The ability of stromally derived L-PGDS factors to suppress tribute to growth-suppressive responses in PPARg-expressing tumor growth also remains to be confirmed in vivo. Specifically, prostate tumor cells (Figs. 2, 3, and 4). Our own PPARg shRNA in vivo studies must be done to determine if the L-PGDS produced data showing that the suppression of PC-3 cell growth by PGD2 is by normal tissue is gradually lost during the later stages of partly dependent on PPARg expression (Fig. 6) strengthen the progression leading to outgrowth of the tumor. Such studies should argument that PPARg can act as a tumor suppressor in the also attempt to determine whether tumor cells can evade the presence of a ligand. suppressive influence of the prostate microenvironment by forming Our study shows that prostatic stromal cells generate L-PGDS metastases. Shifting the equilibrium between stromal-epithelial and PPARg ligands, which suppress the growth of tumor cells that interactions selectively toward growth suppression of prostate express PPARg. We suggest that L-PGDS and PPARg are promising tumor cells by prostaglandins or PPARg agonists may therefore be molecular targets for the development of chemopreventive or useful in future designs of cancer therapeutic agents, adjuvant chemotherapeutic agents that can specifically affect prostate therapy, or preventive measures. cancer cells, while sparing normal prostate cells, and the tumor- Gene-targeting studies illustrate the importance of PPAR suppressive effects of L-PGDS and PPARg. receptors to fatty acid and lipoprotein homeostasis and the need for tissue-specific targeting models to understand the unique role Acknowledgments of individual receptors in each particular organ site (50). PPARg is Received 12/17/2004; revised 3/26/2005; accepted 4/29/2005. critical to survival because null embryos die at 10 days’ gestation Grant support: American Cancer Society grant TPRN-99-240-01-CNE-1; National (51). When PPARc (+/À) heterozygous mice (51) were crossed with Cancer Institute grants P01 CA-91844, P50-CA90270-04, P01 CA-106451, R21 CA-10241, mice that had transgenic adenocarcinoma of the mouse prostate and R21 CA-102145; National Institute of Environmental Health Sciences Center grant ES07784; and Kadoorie Charitable Foundation Prostate Cancer Research Fellowship. (TRAMP; ref. 52), there was no effect on the development of The costs of publication of this article were defrayed in part by the payment of page prostate cancer (53). The TRAMP model, however, incorporates a charges. This article must therefore be hereby marked advertisement in accordance minimal probasin promoter driving large T SV40 virus antigen, with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. R. Evans (The Salk Institute for Biological Studies, La Jolla, CA) for which is not highly expressed in all lobes of the mouse prostate providing the pCMX-Gal-L-mPPARg and tk-MH100X4-Luc plasmids, Dr. C.K. Glass and may bypass some of the signaling pathways associated with (University of California, San Diego, CA) for providing (acyl-CoA oxidase-PPRE)3- thymidine kinase-luciferase reporter plasmid, Dr. D. Takai for providing the human typical prostate cancer progression that are not mediated by viral U6-tetO vector, and Dr. E.M. McDonald (Department of Scientific Publications, The proteins. In other mouse studies not involving up-regulated viral University of Texas M.D. Anderson Cancer Center) for editorial expertise.

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