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Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan
¹ Department of Molecular Medical Technology, Tohoku University School of Medicine, Sendai, Japan
² Department of Medicine, Tohoku University School of Medicine, Sendai, Japan
³ Department of Surgery, Tohoku University School of Medicine, Sendai, Japan

(Requests for offprints should be addressed to T Suzuki; Email: t-suzuki{at}patholo2.med.tohoku.ac.jp)

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
It has been reported that agonists of peroxisome proliferator-activated receptor (PPAR) inhibit proliferation of breast carcinoma cells, but the biological significance of PPAR remains undetermined in human breast carcinomas. Therefore, we immunolocalized PPAR in 238 human breast carcinoma tissues. PPAR immunoreactivity was detected in 42% of carcinomas, and was significantly associated with the status of estrogen receptor (ER) , ERß, progesterone receptor, retinoic X receptors, p21 or p27, and negatively correlated with histological grade or cyclooxygenase-2 status. PPAR immunoreactivity was significantly associated with an improved clinical outcome of breast carcinoma patients by univariate analysis, and multivariate analysis demonstrated that PPAR immunoreactivity was an independent prognostic factor for overall survival in ER-positive patients. We then examined possible mechanisms of modulation by PPAR on estrogenic actions in MCF-7 breast carcinoma cells. A PPAR activator, 15-deoxy-^12,14- prostaglandin J₂ (15d-PGJ₂), significantly inhibited estrogen-responsive element-dependent transactivation by estradiol in MCF-7 cells, which was blocked by addition of a PPAR antagonist GW9662. Subsequent study, employing a custom-made microarray focused on estrogen-responsive genes, revealed that mRNA expression was significantly regulated by estradiol in 49 genes, but this significance vanished on addition of 15d-PGJ₂ in 16 out of 49 (33%) genes. These findings were confirmed by real-time PCR in 11 genes. 15d-PGJ₂ significantly inhibited estrogen-mediated proliferation of MCF-7 cells, and caused accumulation of p21 and p27 protein. These results suggest that PPAR is mainly expressed in well-differentiated and ER-positive breast carcinomas, and modulates estrogenic actions.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Peroxisome proliferator-activated receptor (PPAR) is a member of the nuclear hormone receptor superfamily, and has also been designated NR1C3 (Lemberger et al. 1996, Schoonjans et al. 1996). PPAR functions as a transactivation factor following heterodimerization with retinoic X receptors (RXRs), and binds to its specific response elements termed peroxisome proliferating responsive elements (PPREs) of various target genes (Mangelsdorf & Evans 1995). PPAR is one of the ligand-activated transcription factors, and 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) is currently considered a naturally occurring PPAR ligand, which activates PPAR at µM concentrations in the human (Koeffler 2003).

It is well known that PPAR plays essential roles in adipogenesis (Tontonoz et al. 1994), insulin resistance (Celi & Shuldiner 2002), and development of various organs (Barak et al. 1999). In addition, various in vitro studies have demonstrated that PPAR ligands have a potent antiproliferative activity against a wide variety of neoplastic cells (Koeffler 2003). For instance, PPAR agonist inhibits the proliferation of human breast cancers (Elstner et al. 1998, Mueller et al. 1998, Yee et al. 1999), and a phase II clinical trial using PPAR ligands has been recently performed as a novel therapy for advanced breast cancer patients (Burstein et al. 2003).

It then becomes very important to obtain a better understanding of the clinical and/or biological roles of PPAR in breast cancer tissues in order to improve the potential clinical efficiency of PPAR ligand therapy for breast cancer patients. Mueller et al. (1998) previously demonstrated the expression of PPAR in human primary and metastatic cancers, and Wang et al. (2004) reported higher amounts of PPAR expression in breast carcinoma cells than in normal human mammary epithelial cells. However, it has been also demonstrated that PPAR expression is significantly lower in breast cancer tissues at both mRNA (Jiang et al. 2003) and protein (Watkins et al. 2004) levels than that in normal tissues. Expression of PPAR has been examined in human breast carcinomas by several groups, but little information is available on the clinicopathological features of PPAR-positive breast cancers. Therefore, the biological significance of PPAR remains largely undetermined in human breast carcinoma. In this study, we examined immunolocalization of PPAR in 238 cases of human breast carcinoma patients, and correlated these findings with various clinicopathological parameters. As the results of immunohistochemistry demonstrated a strong association between PPAR and estrogen receptor (ER) in breast carcinomas, we also examined a possible modulation by PPAR on estrogenic actions in breast cancer cells for further characterization of PPAR in human breast carcinoma.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Patients and tissues

Two hundred and thirty-eight surgical pathology specimens of invasive ductal carcinoma of the breast were retrieved from pathology archives of the Department of Surgery, Tohoku University Hospital, Sendai, Japan. Breast tissue specimens were obtained from female patients who underwent mastectomy from 1982 to 1992 with a mean age of 54.1 years (range 22–82). The patients did not receive chemotherapy or irradiation prior to surgery. Review of the charts of patients revealed that 194 patients received adjuvant chemotherapy, and 43 patients received tamoxifen therapy after surgery. The mean follow-up time was 102 months (range 2–157 months). The histological grade of each specimen was evaluated based on the method of Elston & Ellis (1991). All specimens were fixed with 10% formalin and embedded in paraffin wax. Research protocols for this study were approved by the Ethics Committee at Tohoku University School of Medicine (approved number: 2000-142).

Antibodies

Rabbit polyclonal antibody for PPAR was raised against a synthetic peptide corresponding to amino acids 60–79 of mouse PPAR1 (accession number; AAA62110 [GenBank] ), which also corresponds to amino acids 62–81 of human PPAR1 (CAA62152 [GenBank] ) or 90–109 of human PPAR2 (AAB04028 [GenBank] ). This antibody therefore recognizes both human PPAR1 and 2. The characterization of this antibody has been previously confirmed by both immunoblotting and immunohistochemistry (Sato et al. 2004). The characteristics of polyclonal antibodies for RXR, RXRß and RXR have been previously reported by the authors (Sugawara et al. 1995, Suzuki et al. 2001). Monoclonal antibodies for ER (ER1D5), progesterone receptor (PR; MAB429), Ki-67 (MIB1), p21 (6B6), p27 (1B4), c-Myc (1-6E10), pS2 (M7184), and cyclin D1 (P2D11F11) were purchased from Immunotech (Marseille, France), Chemicon (Temecula, CA, USA), DAKO (Carpinteria, CA, USA), Pharmingen (San Diego, CA, USA), Novocastra Laboratories (Newcastle, UK), Cambridge Research Biochemical (Cambridge, UK), DAKO and Novocastra Laboratories respectively. Rabbit polyclonal antibodies for ERß (06-629), HER2 (A0485) and cathepsin D (A0561) were obtained from Upstate Biotechnology (Lake Placid, NY, USA), DAKO and DAKO respectively. Goat polyclonal antibody for cyclooxygenase-2 (COX2) (C-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunohistochemistry

A Histofine Kit (Nichirei, Tokyo, Japan), which employs the streptavidin-biotin amplification method was used in this study. Antigen retrieval for PPAR, ER,ß, PR, RXR,ß,, HER2, Ki-67, p21, p27 and cyclin D1 immunostaining was performed by heating the slides in an autoclave at 120 °C for 5 min in citric acid buffer (2 mM citric acid and 9 mM trisodium citrate dehydrate, pH6.0), and antigen retrieval for COX2 and pS2 immunostaining was done by heating the slides in a microwave oven for 15 min in the citric acid buffer. Dilutions of primary antibodies used in this study were as follows: PPAR; 1/1500, ER; 1/50, ERß; 1/50, PR; 1/30; RXR; 1/4000, RXRß; 1/4000, RXR; 1/2000, COX2; 1/500, HER2; 1/200, Ki-67; 1/50, p21; 1/250, p27; 1/150, c-Myc 1/600, pS2; 1/30, cyclin D1; 1/40 and cathepsin D; 1/300. The antigen–antibody complex was visualized with 3,3'-diaminobenzidine solution (1 mM, in 50 mM Tris–HCl buffer (pH 7.6) and 0.006% H₂O₂), and counterstained with hematoxylin. As a negative control, normal rabbit, mouse or goat IgG was used instead of the primary antibodies. For PPAR immunohistochemistry, a preabsorption test was also performed as a negative control.

Scoring of immunoreactivity and statistical analysis

PPAR, ER,ß, PR, RXR,ß,, Ki-67, p21, p27, c-Myc and cyclin D1 immunoreactivity was detected in the nucleus, and the immunoreactivity was evaluated in more than 1000 carcinoma cells for each case, and subsequently the percentage of immunoreactivity, i.e. labeling index (LI), was determined. Inter-observer differences were less than 5%, and the mean of the three values was obtained. Cases with PPAR, ER or c-Myc LIs of more than 10% were considered PPAR-, ER- or c-Myc-positive breast carcinomas in this study, according to a report for ER by Allred et al. (1998). For p21 and p27 immunohistochemistry, the cut-off values used were 5 and 50% respectively, according to previous reports (Barbareschi et al. 2000, Pellikainen et al. 2003). Immunoreactivity for COX2 and cathepsin D was detected in the cytoplasm, and cases that had more than 10% of positive carcinoma cells were considered positive.

An association between immunoreactivity for PPAR and clinicopathological factors was evaluated using a one-way ANOVA and a Bonferroni test or a cross-table using the chi-square test. Overall and disease-free survival curves were generated according to the Kaplan–Meier method and statistical significance was calculated using the log-rank test. An association between ER LI and PR or cyclin D1 LI was performed utilizing a correlation coefficient (r) and regression equation. Univariate and multivariate analyses were evaluated by a proportional hazard model (Cox) using PROC PHREG in our SAS software. P values less than 0.05 were considered significant in this study.

Cell line, plasmids and chemicals

MCF-7 human breast cancer cell line was cultured in RPMI-1640 (Sigma-Aldrich, St Louis, MO, USA) with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA). MCF-7 cells were cultured with phenol red-free RPMI 1640 medium containing 10% dextran-coated charcoal (DCC)-FBS for 3 days before treatment in the experiment. In this study, we used estrogen-responsive reporter plasmids pERE-Luc, containing Xenopus vitellogenin A2 estrogen-responsive element (ERE) (Saji et al. 2001). The pRL-TK vectors were purchased from Promega (Madison, WI, USA). 15d-PGJ₂, ciglitazone and PGF₂ were purchased from Biomol Research Laboratories (Butler Pike, PA, USA), and GW1929 and GW9662 were purchased from Sigma-Aldrich.

Luciferase assay

The luciferase assay was performed according to a previous report (Sakamoto et al. 2002) with some modifications. Briefly, 1 µg ptk-ERE-Luc plasmids and 200 ng pRL-TK control plasmids were used to measure the transcriptional activity of endogenous ER. Transient transfections were carried out using TransIT-LT Transfection Reagents (TaKaRa, Tokyo, Japan) in MCF-7 cells, and the luciferase activity of lysates was measured using a Dual-Luciferase Reporter Assay system (Promega) and Luminescencer-PSN (AB-2200) (Atto Co., Tokyo, Japan) after incubation with growth medium with the indicated concentrations of estradiol and/or 15d-PGJ₂ for 24 h. The cells were also treated with the same volume of ethanol (final dilution – 0.1%) for 24 h as controls. The transfection efficiency was normalized against Renilla luciferase activity using pRL-TK control plasmids, and the luciferase activity for each sample was evaluated as a ratio (%) compared with that of controls. The statistical analyses were performed using a one-way ANOVA and Bonferroni test.

Immunoblotting

The cell protein was extracted in triple detergent lysis buffer (LK-18) at 4 °C. Twenty micrograms of the protein (whole cell extracts) were subjected to SDS-PAGE (10% acrylamide gel). Following SDS-PAGE, proteins were transferred onto Hybond P polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ, USA). The blots were blocked in 5% non-fat dry skim milk for 1 h at room temperature, and were then incubated with a primary antibody for ER, p21, p27 or ß-actin (Sigma-Aldrich) for 18 h at 4 °C. After incubation with anti-mouse IgG horseradish peroxidase (Amersham Biosciences) for 1 h at room temperature, antibody–protein complexes on the blots were detected using ECL-plus western blotting detection reagents (Amersham Biosciences). Immunointensity of specific bands was measured by an LAS-1000 imaging system (Fuji Photo Film, Tokyo, Japan), and relative immunointensity of ER, p21 or p27 was evaluated as a ratio (%) of ß-actin immunointensity.

Microarray analysis

In this study, we used a custom-made microarray named EstrArray (InfoGenes, Tsukuba, Japan), which contains 175 estrogen-responsive genes identified in MCF-7 cells (Inoue et al. 2002, Hayashi et al. 2003). MCF-7 cells were cultured with phenol red-free RPMI 1640 medium containing 10% DCC-FBS for 3 days, and subsequently treated with estradiol (10 nM) with or without 15d-PGJ₂ (5 µM) for 72 h. The MCF-7 cells used as references were treated with the same volume of ethanol (final dilution – 0.1%) for 72 h.

Total RNA was isolated using TRIzol reagent (InVitrogen Life Technologies, Inc., Gaitherburg, MD, USA). Two micrograms of mRNA were reverse-transcribed with Cy3- or Cy5-dUTP (Amersham Biosciences, Bucks, UK) using a SUPER-SCRIPT II Preamplification system (Gibco-BRL, Grand Island, NY, USA). Cy3- and Cy5-labeled cDNA probes were hybridized on the microarray slide for 16 h at 65 °C. The fluorescent signals were scanned by a GenePix 4000A (Axon Instruments, Foster City, CA, USA), and the ratio of Cy3 and Cy5 signal intensity of each spot was quantitatively calculated using GenePixPro 5.0 (Axon Instruments). The duplicated sets of values were averaged and normalized by subtracting the average of values for internal genes. The data from insufficient hybridization (signal areas below 100) were excluded from the analysis. Genes which showed a value of more than 2.0 or less than 0.5 were evaluated as significantly up-regulated or down-regulated respectively, in this study.

Real-time PCR

MCF-7 cells were cultured with phenol red-free RPMI 1640 medium containing 10% DCC-FBS for 3 days, and subsequently treated with the indicated concentration of estradiol and/or 15d-PGJ₂ for 72 h. As controls for the experiments, the cells were treated with the same volume of ethanol (final dilution – 0.1%) for 72 h. Total RNA was extracted using TRIzol reagent (InVitrogen Life Technologies, Inc.), and a reverse transcription kit (SUPERSCRIPT II Preamplification system (Gibco-BRL) was used in the synthesis of cDNA.

The Light Cycler System (Roche Diagnositics GmbH, Mannheim, Germany) was used to semi-quantify the mRNA expression levels by real-time PCR (Dumoulin et al. 2000). Characteristics of the primer sequences used in this study are summarized in Table 1 (Colombel et al. 1999, Schonherr et al. 2001, Vandesompele et al. 2002, Kao et al. 2003, Paruthiyil et al. 2004, Yoshida et al. 2004). Settings for the PCR thermal profile were: initial denaturation at 95 °C for 1 min followed by 40 amplification cycles of 95 °C for 1 s, annealing at 59 °C (PR), 60 °C (pS2, PDZ domain-containing protein (PDZK1), cathepsin D, selenium-binding protein 1 (SELENBP1), tumor protein D52-like 1 (TPD52L1), insulin-like growth factor-binding protein-5 (IGFBP-5), PDZ domain-containing- protein (PDZK1), and p21), 64 °C (cyclin D1, and tumor-associated antigen L6 (TAL6)), 66 °C (solute carrier family 7, member 5 (SLC7A5), and p27), or 68 °C (ribosomal protein L 13a (RPL13A)) for 15 s, and elongation at 72 °C for 15 s. To verify amplification of the correct sequences, PCR products were purified and subjected to direct sequencing. Negative control experiments lacked cDNA substrate to check for the possibility of exogenous contaminant DNA. The mRNA levels were summarized as a ratio of RPL13A, and subsequently evaluated as a ratio (%) compared with that of controls. The statistical analyses were performed using a one-way ANOVA and Bonferroni test.

The status of cell proliferation of MCF-7 cells was measured using a WST-8 (2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2- H-tetrazolium, monosodium salt) method (Cell Counting Kit-8; Dojindo Inc., Kumamoto, Japan)) (Isobe et al. 1999). We also examined apoptosis status of MCF-7 cells using an apoptosis screening kit (Wako, Osaka, Japan), which employed a modified TdT-mediated dUTP nick-end labeling (TUNEL) method (Gavrieli et al. 1992). Optical densities (OD = 450 nM for cell proliferation assay, and OD = 490 nM for apoptosis analysis) were obtained with a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The cell number and apoptosis index were calculated according to the equation (Cell OD value after test material treatment)/(Vehicle control cell OD value), and subsequently evaluated as a ratio (%) compared with that of controls.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Immunohistochemistry for PPAR in breast carcinoma tissues

Immunoreactivity for PPAR was detected in the nuclei of invasive ductal carcinoma cells (Fig. 1A and B). A mean value of PPAR LI in 238 breast carcinoma cases examined was 15% (range 0–74%), and the number of PPAR-positive breast carcinomas (i.e. PPAR LI 10%) was 99 out of 238 cases (42%). Immunoreactivity of PPAR was also detected in epithelia of morphologically normal mammary glands (Fig. 1C), and adipocytes (Fig. 1D).

View larger version (121K): [in this window] [in a new window]   Figure 1 Immunohistochemistry for PPAR in invasive ductal carcinoma. (A) PPAR immunoreactivity was detected in the nuclei of carcinoma cells. (B) No significant immunoreactivity of PPAR was detected in the sections where an immunohistochemical preabsorption test was performed as a negative control. (C) Immunoreactivity for PPAR was detected in the nuclei of epithelial cells in morphologically normal mammary glands. (D) PPAR immunoreactivity was positive in the nuclei of adipocytes. Bar=50 µm.

View this table: [in this window] [in a new window]   Table 2 Association between PPAR immunoreactivity and clinicopathological parameters in 238 breast carcinomas

View this table: [in this window] [in a new window]   Table 3 Statistical association between PPAR immunoreactivity and clinicopathological parameters according to the ER status in 238 breast carcinomas

Influence of PPAR immunoreactivity on association between ER and estrogenresponsive genes in breast cancer tissues

pS2, cyclin D1, PR and cathepsin D are all well recognized as estrogen-responsive genes in human breast cancers. As shown in Table 4, a significant positive association was detected between ER LI and the status of these immunoreactivity in the 238 breast cancer tissues examined (P<0.0001), which is in good agreement with previous immunohistochemical studies (Horwitz & McGuire 1978, Barbareschi et al. 1997, Gillesby & Zacharewski 1999, Ioachim et al. 2003). When the breast cancers were classified into two groups according to their PPAR status, no significant association was detected between ER LI and pS2 (P=0.3785) or cyclin D1 LI (P=0.1978) in PPAR-positive breast carcinomas, although significant association (P<0.0001 for pS2, and P=0.0018 for cyclin D1) was detected in PPAR-negative breast carcinomas. On the other hand, ER LI was significantly associated with PR LI (P=0.0008 in PPAR-positive cases, and P<0.0001 in PPAR-negative cases) or cathepsin D (P=0.0006 in PPAR-positive cases, and P=0.0003 in PPAR-negative cases) regardless of the PPAR status in the breast carcinoma cases examined.

View this table: [in this window] [in a new window]   Table 4 Correlation between ER and estrogen-responsive gene immunoreactivity associated with PPAR status in 238 breast carcinomas

Correlation between PPAR immunoreactivity and clinical outcome of the patients

No significant association was detected between PPAR immunoreactivity and a risk of recurrence (P=0.8715) (Fig. 2A). PPAR immunoreactivity was significantly associated with a better clinical outcome of the 238 breast cancer patients (P=0.0257) (Fig. 2B). This significant association was detected in the ER-positive group (P=0.0057) (Fig. 2C), but not in the ER-negative group (P=0.6405) (Fig. 2D). The significant correlation between PPAR immunoreactivity and overall survival of ER-positive breast cancer patients was not influenced by tamoxifen therapy after the surgery (Fig. 2E and F).

View larger version (28K): [in this window] [in a new window]   Figure 2 Disease-free (A) and overall (B–F) survival of 238 patients with breast carcinoma according to PPAR immunoreactivity (Kaplan–Meier method). PPAR immunoreactivity was not correlated with a risk of recurrence (P=0.8715 in log-rank test) (A), but significantly associated with an improved overall survival (P=0.0257 in log-rank test) (B). The significant association was detected in the ER-positive group (n=174; P=0.0057 in log-rank test) (C), but not in the ER-negative cases (n=64; P=0.6405 in log-rank test) (D). PPAR immunoreactivity was significantly associated with an improved prognosis regardless of tamoxifen therapy after surgery in the ER-positive breast cancer patients (E, F). P values less than 0.05 are in boldface.

View this table: [in this window] [in a new window]   Table 6 Univariate and multivariate analyses of overall survival in 174 ER -positive breast cancer patients examined

Effects of PPAR activator 15d-PGJ₂ on estrogen-mediated transcription in MCF-7 cells in luciferase assay

Results of PPAR immunoreactivity demonstrated a strong association between PPAR and ER in breast carcinoma tissues, suggesting a possible interaction of these two nuclear receptors in human breast carcinoma cells. Previously, Keller et al. (1995) reported that PPAR/RXRß heterodimer can bind to ERE and possibly modulate the ER-signaling pathway, but this has not been examined in breast cancers.

In order to examine this hypothesis, we used MCF-7 breast cancer cells in the following in vitro experiments, because MCF-7 cells were associated with expression of ER, PPAR, and RXR, ß, (data not shown). When MCF-7 cells were transiently transfected with ptk-ERE-Luc plasmids and treated with 10 nM estradiol, the luciferase activity of the cells was 17-fold increased compared with their basal level (Fig. 3A). PPAR activator 15d-PGJ₂ significantly inhibited ERE-dependent transactivation by estradiol in a dose-dependent manner, and the luciferase activity of MCF-7 cells treated with 10 nM estradiol and 5 µM 15d-PGJ₂ was decreased to 53% of that treated with 10 nM estradiol alone (P<0.001). 15d-PGJ₂ (5 µM) alone did not significantly change the luciferase activity compared with their basal level (P=0.8837). 15d-PGJ₂, however, did not significantly inhibit the ERE-dependent transactivation by estradiol, when these cells were treated with a potent PPAR antagonist GW9662 (Leesnitzer et al. 2002).

View larger version (33K): [in this window] [in a new window]   Figure 3 Effects of PPAR activator 15d-PGJ2 on ERE-dependent transactivation in MCF-7 cells. (A) MCF-7 cells were transiently transfected with pERE-Luc plasmids, and treated with the indicated concentrations of estradiol, 15d-PGJ2 and GW9662 (PPAR antagonist) for 24 h. The luciferase activity was evaluated as a ratio (%) compared with that of controls (treatment without estradiol, 15d-PGJ2 or GW9662 for 24 h (left column)). Data are presented as means ± S.D. (n=3). **P<0.01 and ***P<0.001 vs 10 nM estradiol alone. (B) MCF-7 cells transfected with pERE-Luc plasmids were treated with the indicated concentrations of estradiol and GW1929 (PPAR agonist), ciglitazone (PPAR agonist), or PGF2 (no activator of PPAR) for 24 h. Data are presented as means ± S.D. (n=3). **P<0.01 and ***P<0.001 vs 10 nM estradiol alone respectively. (C) Immunoblotting for ER in MCF-7 cells. ER and ß-actin immunoreactivities were detected as specific bands (approximately 66 and 42 kDa respectively); 20 µg of protein were loaded in each lane. Data of immunointensity ratio (ER/ß-actin) are presented as means ± S.D. (n=3). No significant association was detected.

2

Effects of 15d-PGJ₂ on estrogen-mediated transcription in MCF-7 cells in microarray analysis

We further examined the effects of 15d-PGJ₂ on estrogen-mediated transcription in MCF-7 cells using a custom-made microarray. Significant alterations of mRNA expression by estradiol treatment (10 nM for 72 h) were detected in 49 genes in this study, and these genes were classified into the following four groups (Table 7): Group A; mRNA expression was significantly up-regulated by estradiol, but the significance vanished on addition of 15d-PGJ₂ (5 µM) (nine genes; Table 8), Group B; mRNA expression was significantly up-regulated by estradiol with or without 15d-PGJ₂ (23 genes; Table 9), Group C; mRNA expression was significantly down-regulated by estradiol, but the significance vanished on addition of 15d-PGJ₂ (seven genes; Table 10), and Group D; mRNA expression was significantly down-regulated by estradiol with or without 15d-PGJ₂ (ten genes; Table 11).

In order to confirm the results from microarray analysis, we performed real-time PCR analyses for the representative 11 genes in MCF-7 cells (Fig. 4A–K). mRNA expression of pS2 (Fig. 4A), PDZK1 (Fig. 4B) cyclin D1 (Fig. 4C) and IGFBP-4 (Fig. 4D), which were tentatively classified into Group A in microarray analysis as above, was significantly (P<0.001) increased by estradiol treatment (10 nM, for 72 h) compared with the control (neither estradiol nor 15d-PGJ₂), but not by treatment with estradiol (10 nM) with 15d-PGJ₂ (5 µM). mRNA expression of SLC7A5 (Fig. 4E), TPD52L1 (Fig. 4F), PR (Fig. 4G), and cathepsin D (Fig. 4H) in Group B was, however, significantly up-regulated by the treatment with estradiol with or without 15d-PGJ₂ (1 or 5 µM). Estradiol-mediated mRNA expression of PR was also demonstrated to be inhibited by addition of 15d-PGJ₂ in a dose-dependent manner (P<0.01, between estradiol alone and estradiol with 15d-PGJ₂ (5 µM)). The mRNA level of TAL6 in Group C (Fig. 4I) was significantly lower (P<0.001) in estradiol alone than that in the control group, but was not significantly different under treatment with estradiol with 15d-PGJ₂ (5 µM). mRNA expression of IGFBP-5 (Fig. 4J) and SELENBP1 (Fig. 4K) in Group D was significantly down-regulated by the treatment with estradiol (10 nM) with or without 15d-PGJ₂ (1 or 5 µM). mRNA expression in these 11 genes was not significantly altered by treatment with 15d-PGJ₂ (5 µM) alone in this study.

View larger version (41K): [in this window] [in a new window]   Figure 4 Effects of estrogen and 15d-PGJ2 on mRNA expression of estrogen-responsive genes in MCF-7 cells by real-time PCR. (A) pS2, (B) PDZK1, (C) cyclin D1, (D) IGFBP-4, (E) SLC7A5, (F) TPD52L1, (G) PR, (H) cathepsin D, (I) TAL6, (J) IGFBP-5 and (K) SELENBP1. MCF-7 cells were treated with the indicated concentrations of estradiol and/or 15d-PGJ2 for 72 h, and mRNA expression was evaluated by real-time PCR. The mRNA level was summarized as a ratio of RPL13A, and subsequently evaluated as a ratio (%) compared with that of controls (treatment without estradiol or 15d-PGJ2 for 72 h (left column)). Data are presented as means ± S.D. (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs controls

The number of MCF-7 cells was significantly increased after the treatment with estradiol (10 nM) in a time-dependent manner, and was 1.4-fold higher than the basal level (control: no treatment with estradiol or 15d-PGJ₂) at 5 days after the treatment (Fig. 5A). The estrogen-mediated proliferation of MCF-7 cells was significantly inhibited by addition of 5 µM 15d-PGJ₂ (P<0.05 and P<0.001 for 3 and 5 days respectively). The apoptosis index of MCF-7 cells was not significantly altered under the same treatments for 3 days (Fig. 5B). The treatment with 5 µM 15d-PGJ₂ alone did not significantly influence the proliferation or apoptosis of MCF-7 cells compared with the basal level (data not shown).

View larger version (43K): [in this window] [in a new window]   Figure 5 Effects of 15d-PGJ2 on estrogen–mediated proliferation in MCF-7 cells. (A) MCF-7 cells were treated with the indicated concentrations of estradiol and 15d-PGJ2 for 0, 3 or 5 days, and the status of cell proliferation was measured using a WST-8 method. The cell number was evaluated as a ratio (%) compared with that at 0 day after the treatment. Control; no treatment with estradiol or 15d-PGJ2. Data are presented as means ± S.D. (n=3). *P<0.05 and ***P<0.001 vs 10 nM estradiol alone respectively. (B) MCF-7 cells were treated with the indicated concentrations of estradiol and 15d-PGJ2 for 3 days, and apoptosis was evaluated by an apoptosis screening kit. The apoptosis index was evaluated as a ratio (%) compared with that of controls (no treatment with estradiol or 15d-PGJ2 for 3 days (left column)). Data are presented as means ± S.D. (n=3). No significant association was detected. (C) Real-time PCR for p21 and p27 in MCF-7 cells. MCF-7 cells were treated with the indicated concentrations of 15d-PGJ2 for 72 h. The mRNA level was summarized as a ratio of RPL13A, and subsequently evaluated as a ratio (%) compared with that of controls (no treatment with 15d-PGJ2 for 72 h (left column)). Data are presented as means ± S.D. (n=3). ***P<0.001 vs controls). (D) Immunoblotting for p21 and p27 in MCF-7 cells. MCF-7 cells were treated with the indicated concentrations of 15d-PGJ2 for 72 h; 20 µg of protein were loaded in each lane. Data of immunointensity ratio (p21 or p27/ß-actin) are presented as means ± S.D. (n=3). *P<0.05 and **P<0.01 vs controls (no treatment with 15d-PGJ2 for 72 h).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
In our present study, PPAR immunoreactivity was detected in carcinoma cells in 99 out of 238 human breast carcinomas (42%), and was significantly associated with the histological grade or ER status of the cases. Expression of PPAR has been previously reported in breast cancer cases by several groups (Mueller et al. 1998, Jiang et al. 2003, Watkins et al. 2004). Mueller et al. (1998) reported that ligand-activated PPAR in cultured breast cancer cells resulted in extensive lipid accumulation, and transformed the breast epithelial gene expression to a more differentiated and less-malignant state. In addition, both Jiang et al. (2003) and Watkins et al. (2004) reported that PPAR expression was significantly lower in breast cancer tissues than in normal tissues, suggesting that PPAR has a possible protective role against development of breast cancers (Jiang et al. 2003, Koeffler 2003). Results of our present study are generally consistent with these previously reported findings, and PPAR may be mainly expressed in well-differentiated breast carcinomas with hormonal regulatory mechanisms maintained.

It then becomes important to know whether PPAR is colocalized with RXRs and its natural ligands or not in breast cancers, because these factors play essential roles in activation of PPAR function. PPAR immunoreactivity was significantly associated with all the subtypes of RXR. RXR and RXRß are known to be major subtypes of RXR in breast cancer tissues (Suzuki et al. 2001), and PPAR/RXR heterodimer was reported to be biologically active in human breast cancer cells (Crowe & Chandraratna 2004). Therefore, PPAR is mainly expressed in RXR-overexpressing breast cancer tissues, and possibly heterodimerizes with RXR and/or RXRß in breast cancer cells. Previously, Badawi & Badr (2003) reported that concentration of 15d-PGJ₂, which is considered a natural ligand of PPAR, was inversely correlated with mRNA expression of COX2 or concentration of PGE₂, and was marginally associated (P=0.081) with PPAR mRNA levels in breast cancer tissues. In addition, estrogen is known to influence PG synthesis in estrogen target tissues (Ham et al. 1975). Ma et al. (1998a) reported that estrogen induced enzymatic conversion of PGD₂ and the metabolites of PGD₂ potently activated PPAR, although estrogen did not directly induce the mRNA expression of PPAR (Ma et al. 1998b). In our present study, PPAR immunoreactivity was inversely associated with COX2 immunoreactivity and positively associated with ER, although we could not examine the tissue concentrations of natural PPAR ligands in breast cancer tissues. These data suggest that PPAR is biologically activated in human breast cancer tissues.

Estrogens are well-known to contribute immensely to the development of hormone-dependent breast carcinomas, and biological estrogenic actions are mainly mediated by ER (Korach 1994). Estrogens stimulate the transactivation of activation function 2 domain of ER in a ligand-dependent manner (Kumar et al. 1987), and subsequently ERs activate transcription of various target genes by direct DNA interaction through EREs or by tethering to other transcription factors (Tsai & O’Malley 1994, Acconcia & Marino 2003). Previously, Keller et al. (1995) demonstrated that PPAR/RXRß heterodimer could bind to ERE using the artificial promoter context. In this report, PPAR/RXRß strongly bound with EREs of pS2 and vitellogenin A2 (vitA2) genes, but did not induce these ERE-dependent transactivations (Keller et al. 1995). The binding affinity between PPAR/RXRß and ERE of very-low-density apolipoprotein II gene was, however, very low, despite containing the same ERE consensus sequence as vitA2. In our study, ligand-mediated PPAR activation significantly inhibited estrogen-mediated ERE transactivation in MCF-7 cells. These data are in good agreement with the report by Keller et al. (1995), and suggest that PPAR suppresses the estrogen-signaling pathway through inhibition of the binding of ERs with the target genes in breast cancer cells. In our microarray analysis, inhibition of estrogen-mediated mRNA expression by PPAR was detected in 33% of estrogen-responsive genes, including ERE-containing genes such as pS2 (Stack et al. 1988) and early growth response 3 (Bourdeau et al. 2004). However, 15d-PGJ₂ did not significantly regulate the estrogen-mediated transactivation of a proportion of ERE-containing genes, such as SLC7A5 (Bourdeau et al. 2004), cathepsin D (Wang et al. 1997), retinoblastoma-binding protein 8 (Bourdeau et al. 2004), and Fos-like antigen 2 (Bourdeau et al. 2004). On the other hand, 15d-PGJ₂ inhibited the estrogen-mediated expression of cyclin D1 and IGFBP-4 (Group A), in which functional ERE has not been identified and indirect gene regulation by ER is suggested (Qin et al. 1999, Acconcia & Marino 2003, O’Lone et al. 2004). Therefore, inhibition of PPAR in estrogen-mediated transactivation is considered to vary among the target genes, and may influence not only ERE-containing genes but also some genes which are induced by an interaction between ER and other DNA-binding transcription factors.

In our immunohistochemical analysis (Table 4), significant associations were detected between ER and estrogen-responsive genes, such as pS2, cyclin D1, PR and cathepsin D, as reported previously (Barbareschi et al. 1997, Gillesby & Zacharewski 1999, Ioachim et al. 2003). However, the significant association between ER and pS2 or cyclin D1 was not detected in the group of PPAR-positive breast cancers, while correlation between ER and PR or cathepsin D was not influenced by PPAR status in those breast cancer patients examined. These data are in good agreement with our results of microarray and real-time PCR analyses. Recently, Qin et al. (2003) reported that PPAR agonists induced proteasome-dependent degradation of cyclin D1, which may be partly involved in the present immunohistochemical results of cyclin D1.

In this study, PPAR immunoreactivity was correlated with immunoreactivity of p21 and p27 in breast carcinoma tissues, and expression of p21 and p27 was significantly induced by 15d-PGJ₂ at mRNA and/or protein levels in MCF-7 cells. Previous studies demonstrated that PPAR ligands induced cyclin-dependent kinase inhibitors such as p21 and p27 in various types of cancer cells (Chung et al. 2002, Han et al. 2004, Motomura et al. 2004), and Lapillonne et al. (2003) reported the induction of p21 by a novel synthetic ligand for PPAR (2-cyano-3,12-dioxooleana- 1,9-dien-28-oic acid) in breast carcinoma cells. A potential conserved consensus PPRE was detected in the promoter region of p21 gene (Lapillonne et al. 2003, Qin et al. 2003), and Motomura et al. (2004) have reported that accumulation of p27 by ligand-activated PPAR was caused by induction of ubiquitination of p27 and reduction of degradation activity of p27 by proteasomes in hepatocellular carcinoma cells. Results of our present study are consistent with these previous reports, and suggest that PPAR regulates the expression of p21 and p27 in breast cancer tissues.

PPAR immunoreactivity was demonstrated as an independent improved prognostic factor for overall survival in ER-positive breast carcinoma patients in our study, although it may not be as robust as lymph node status, a well-established diagnostic modality (Dowlatshahi et al. 1997). In addition, 15d-PGJ₂ significantly inhibited the estrogen-mediated proliferation in MCF-7 cells. Recently, Jiang et al. (2003) reported that mRNA levels of PPAR in patients with local recurrence or those who died of breast cancer were significantly lower than those who remained disease free, which is generally consistent with our immunohistochemical results. An antiproliferative effect of PPAR is considered to be, at least in part, due to overexpression of p21 and/or p27 in carcinoma cells, but this mechanism still remains largely unknown. Immunoreactivities of p21 and p27 are not necessarily associated with improved clinical outcomes of breast cancer patients (Barbareschi et al. 2000, Pellikainen et al. 2003), which is consistent with the findings in our present study (Table 5). PPAR modulates estrogenic actions in breast carcinoma cells, through the suppression of a part of estrogen-mediated transactivation as described above, which may be also involved in an improved prognosis in breast carcinoma patients positive for PPAR and ER. Further examinations are required to clarify detailed functions of PPAR as a modulator of estrogenic actions in breast carcinoma tissues.

In summary, PPAR immunoreactivity was detected in carcinoma cells in 42% of breast cancer tissues. PPAR immunoreactivity was positively associated with ERs, PR, RXRs, p21, or p27, and negatively correlated with histological grade or COX2. Moreover, PPAR immunoreactivity was a better independent prognostic factor in ER-positive breast carcinoma patients. Ligand-mediated PPAR activation caused the suppression of a portion of estrogen-mediated transactivation or inhibition of estrogen-mediated proliferation in MCF-7 cells. These findings suggest that PPAR is mainly expressed in well-differentiated and ER-positive breast cancers, and in part, plays a role as a modulator of estrogenic actions.

	Acknowledgements

 
We appreciate the skillful technical assistance of Ms Chika Kaneko, Mr Katsuhiko Ono, Mr Masao Nakabayashi and Ms Toshie Suzuki (Department of Pathology, Tohoku University School of Medicine). This work was supported by a Grant-in-aid for Health and Labor Sciences Research for Food and Chemical Saftey (H13-Seikatsu-013) from the Ministry of Health, Labor and Welfare, Japan. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	References

Top Abstract Introduction Materials and methods Results Discussion References

 
Acconcia F & Marino M 2003 Synergism between genomic and non genomic estrogen action mechanisms. IUBMB Life 55 145–150.[ISI][Medline]

Allred DC, Harvey JM, Berardo M & Clark GM 1998 Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Modern Pathology 11 155–168.[ISI][Medline]

Badawi AF & Badr MZ 2003 Expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-gamma and levels of prostaglandin E2 and 15-deoxy-delta12,14- prostaglandin J2 in human breast cancer and metastasis. International Journal of Cancer 103 84–90.[CrossRef][ISI][Medline]

Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR, Koder A & Evans RM 1999 PPAR gamma is required for placental, cardiac, and adipose tissue development. Molecular Cell 4 585–595.[CrossRef][ISI][Medline]

Barbareschi M, Pelosio P, Caffo O, Buttitta F, Pellegrini S, Barbazza R, Dalla Palma P, Bevilacqua G & Marchetti A 1997 Cyclin-D1-gene amplification and expression in breast carcinoma: relation with clinicopathologic characteristics and with retinoblastoma gene product, p53 and p21WAF1 immunohistochemical expression. International Journal of Cancer 74 171–174.[CrossRef][Medline]

Barbareschi M, van Tinteren H, Mauri FA, Veronese S, Peterse H, Maisonneuve P, Caffo O, Scaioli M, Doglioni C, Galligioni E et al. 2000 p27 (kip1) expression in breast carcinomas: an immunohistochemical study on 512 patients with long-term follow-up. International Journal of Cancer 89 236–241.[CrossRef][ISI][Medline]

Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, Gannon F, White JH & Mader S 2004 Genome-wide identification of high-affinity estrogen response elements in human and mouse. Molecular Endocrinology 18 1411–1427.[Abstract/Free Full Text]

Burstein HJ, Demetri GD, Mueller E, Sarraf P, Spiegelman BM & Winer EP 2003 Use of the peroxisome proliferator-activated receptor (PPAR) gamma ligand troglitazone as treatment for refractory breast cancer: a phase II study. Breast Cancer Research and Treatment 79 391–397.[CrossRef][Medline]

Celi FS & Shuldiner AR 2002 The role of peroxisome proliferator-activated receptor gamma in diabetes and obesity. Current Diabetes Reports 2 179–185.[Medline]

Chung SH, Onoda N, Ishikawa T, Ogisawa K, Takenaka C, Yano Y, Hato F & Hirakawa K 2002 Peroxisome proliferator-activated receptor gamma activation induces cell cycle arrest via the p53-independent pathway in human anaplastic thyroid cancer cells. Japanese Journal of Cancer Research 93 1358–1365.[CrossRef][ISI][Medline]

Colombel M, Dante R, Bouvier R, Ribieras S, Pangaud C, Marechal JM & Lasne Y 1999 Differential RNA expression of the pS2 gene in the human benign and malignant prostatic tissue. Journal of Urology 162 927–930.[CrossRef][Medline]

Crowe DL & Chandraratna RA 2004 A retinoid X receptor (RXR)-selective retinoid reveals that RXR-alpha is potentially a therapeutic target in breast cancer cell lines, and that it potentiates antiproliferative and apoptotic responses to peroxisome proliferator-activated receptor ligands. Breast Cancer Research 6 R546–R555.[CrossRef][Medline]

Dowlatshahi K, Fan M, Snider HC & Habib FA 1997 Lymph node micrometastases from breast carcinoma: reviewing the dilemma. Cancer 80 1188–1197.[CrossRef][ISI][Medline]

Dumoulin FL, Nischalke HD, Leifeld L, von dem Bussche A, Rockstroh JK, Sauerbruch T & Spengler U 2000 Semi-quantification of human C-C chemokine mRNAs with reverse transcription/real-time PCR using multi-specific standards. Journal of Immunological Methods 241 109–119.[CrossRef][ISI][Medline]

Elstner E, Muller C, Koshizuka K, Williamson EA, Park D, Asou H, Shintaku P, Said JW, Heber D & Koeffler HP 1998 Ligands for peroxisome proliferator-activated receptor gamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. PNAS 95 8806–8811.[Abstract/Free Full Text]

Elston CW & Ellis IO 1991 Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer. Experience from a large study with long-term follow-up. Histopathology 19 403–410.[ISI][Medline]

Gavrieli Y, Sherman Y & Ben-Sasson SA 1992 Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology 119 493–501.[Abstract/Free Full Text]

Gillesby BE & Zacharewski TR 1999 pS2 (TFF1) levels in human breast cancer tumor samples: correlation with clinical and histological prognostic markers. Breast Cancer Research and Treatment 56 253–265.[CrossRef][ISI][Medline]

Ham EA, Cirillo VJ, Zanetti ME & Kuehl FA Jr 1975 Estrogen-directed synthesis of specific prostaglandins in uterus. PNAS 72 1420–1424.[Abstract/Free Full Text]

Han S, Sidell N, Fisher PB & Roman J 2004 Up-regulation of p21 gene expression by peroxisome proliferator-activated receptor gamma in human lung carcinoma cells. Clinical Cancer Research 10 1911–1919.[Abstract/Free Full Text]

Hayashi SI, Eguchi H, Tanimoto K, Yoshida T, Omoto Y, Inoue A, Yoshida N & Yamaguchi Y 2003 The expression and function of estrogen receptor alpha and beta in human breast cancer and its clinical application. Endocrine-Related Cancer 10 193–202.[Abstract]

Horwitz KB & McGuire WL 1978 Estrogen control of progesterone receptor in human breast cancer. Correlation with nuclear processing of estrogen receptor. Journal of Biological Chemistry 253 2223–2228.[Free Full Text]

Inoue A, Yoshida N, Omoto Y, Oguchi S, Yamori T, Kiyama R & Hayashi S 2002 Development of cDNA microarray for expression profiling of estrogen-responsive genes. Journal of Molecular Endocrinology 29 175–192.[Abstract]

Ioachim E, Tsanou E, Briasoulis E, Batsis C, Karavasilis V, Charchanti A, Pavlidis N & Agnantis NJ 2003 Clinicopathological study of the expression of hsp27, pS2, cathepsin D and metallothionein in primary invasive breast cancer. Breast 12 111–119.[CrossRef][Medline]

Isobe I, Michikawa M & Yanagisawa K 1999 Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid beta-protein and chloroquine in cultured rat astrocytes. Neuroscience Letters 266 129–132.[CrossRef][Medline]

Jiang WG, Douglas-Jones A & Mansel RE 2003 Expression of peroxisome-proliferator activated receptor-gamma (PPARgamma) and the PPARgamma co-activator, PGC-1, in human breast cancer correlates with clinical outcomes. International Journal of Cancer 106 752–757.[CrossRef][ISI][Medline]

Kao YR, Shih JY, Wen WC, Ko YP, Chen BM, Chan YL, Chu YW, Yang PC, Wu CW & Roffler SR 2003 Tumor-associated antigen L6 and the invasion of human lung cancer cells. Clinical Cancer Research 9 2807–2816.[Abstract/Free Full Text]

Keller H, Givel F, Perroud M & Wahli W 1995 Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements. Molecular Endocrinology 9 794–804.[Abstract]

Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC & Lehmann JM 1995 A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 83 813–819.[CrossRef][ISI][Medline]

Koeffler HP 2003 Peroxisome proliferator-activated receptor gamma a