REPORT

A Ligand of Peroxisome Proliferator-Activated Receptor {gamma}, Retinoids, and Prevention of Preneoplastic Mammary Lesions

Rajendra G. Mehta, Elizabeth Williamson, Minu K. Patel, H. Phillip Koeffler

Affiliations of authors: R. G. Mehta, M. K. Patel, Department of Surgical Oncology, College of Medicine, University of Illinois, Chicago; E. Williamson, H. P. Koeffler, Hematology/Oncology Division, Cedars-Sinai Medical Center/University of California at Los Angeles School of Medicine.

Correspondence to: Rajendra G. Mehta, Ph.D., Department of Surgical Oncology, University of Illinois at Chicago, College of Medicine, 840 S. Wood St. (M/C 820), Chicago, IL 60612 (e-mail: raju{at}uic.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Chemoprevention of breast cancer is an active area of investigation. Recent in vivo and in vitro studies have shown that thiazolidinediones (e.g., troglitazone) and retinoids are able to inhibit the growth of breast cancer cells. Troglitazone mediates its action via peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). We evaluated the ability of troglitazone, alone or in combination with retinoids, to prevent the induction of preneoplastic lesions by 7,12-dimethylbenz[a]anthracene (DMBA) in a mouse mammary gland organ culture model. METHODS: Mammary glands of BALB/c mice were treated with DMBA (2 µg/mL) to induce preneoplastic lesions in organ culture. Effects of troglitazone, all-trans-retinoic acid (retinoic acid; ligand for retinoic acid receptor [RAR] {alpha}), and LG10068 (ligand for retinoid X receptors [RXRs]), singly or in combination, on the development of lesions were evaluated. Expression of retinoid receptors (RAR{alpha} and RXR{alpha}) and PPAR{gamma} was determined by western blot analysis. Statistical significance was determined by generalized chi-squared analysis using the GENCAT software program and Bonferroni correction. All P values are two-sided. RESULTS: Troglitazone (at 10-5 M) or retinoic acid (at 10-6 M) markedly inhibited the development of mammary lesions (both P values <.05); however, together they did not enhance the effectiveness of the other. In contrast, LG10068 (at 10-7 M or 10-8 M) alone had very little ability to inhibit development of these lesions, but a combination of LG10068 (at 10-8 M) and troglitazone (at 10-5 M or 10-6 M) almost completely inhibited (by 85% and 100%, respectively; both P values <.05) the development of mammary lesions. The expression of PPAR{gamma} and RXR{alpha} remained unchanged with the various treatments, whereas the expression of RAR{alpha} was substantially reduced after treatment with the combination of retinoic acid and troglitazone. CONCLUSIONS: To our knowledge, this is the first report showing the possibility of a PPAR{gamma} ligand having chemopreventive activity. Furthermore, an RXR-selective retinoid, LG10068, appears to enhance this activity.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Breast cancer is one of the leading causes of cancer-related deaths. Clearly, the prevention of breast cancer is the best approach to this disease. For example, blockers of estrogen receptors (ERs), including tamoxifen and raloxifene, appear to diminish the frequency of breast cancer by about 50% in postmenopausal women (1). We and other investigators (2,3) have shown that activation of the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear hormone receptor superfamily, by thiazolidinediones including the synthetic ligand troglitazone inhibited proliferation of cultured breast cancer cells. Furthermore, all-trans-retinoic acid (hereafter referred to as retinoic acid), a ligand for another nuclear hormone receptor, i.e., retinoic acid receptor (RAR), enhanced the inhibition of proliferation of breast cancer cells. The combination of troglitazone and retinoic acid caused marked apoptotic cell death of tumors induced by MCF-7 breast cancer cells in immunodeficient mice without causing toxic effects in these animals (3).

The PPAR{gamma} is a member assigned to the subfamily of nuclear hormone receptors that includes receptors for retinoic acid and thyroid hormone (4). The PPAR{gamma} heterodimerizes with retinoid X receptor (RXR) and binds to DNA by recognizing target sequences that have a direct repeat of core recognition motifs (AGGTCA) spaced by one nucleotide. Activation of PPAR{gamma} results in expression of genes associated with many different aspects of differentiation, cellular development, and general physiology, including differentiation of adipocytes, lipid metabolism, and glucose homeostasis (5,6). The natural PPAR{gamma} ligand appears to be 15-deoxy-{Delta}-12,14 prostaglandin J2, but a variety of polyunsaturated fatty acids including linoleic acid can also activate PPAR{gamma} (7-9). Furthermore, nonsteroidal anti-inflammatory agents, such as indomethacin, can bind and activate PPAR{gamma} (10). A series of thiazolidinediones, including troglitazone and plioglitazone, are useful for the treatment of type II adult-onset diabetes. Troglitazone has been used for the treatment of nearly one million individuals with diabetes. The mechanism by which this class of agents lowers blood glucose is unclear, although these agents may enhance differentiation of adipocytes associated with increased function of their glucose pumps.

Retinoids mediate their activity through the RAR and RXRs, both of which are expressed in breast cancer cells, although RARß may not be expressed in all breast cancers (11). The RAR and RXRs bind to specific retinoic acid-response elements and regulate the transcription of a variety of target genes in a ligand-dependent manner (12,13). The all-trans-retinoic acid, an RAR-specific ligand, selectively inhibits the growth of human ER-positive breast cancer cells (14-16). These cells appear to express higher levels of RAR{alpha} than ER-negative cell lines (16,17). Inhibition of growth of ER-positive human breast cancer cells by retinoids requires transactivation of retinoid-responsive genes (18). Often the inhibition of growth of breast cancer cells by retinoids is reversible with the removal of the ligand (19). These compounds also are effective in preventing mammary carcinogenesis in rodents (20).

The murine mammary gland organ culture model system has been effectively used to evaluate the ability of potential chemopreventive agents to prevent the development of preneoplastic lesions (21-24). Mammary glands of BALB/c mice are placed in organ cultures containing a variety of growth-promoting hormones and are treated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneoplastic lesions (24,25). The mammary epithelial cells isolated from these lesions, when placed into syngeneic hosts, develop into adenocarcinoma (26). With the use of this technique, more than 150 potential chemopreventive agents have been tested (27). Effective chemopreventive agents, such as retinoids, selenium, oltipraz, limonene, and vitamin D3 analogues, are able to inhibit the formation of these lesions. The assay is highly reproducible and provides a good association with the efficacy of an effective chemopreventive agent in the two-stage skin carcinogenesis in vivo assay and in prevention of chemically induced mammary tumors in vivo (21,23,28). In this report, we have analyzed the efficacy of troglitazone, with or without a retinoid, in preventing the formation of DMBA-induced mammary lesions in a murine mammary gland organ culture model.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Reagents. Troglitazone was dissolved in a solution containing 50% dimethyl sulfoxide (DMSO) and 50% ethanol. The all-trans-retinoic acid (Sigma Chemical Co., St. Louis, MO) and LG10068 (Ligand Pharmaceuticals, Inc., San Diego, CA) were dissolved in 100% ethanol and added to organ cultures at a concentration of less than 0.1%. (Exact concentrations used are shown with the appropriate experiments.)

Protein extraction and western blot analysis. Murine mammary glands were homogenized in Triton X-100 containing lysis buffer. (The lysis buffer mixture contained 20 mM Tris buffer [pH 8.0], 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, and protease inhibitors and was obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN.) Protein lysate was separated on a 10%-20% gradient polyacrylamide Ready gel (BioRad Laboratories, Hercules, CA), and western blotting was performed on polyvinylidene fluoride membranes (Immobilon; Millipore Corp., Bedford, MA). The western blots were probed with an antibody raised against PPAR{gamma}-amino acids 2-20 (N-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA) used at a 1 : 2000 dilution and subsequently with an antibody against mouse actin (Oncogene Research Products, San Diego, CA) used at a 1 : 1000 dilution. The membranes were stripped and probed sequentially with RAR{alpha}, RXR{alpha}, RXRß, or RXR{gamma} (Santa Cruz Biotechnology Inc.; 1 : 200 dilution). Results were visualized after reaction with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Amersham Life Science Inc., Arlington Heights, IL).

Induction of preneoplastic lesions in mammary glands and their prevention by ligands of nuclear hormone receptors. Young, virgin BALB/c female mice, 3-4 weeks of age, were obtained from Charles River Laboratories, Wilmington, MA. All of the animal studies were approved by the University of Illinois Animal Review Board and were performed in accordance with institutional guidelines. The entire culture procedure has been described in detail previously (21-25). Briefly, the mice were pretreated for 9 days with 17ß-estradiol (1 µg in 0.1 mL of saline/animal) and progesterone (1 mg in 0.1 mL of saline/animal). They were then killed by cervical dislocation, and the thoracic pair of mammary glands was removed, placed on silk rafts, and incubated for 10 days in serum-free Waymouth MB752 medium (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) containing the following growth-promoting hormones: insulin (5 µg/mL), prolactin (5 µg/mL), aldosterone (1 µg/mL), and hydrocortisone (1 µg/mL). The carcinogen DMBA at a dose of 2 µg/mL in DMSO was added to the medium on day 3 for a duration of 24 hours to induce mammary lesions. The DMBA-containing medium was removed, and the mammary glands were incubated for an additional 14 days with medium containing only insulin. This procedure allowed the normal glands to undergo structural regression in which all the normal alveolar structures were disintegrated. However, the alveolar lesions in the carcinogen-treated glands behave differently. They acquired altered hormone responsiveness and continued to grow. The nuclear hormone receptor ligand analogues were included in the medium during the first 10 days of the in vitro culture to determine if they lowered the incidence of formation of mammary lesions. Throughout the culture period, the glands were maintained at 37 °C in an environment of 95% O2 and 5% CO2. At the end of the culture period, the glands were fixed in formalin, stained in alum-carmine solution, and evaluated for the presence or absence of mammary lesions. All hormones and chemicals were purchased from Sigma Chemical Co.

Statistical analysis. The statistical significance was determined with the use of the GENCAT computer software program specifically written for the generalized chi-squared analysis of categorical data using weighted least squares (29). The P values were further subjected to Bonferroni correction. All P values are two-sided.


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
To examine the efficacy of troglitazone and/or a retinoid in preventing mammary lesions, we incubated 10-20 mammary glands per group (239 glands in total) from BALB/c mice with appropriate growth hormones and exposed them for 24 hours to DMBA on day 3 of culture. Fig. 1, A, shows the presence of mammary alveolar lesions in DMBA-treated glands. Mammary glands were cultured for 10 days with troglitazone with or without a retinoid. The incidence of mammary lesions was calculated for each group and was reported as a ratio of the number of mammary glands showing lesions compared with the total number of mammary glands at risk. The percent inhibition of formation of lesions for each treatment group was calculated by the comparison of the incidence of lesions between the control and the treatment groups. A dose-related decrease in the number of glands exhibiting lesions occurred in both the troglitazone-treated group and the retinoic acid-treated group (Table 1). Troglitazone at 10-6 M and 10-5 M inhibited mammary alveolar lesions by 31% and 60%, respectively. Glands treated with troglitazone (10-5 M) contained very few alveolar lesions, and no extensive dilation of ducts was evident. Toxicity in organ cultures is characterized by extensive dilation of mammary ducts, which results in disintegration of the gland structure (24). Thus, troglitazone was not toxic at these concentrations.



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Fig. 1. Effects of nuclear hormone receptor ligands on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary lesions. Mammary glands were incubated with insulin, prolactin, aldosterone, and hydrocortisone for 10 days either alone or with troglitazone and/or a retinoid. The glands were treated with DMBA on day 3, for 24 hours, during the initial 10 days of culture. The glands were further cultured for an additional 14 days with insulin alone. At that time, the glands were fixed in formalin and processed for morphologic evaluation. Panel A: mammary gland cultured with DMBA (24 hours) in the absence of chemopreventive agents. Representative mammary lesions (MAL) are shown. Panel B: representative photograph of the nearly normal ducts in a gland after culture with DMBA (24 hours) on day 3 of the culture plus troglitazone (10-5 M) and an RXR ligand (LG10068, 10-8 M) added to the culture from days 1 through 10 of culture (original magnification x40).

 

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Table 1. Effects of troglitazone and/or all-trans-retinoic acid on development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced lesions in mouse mammary glands*

 
The retinoic acid at 10-8 M, 10-7 M, and 10-6 M inhibited formation of lesions by 43%, 54%, and 77% (all P values <.05), respectively (Table 1). To evaluate the effects of the combination of troglitazone and retinoic acid, we used various concentrations of troglitazone (10-6 M to 10-5 M) and retinoic acid (10-8 M to 10-6 M). Inhibition of lesions with both ligands was similar to that with retinoic acid alone, with the exception of the combination of 10-5 M troglitazone and 10-7 M retinoic acid, which resulted in 100% inhibition of the lesions as compared with 66% by 10-5 M troglitazone or 54% inhibition mediated by 10-7 M retinoic acid. Since both troglitazone and retinoic acid were independently very active, the combined effect did not appear to be synergistic. There was no dilation of ducts or no noticeable toxicity observed with retinoic acid or with the combination of retinoic acid and troglitazone.

The PPAR{gamma} heterodimerizes with RXR, and each can simultaneously bind to its ligand, resulting in enhanced activity of this activated receptor complex. Thus, we examined the effect of troglitazone combined with an RXR ligand (LG10068). The RXR ligand (10-7 M to 10-8 M) was unable to inhibit DMBA-induced mammary lesions, and troglitazone (10-6 M) in this series of experiments inhibited mammary lesions by approximately 14% (Table 2). However, when the two were combined, the percent inhibition of development of alveolar lesions in mammary gland cultures was 85% or more (100%) showing that the two ligands together were clearly more effective than either alone (Table 2). The effect of the combination appears to be much enhanced compared with that of the individual compounds and may be synergistic. As shown in Fig. 1, A, DMBA induced mammary alveolar lesions in the absence of chemopreventive agents. However, the presence of the combination of troglitazone and LG10068, an RXR ligand, inhibited the development of DMBA-induced mammary lesions (Fig. 1, B).


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Table 2. Effects of troglitazone and/or the retinoid X receptor ligand LG10068 on development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced lesions in mouse mammary glands*

 
In the same experiment, the effects of troglitazone on the initiation and promotion of lesions were investigated. When troglitazone (10-5 M) was present in the culture for only the first 4 days, we observed 71% inhibition of DMBA-induced mammary lesions. During the first 4 days of culture, DMBA was present for 24 hours on day 3 in the culture medium. Likewise, troglitazone, when present from days 4 through 10 of culture, was similarly able to inhibit development of mammary lesions (71% inhibition). During that period, DMBA was no longer present. Taken together, the results showed that troglitazone could inhibit both initiation and promotion of lesions of the mammary gland.

Expressions of RAR{alpha}, RXR{alpha}, and PPAR{gamma} were examined in the control and experimental glands (Fig. 2). Results showed that RXR{alpha} and PPAR{gamma} were expressed in all glands; when normalized for the expression of actin, little change occurred in levels of RXR{alpha} or PPAR{gamma} with the various treatments. Mammary glands following the organ culture did not express RXRß or RXR{gamma} (data not shown). However, RAR{alpha} was expressed in all glands. Results showed that there was a 25%-30% reduction in the RAR{alpha} expression in the glands treated with retinoic acid (10-6 M) or troglitazone (10-6 M). The expression of RAR{alpha} was further reduced by 60% when the glands were treated with a combination of troglitazone (10-6 M) and retinoic acid (10-6 M).



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Fig. 2. Western blot, showing levels of expression of nuclear hormone receptors, e.g., peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), retinoic acid receptor {alpha} (RAR{alpha}), and retinoid X receptor {alpha} (RXR{alpha}), in mammary glands after different treatments: troglitazone (TROG; 10-5 M or 10-6 M), with or without all-trans-retinoic acid (ATRA; 10-6 M), as outlined in the text (see the "Materials and Methods" section and Table 1). Expression of actin controls is also shown.

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Chemoprevention of cancer is clearly of great benefit to the individual and is less costly to society than is the treatment of established cancer. This one approach of cancer prevention is known as chemoprevention (30). Compounds that can arrest either initiation or progression of breast carcinogenesis include ER antagonists, retinoids, monoterpenes, isoflavonoids, thiols, inhibitors of polyamine synthesis as well as prostaglandins, and vitamin D3 analogues (31-34). The synthetic ligand of PPAR{gamma}, troglitazone, has been shown to be effective against the proliferation of breast cancer cells; however, it has not been evaluated for its possible activity as a chemopreventive agent. In this study, troglitazone was able to inhibit the development of DMBA-induced mammary lesions, and this activity was potentiated by an RXR ligand, although by itself this ligand had no chemopreventive effects. On the other hand, unlike the RXR ligand, retinoic acid inhibited the development of mammary alveolar lesions in culture. The combination of retinoic acid and troglitazone resulted in additive chemopreventive activity. These experiments represent one of the initial steps toward the long-range goal of identifying effective chemopreventive agents for breast cancer. The lack of toxicity for most individuals receiving troglitazone for adult-onset diabetes, as well as the lack of adverse effects of several RXR ligands, including 9-cis-retinoic acid, makes the combination of troglitazone and an RXR analogue attractive for in vivo chemopreventive trials. Furthermore, several new thiazolidinediones are now available, and these compounds do not appear to have the idiosyncratic liver toxicity that occurs rarely with the administration of troglitazone.

How troglitazone, the synthetic ligand of PPAR{gamma}, inhibits transformation of mammary tissue is unclear. PPAR{gamma} is an important regulator of metabolism and storage of lipid (35,36). This study and previous studies (2,36,37) showed that breast tissue expressed PPAR{gamma}. The amount of PPAR{gamma} expressed in normal breast epithelium appears to be less than that expressed in breast cancer (3). Furthermore, the level of expression of PPAR{gamma} varies in normal mammary ducts, depending on whether the breasts are examined during the lactogenic period or the nonlactogenic period. We previously showed that, after culture with troglitazone and other natural and synthetic ligands of PPAR{gamma}, the MCF-7 breast cancer cells had an increased accumulation of fat and an increased expression of CD36 protein that was associated with active metabolism and storage of lipid (3). However, these cancer epithelial cells did not cross-differentiate to adipocytes, as shown by their lack of expression of a number of key markers, such as aP2, lipoprotein lipase, and adipsin (2,3). Our present data also showed that troglitazone had no marked effect on the level of expression of PPAR{gamma}, as shown by western blot analysis (Fig. 2). In addition, examination of these mammary cells by light microscopy revealed no increase in the level of apoptotic cell death, with the cells appearing perfectly normal. Studies have shown that PPAR{gamma} ligands can induce cell cycle arrest; the mechanism for this effect is unclear, but it may be a result of the receptor's reported involvement in inhibiting the activity of the E2F/DP family of transcriptional factors implicated in the initiation of the S phase of the cell cycle (38) and/or to antagonize the activity of the secondary signal proteins AP-1, STAT, and NF-{kappa}B (39,40).

Thiazolidinediones can inhibit proliferation of a variety of cancer cells (6,41,42). Activation of PPAR{gamma} promotes the differentiation and cessation of proliferation of human liposarcoma cells (6). PPAR{gamma} ligands also induce differentiation and reverse the malignant phenotype of colon cancer cells (41). Furthermore, PPAR{gamma} ligands can inhibit the proliferation of prostate cancer cells in vitro and in laboratory animals (42). Clinical studies in which troglitazone is given to individuals after radical prostatectomy for prostate cancer but who are still having a detectable serum prostate-specific antigen are now ongoing both in Los Angeles, CA, and Boston, MA. PPAR{gamma} ligands have also been shown to induce differentiation of myeloid leukemia cells (43).

Previous studies by others and us (32,33) have shown that, after activation of the PPAR{gamma} receptor for several days, troglitazone could be removed and the breast cancer cell still had a markedly reduced capacity for clonogenic growth. Similarly, we showed here that exposure to troglitazone during either the first 4 days, i.e., initiation phase, or the final 6 days of the growth-promoting phase of culture suppressed transformation of the cells by DMBA (Table 2). In this experiment, the cells were pulse-exposed to the carcinogen only on day 3 of culture; therefore, the troglitazone appeared capable of inhibiting both the initiation and the promotion of cellular transformation.

The retinoic acid (RAR-specific ligand) also inhibited the carcinogen-induced development of mammary lesions. Previous studies have shown that the AP-1 transcriptional factor can be inhibited by retinoic acid (44) and may be responsible for the antitumor-promoting activity of retinoic acid (45). Therapy with retinoic acid is strikingly successful for acute promyelocytic leukemia by inducing terminal differentiation of these leukemic cells (46,47). Moreover, clinical trials have shown that N-4-hydroxyphenyl retinamide, a retinoid, may provide an effective therapy for some breast cancer patients (32,33).

In this study, we found that combining a PPAR{gamma} ligand with a ligand specific for RXR (LG10068) enhanced the suppression of development of mammary lesions. A previous study (48) has shown that simultaneous activation of both receptors can result in synergistic activity in several assays of cultured cells as well as in augmented in vivo antidiabetic activity. Furthermore, we have previously shown that a PPAR{gamma} ligand and an RXR ligand can have enhanced antiproliferative effects against breast and prostate cancer cells (3,42). Another study (49) has also shown that an RXR-specific agonist (LG10069) had chemopreventive activity against chemically induced rat mammary tumors; however, such activity for LG10068 has not been reported. Further studies are required to determine the target genes associated with this anticancer activity.

As far as we know, this is the first report showing the possibility of troglitazone, a PPAR{gamma} ligand, having chemopreventive activity. Troglitazone is a relatively nontoxic compound at a wide range of concentrations, but it is a potent inhibitor of the development of preneoplastic lesions of the mammary gland in organ culture. Also, an RXR- or an RAR-selective retinoid appears to enhance this chemopreventive activity; thus, the combination of a thiazolidedione and a retinoid, such as either retinoic acid or LG10068, may be a good candidate for an in vivo breast cancer chemoprevention study. Because of advances in the knowledge of genetics and epidemiology of breast cancer, individuals at high risk for developing breast cancer can be identified; these are the individuals who may receive the benefit from a chemoprevention regimen containing a PPAR{gamma} ligand combined with an appropriate RXR-selective retinoid.


    NOTES
 
Supported by Public Health Service grant CA26038-20 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by U.S. Army grant PC970577; by the California Breast Cancer Research Program; by the Parker Hughes Trust; and by the C. and H. Koeffler Fund.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received June 6, 1999; revised December 10, 1999; accepted December 27, 1999.


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