PPAR
influences susceptibility to DMBA-induced mammary, ovarian and skin carcinogenesis
Christopher J. Nicol1,
Michung Yoon1,4,
Jerrold M. Ward2,
Masamichi Yamashita1,
Katsumi Fukamachi1,
Jeffrey M. Peters3 and
Frank J. Gonzalez1,5
1 Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, 2 Veterinary and Tumor Pathology Section, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702 and 3 Department of Veterinary Science and Center for Molecular Toxicology, The Pennsylvania State University, University Park, PA 16802, USA
4 Present address: Department of Life Sciences, Mokwon University, Taejon 302-729, Korea
5 To whom correspondence should be addressed Email: fjgonz{at}helix.nih.gov
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Abstract
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Peroxisome proliferator-activated receptor
(PPAR
), a member of the nuclear receptor superfamily, plays a role in adipocyte differentiation, type II diabetes, macrophage response to inflammation and is suggested to influence carcinogen-induced colon cancer. Studies done in vitro and in vivo also revealed that PPAR
ligands might promote differentiation and/or regression of mammary tumors. To directly evaluate the role of PPAR
in mammary carcinogenesis, PPAR
wild-type (+/+) or heterozygous (+/) mice were administered 1 mg 7,12-dimethylbenz[a]anthracene (DMBA) by gavage once a week for 6 weeks and followed for a total of 25 weeks. Compared with congenic PPAR
(+/+) littermate controls, PPAR
(+/) mice had early evidence for increased susceptibility to DMBA-mediated carcinogenesis based on a 1.6-fold increase in the percentage of mice with skin papillomas, as well as a 1.7-fold increase in the numbers of skin papillomas per mouse (P < 0.05). Similarly, PPAR
(+/) mice also had a 1.5-fold decreased survival rate (P = 0.059), and a 1.7-fold increased incidence of total tumors per mouse (P < 0.01). Moreover, PPAR
(+/) mice had an almost 3-fold increase in mammary adenocarcinomas (P < 0.05), an over 3-fold increase in ovarian granulosa cell carcinomas (P < 0.05), an over 3-fold increase in malignant tumors (P < 0.02) and a 4.6-fold increase in metastatic incidence. These results are the first to demonstrate an increased susceptibility in vivo of PPAR
haploinsufficiency to DMBA-mediated carcinogenesis and suggest that PPAR
may act as a tumor modifier of skin, ovarian and breast cancers. The data also support evidence suggesting a beneficial role for PPAR
-specific ligands in the chemoprevention of mammary, ovarian and skin carcinogenesis.
Abbreviations: MMTV-LTR, mouse mammary tumor virus long terminal repeat; PPAR
, peroxisome proliferator activated receptor
.
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Introduction
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According to the American Cancer Society's 2003 statistics, breast cancer is the second leading cause of cancer death in women, where in the US alone, one in eight women are at risk. Current adjuvant treatments include chemotherapy and hormone therapy, such as with the anti-estrogen tamoxifen. While tamoxifen has proven efficacious in treating tumors, especially among those women at elevated risk, the benefits of this treatment are counter-balanced by problems with its side-effects, as well as a limited long-term efficacy after 5 years of therapy, and perhaps most importantly, the failure of a majority of women to respond to tamoxifen therapy (1,2). Hence, a better understanding of the mechanisms involved in breast cancer is needed to assist with the development of new chemopreventive therapies.
The peroxisome proliferator-activated receptors (PPARs), of which there are three family members (
, ß and
) first discovered in the early 1990 s, are members of the orphan nuclear receptor superfamily (35). PPAR
is expressed in a number of cell types, including adipocytes and macrophages (69). Ligands for PPAR
include not only natural compounds, such as 15-deoxy-
12,14-prostaglandin J2, 9- and 13-hydroxyoctadecanoic acid, 15-hydroxyeicosatetraenoic acid and linoleic acid (4,1012) as well as the newly discovered putative endogenous PPAR
agonist, lysophosphatidic acid (13), but also synthetic insulin-sensitizing thiazolidinedione compounds, such as troglitazone, BRL-49653 (aka rosiglitazone) and pioglitazone (14).
It was suggested that PPAR
ligands may help to promote differentiation and reduce the growth rate of breast adenocarcinoma cell lines in vitro and promote regression or stasis of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated rat mammary tumors in vivo (11,1518). The results from another study to selectively delete PPAR
from mouse mammary tissue, by using the Cre-LoxP system and crossing mice with flanking lox P sites in the PPAR
gene to transgenic mice expressing either the whey acidic protein (WAP) or mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter driven Cre transgenes (19), suggests that the loss of PPAR
neither affects mammary development, nor leads to increased spontaneous tumor formation among mice where both alleles of PPAR
were disrupted (20).
Because classical PPAR
null mice die in utero around gestational day 10 (21), the objective of the current study was to directly evaluate the role of PPAR
in mammary carcinogenesis using a whole mouse model of PPAR
haploinsufficiency. These results show that PPAR
(+/) mice have increased susceptibility toward DMBA-mediated carcinogenesis, and suggest a tumor modifier role for PPAR
in skin, ovarian and mammary tumors.
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Materials and methods
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Chemicals and reagents
9,10-Dimethyl-1,2-benzanthracene, otherwise known as 7,12-dimethylbenz[a] anthracene (DMBA) was purchased from Sigma (St Louis, MO). Ten percent phosphate-buffered formalin was purchased from Fisher Scientific (Fair Lawn, NJ).
Animals
All mice were treated in accordance with protocols approved by the NCI Animal Care and Use Committee, and housed in microisolator cages on a 12 h light/dark cycle, with food and water provided ad libitum. PPAR
wild-type (+/+) and heterozygous (+/) female mice were generated from crosses of PPAR
-floxed mice with the EIIa-Cre transgenic mouse as described previously (22). The mice used in this study are of mixed C57Bl6/NCr;Sv129;FVB/NCr background and were bred using sibling mating for at least 16 generations prior to the start of the study.
PPAR
PCR analysis
PCR analysis to detect PPAR
wild-type and null bands was performed using primers designed to amplify around exon 2 (Figure 1). Briefly, tail DNA (
0.5 µg) was incubated with one forward primer 2F: CTC CAA TGT TCT CAA ACT TAC, and two reverse primers 1R: GAT GAG TCA TGT AAG TTG ACC and H5: GTA TTC TAT GGC TTC CAG TGC ([Final] = 0.25 µM), 10x PCR buffer (Perkin Elmer, Foster City, CA) ([Final] = 1x), dNTPs ([Final] = 0.2 µM), MgCl2 (Perkin Elmer) ([Final] = 2 mM), Taq polymerase (Perkin Elmer) ([Final] = 2.5 U) and autoclaved Millipore-filtered water up to a 25 µl final volume. PCR reactions were run on a thermal cycler at 94°C, 2 min, 33 cycles at (94°C, 1 min, 60°C, 1 min, 72°C, 1:15 min), 72°C, 5 min and 4°C. A 2% agaroseTBE gel electrophoresis followed by ethidium bromide staining was used to detect bands. Expected sizes of PPAR
bands are wild-type allele
250 bp and null allele
450 bp.
DMBA by gavage
DMBA was dissolved in sesame oil to give a 10 mg/ml stock concentration and a total of 44 PPAR
(+/+) and 39 PPAR
(+/) 812-week-old female mice were gavaged p.o. with 0.1 ml (total 1 mg) DMBA once a week for 6 weeks. Gross/clinical examinations of mice were done weekly, for a total of 25 weeks, to monitor body weights, skin papilloma and tumor progression. Mice were killed by cervical dislocation either at the end of the study or earlier if they displayed significant weight loss, signs of distress or palpable tumors >2 cm in diameter.
Pathology
All mice were necropsied. Tissues including tumors were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections were prepared and stained with hematoxylin and eosin, and subjected to a blind review by a pathologist.
Analysis of PPAR
gene expression by real-time quantitative PCR
Total RNA was isolated from untreated tissues of PPAR
(+/) or wild-type mice as described previously (22). Extracted RNA (1 µg) was mixed with chloramphenicol acetyltransferase (CAT) RNA (Invitrogen, Carlsbad, CA) (50 pg) as the external control, and reverse transcribed using Super Script II (Invitrogen) according to the manufacturer's instructions. Quantitative gene expression analysis was performed on an ABI PRISM 7900HT machine (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions, and using the methods of Hoekstra et al. (23), with the following modifications. In 96-well optical plates, 12.5 µl of SYBR green master mix (Applied Biosystems) was added to 50 ng of cDNA template, and 300 nM of forward and reverse primers in water. Plates were heated at 50°C for 2 min and 95°C for 10 min, then run for 45 PCR cycles comprised of 95°C for 15 s and 60°C for 60 s. Samples were then heated at 95°C for 20 min at the end of run. Primers for PPAR
(forward CATGCTTGTGAAGGATGCAAG; reverse TTCTGAAACCGACAGTACTGACAT) (SIGMA Genosys, The Woodlands, TX) were as described previously (23), while primers for CAT (forward ACACGCCACA1TCTTCGCAAT; reverse CACCGTTTTCCATGAGCAAACT) were designed using PrimerExpress software (Applied Biosystems).
Northern blot analysis
RNA isolation from tissues and papillomas, and northern blot analysis for PPAR
mRNA expression versus that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as described elsewhere (22).
Statistical analysis
Statistical significance was determined for the rate of tumor formation in PPAR
wild-type and heterozygous mice with a Student's t-test or Fisher's Exact test where appropriate. Survival was analyzed using a Log Rank test. Significance was accepted for P values <0.05.
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Results
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In the present study, all female PPAR
heterozygous knockout [PPAR
(+/)] and congenic wild-type littermate control [PPAR
(+/+)] mice were treated with DMBA. A PCR based assay was established to confirm the PPAR
genotype of mice and was performed prior to the start of the study (Figure 1). Body weights of PPAR
(+/) and littermate control PPAR
(+/+) mice were not significantly different prior to the start of, or at any time during, the course of this study, suggesting this level of DMBA dosing was not toxic to the animals (Figure 2).
Compared with congenic PPAR
(+/+) littermates treated with DMBA, skin papillomas first started to appear in PPAR
(+/) mice 3 weeks following the last dose of DMBA (week 9 from the start of the study). Skin papillomas were noted in areas ranging from ventral midline to dorsal lateral surfaces, and from sub-ocular to vaginal opening areas, but were not confined to any specific region. By the end of the 25th week from the start of the study, the multiplicity of skin papillomas was significantly greater among PPAR
(+/) (0.87 papillomas/mouse) compared with PPAR
(+/+) controls (0.52 papillomas/mouse) (P < 0.05) (Figure 3A). In fact, upwards of 64% (25 out of 39) of PPAR
(+/) compared with only 41% (18 out of 44) of PPAR
(+/+) controls presented with skin papillomas by the end of the study (P < 0.05) (Figure 3B). By the end of the observation period,
41% (18 out of 44) of PPAR
(+/+) and
61% (24 out of 39) of PPAR
(+/) mice died, or had to be killed due to morbidity resulting from tumor progression, with the majority of cases occurring between weeks 13 and 21 (Figure 4A). Analysis of the survival curve revealed that 50% of PPAR
(+/) mice died between weeks 19 and 20 from the start of the study while PPAR
(+/+) mice did not reach this plateau during the observational period of the study, although the overall survival curve was not statistically different (P = 0.059). Upon pathological analyses, the incidence of PPAR
(+/+) and PPAR
(+/) mice presenting with any type of tumor was similar for both genotypes (93 and 95%, respectively), however the total tumor multiplicity was significantly higher among the PPAR
heterozygotes when compared with wild-type controls, respectively 3.69 (144 total tumors) versus 2.18 (96 total tumors) (P < 0.01) (Figure 4B).
Pathological analysis revealed differences between tumors from wild-type and PPAR
(+/) mice (Figure 5). Representative tumor samples are shown illustrating that compared with those from PPAR
(+/+) mice (Figure 5A, C and E), tumors derived from PPAR
(+/) mice (Figure 5B, D and FH) were in a more advanced state of progression. This was particularly true for mammary glands and ovarian tissues.
Tissue distribution and breakdown of the various tumors identified also revealed differences between the two genotypes (Table I). Notably, while the spectrum of tumors is consistent with the well established pattern of DMBA-initiated carcinogenesis, the ratio of total skin tumors per genotype was significantly higher among PPAR
(+/) [1.21 total skin tumors per PPAR
(+/) mice] compared with wild-type controls [0.64 total skin tumors/PPAR
(+/+) mice] (P = 0.01). Of the total tumors found in the skin, the incidences of not only papillomas (P < 0.05), but also squamous cell carcinomas (P < 0.06), were higher on their own among PPAR
(+/) mice when compared with PPAR
(+/+) littermates, although the latter was not significant on a statistical level. When compared with respective PPAR
(+/+) mice, PPAR
(+/) mice also had a 2.8-fold higher multiplicity of total mammary gland tumors per mouse (respectively, +/+ versus +/: 0.18 versus 0.51, P = 0.07), wherein the number of adenosquamous carcinomas per mouse alone was 3.4-fold higher (respectively, +/+ versus +/: 6 out of 44 versus 18 out of 39, P < 0.05) (Table I). Included within the listing for adenocarcinomas in the mammary gland (Table I), are the previous values for adenosquamous carcinomas, one acinar adenocarcinoma each for both PPAR
(+/+) and PPAR
(+/) mice, as well as one myoepithelial carcinoma for PPAR
(+/) mice. Interestingly, while the multiplicity of total ovarian tumors did not differ between the two genotypes, when the tumors were sorted by class into benign versus malignant, PPAR
(+/) mice had a significantly higher multiplicity of malignant tumors per mouse when compared with wild-type controls (carcinomas out of total ovarian tumors, respectively +/+ versus +/: 3 out of 12 versus 10 out of 13, P < 0.02). A similar trend was also observed for mammary tumors whereby the number of malignant (carcinoma) tumors per mouse in PPAR
(+/) mice was 3.2-fold higher compared with congenic wild-type controls (respectively, +/+ versus +/: 7 out of 44 mice versus 20 out of 39, P < 0.0001). More importantly, when total tumors in any tissue were sorted as a whole into benign or malignant (carcinoma) categories, DMBA-treated PPAR
(+/) mice had a significant 3.2-fold higher proportion of malignant tumors per mouse compared with wild-type controls, respectively 1.23 (48 tumors out 39 mice) versus 0.39 (17 tumors out of 44 mice) (P < 0.05). The metastatic incidence to lung and/or liver was also higher among PPAR
(+/) mice compared with PPAR
wild-type controls, where nine PPAR
(+/) mice had metastases, four cases each originating from ovarian granulosa cell carcinomas or mammary gland adenocarcinomas and one case originating from hepatic hemangiosarcoma, versus only two such metastatic incidences among PPAR
wild-type mice, although this difference was not significant statistically.
A QRT-PCR analysis of PPAR
RNA in several normal tissues and skin papillomas from wild-type and PPAR
heterozygous mice was performed (Table II). From the data, PPAR
RNA expression represented as a ratio of PPAR
/CAT levels is clearly evident in normal tissues from PPAR
(+/+) mice with the highest expression in white and brown adipose tissues and colon, and significantly lower expression in all cases among normal tissues from PPAR
(+/) mice (P < 0.05). Levels of mRNA in the mammary gland were also relatively high, reflective of the high amount of fat in this tissue. However, relative levels of PPAR
mRNA in skin were <4% of that found in adipose tissue. Interestingly, DMBA-induced papillomas from PPAR
(+/+) mice failed to show any detectable PPAR
expression, as compared with either the low expression in normal skin or the relatively high expression among adipose tissue. The data were confirmed by northern blot analysis showing only faint mRNA bands in the epidermis and papillomas from PPAR
(+/+) mice (Figure 6).
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Table II. QRT-PCR analysis of PPAR tissue expression in untreated PPAR heterozygous (+/) and wild-type (+/+) mice, and respective DMBA-mediated skin papillomas
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Discussion
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These studies demonstrate that PPAR
(+/) deficient mice have increased susceptibility toward DMBA-mediated carcinogenesis, suggesting that PPAR
functions to suppress tumorigenesis in mammary tissue, ovaries and skin. Compared with congenic PPAR
(+/+) littermate controls, PPAR
(+/) mice had early evidence for increased susceptibility to DMBA-mediated carcinogenesis based on not only an increase in the incidence of mice with skin papillomas, but also an increase in the multiplicity of skin papillomas. In addition, compared with PPAR
(+/+) controls following DMBA treatment, PPAR
(+/) mice had a decreased survival rate and an increased number of total tumors per mouse. Histological analysis of tumors revealed a pattern of DMBA-mediated carcinogenesis consistent with a previous report using this carcinogen (24) where the majority of tumors were located in the skin, ovaries and mammary glands, but other affected tissues were also noted including uterus, liver, lung, the hematopoietic system and pancreas. Furthermore, when tumors were classified on the basis of benign versus malignant, PPAR
(+/) mice had a significant increase in malignant forms of all tumors identified, as well as an increase in metastatic tumors, when compared with PPAR
(+/+) controls. Hence, PPAR
appears to play a role in suppressing not only tumor initiation, but also progression following carcinogen treatment.
While vehicle-treated controls were not used in this study, vehicle (sesame oil) has been shown in numerous published studies to not only have an absence of pro-carcinogenic activity, but also has the potential, possibly through anti-oxidant properties, to act as a chemopreventive agent (2527). This suggests that the tumors observed in this study were due solely to the carcinogenic effects of DMBA (24,28). In addition, no observed spontaneous carcinogenesis was seen among untreated PPAR
(+/+) or PPAR
(+/) mice, consistent with previous long-term observations using mammary gland directed WAP or MMTV-LTR promoter-mediated Cre recombination of PPAR
on both alleles (20), and our unpublished observations. In the former study, only 2 out of 30 PPAR
MMTV-LTR Cre+ mice and 1 out of 20 control mice had developed breast tumors after 15 months (20); however, the type and stage of these tumors was not analyzed. This indicates that PPAR
might only influence chemically induced, and not spontaneous, cancers. However, recombination of PPAR
was limited exclusively to mammary epithelium by the WAP Cre and, depending on the mouse line examined, to mammary ductal and alveolar epithelium, salivary glands, oocytes, ovarian granlulosa cells, megakaryocytes and B- and T-cells, or several secretory organs and the hematopoietic system by the MMTV-LTR Cre (20).
The results with DMBA-induced tumors are consistent with previous reports suggesting that PPAR
functions to suppress tumorigenesis in the colon following azoxymethane treatment (2931). Previous studies revealed loss of function somatic mutations in the PPAR
gene in sporadic human colon cancers (29). More recently, PPAR
was shown to influence colon cancer in the mouse (31). Following treatment with the colon carcinogen azoxymethane, PPAR
(+/) mice alone were found to be more sensitive than wild-type mice as reflected by an increased formation of colon polyps. However, when PPAR
(+/) were crossed to a model of familial adenomatous polyposis, Apc+/1638 mice, PPAR
haploinsufficiency did not influence polyp formation. Thus, this receptor does appear to act as an early tumor suppressor gene in the colon, albeit this effect was shown to be limited once the initiating effect had occurred as is found in Apc+/1638 mice (31). In addition, recent work has added further evidence to support the notion that PPAR
acts as a tumor suppressor gene in the colon based on reduction in azoymethane-induced aberrant crypt foci formation in Balb/c mice following treatment with PPAR
ligands (30).
Interestingly, the data here suggest that PPAR
also may play a role in progression given the advanced stage of tumors in the heterozygotes compared with wild-type controls. An anti-proliferative role for PPAR
is supported by several in vitro and in vivo studies demonstrating that both natural and synthetic PPAR
ligands block growth and/or induce regression in chemically induced prostate carcinomas (32,33). One previous report has shown that human microvascular endothelial cells can promote proliferation of neighboring pre-adipocytes via as yet unidentified paracrine signaling (34). This may help to explain the differences seen between colon and mammary gland. In light of the extensive vascularization present in mammary as compared with colon tissue, it is possible that PPAR
(+/) mice may have enhanced signaling between endothelial cells and those DMBA-initiated cells present in mammary gland, thus promoting tumor growth as well, although this remains speculative.
The relative lower level of PPAR
RNA expression in all tissues from PPAR
(+/) mice compared with PPAR
wild-type controls is consistent with a previous study showing decreased PPAR
protein expression in colons from PPAR
(+/) compared with wild-type controls (31). However, the lack of detectable levels of PPAR
expression among DMBA-induced papillomas from either PPAR
(+/) or wild-type mice is consistent with published reports of trace to undetectable levels of PPAR
mRNA in both mouse and human normal or lesional skin (3537). Similarly, several reports have shown decreased expression of PPAR
protein in human breast and colon cancers, where expression is highest in normal tissue and progressively decreases among samples from benign to malignant states of the disease (11,38,39). In a recent study, PPAR
mRNA and protein was also shown to be lowest among MNU-induced rat mammary tumors as compared with the higher expression seen in normal tissues (40). Immunostaining for PPAR
among DMBA-initiated tumors from PPAR
(+/) and wild-type mice from this study (data not shown) demonstrated a pattern of protein expression consistent with that reported above. Since the mRNA and protein is low to undetectable among tumors from PPAR
(+/+) mice, analysis of PPAR
in tumors from PPAR
(+/) mice to examine loss of heterozygosity was not feasible.
The relatively low expression of PPAR
in skin as described herein and elsewhere, suggests that the receptor is not acting as a direct tumor suppressor in the epidermal target tissue. However, there are other possible mechanisms to explain the increased DMBA-mediated skin tumors among PPAR
(+/) mice as compared with PPAR
(+/+) controls. Given that cytochrome P450 (CYP) 1B1-null mice were reportedly protected from skin tumors compared with wild-type controls (24), and that skin is well known to express CYP1B1 (41), the increased incidence of skin tumors among PPAR
(+/) mice compared with wild-type controls seen in this study suggests a possible role of PPAR
as a regulator of CYP1B1-mediated bioactivation of DMBA to its carcinogenic metabolites resulting in the initiation of transformed epidermal cells into latent neoplastic cells, although PPAR
-mediated regulation of detoxifying/cytoprotective and/or DNA repair pathways cannot be discounted and warrant further study. Furthermore, promotion and progression of initiated neoplastic cells may result from the influence of an increased inflammatory response among PPAR
haploinsufficient mice. Several reports have shown previously that an increase in inflammatory cell infiltrates following UV radiation promoted skin tumor growth, in an inflammatory-dependent but immunosuppression-independent manner, suggesting a paracrine role for macrophages (42,43). Similarly, reports that antibodies to tumor necrosis factor alpha (TNF
) were able to block DMBA-mediated skin tumors, and that TNF
-null mice were resistant to chemically mediated skin tumors, suggest that early induction of TNF
is critical for skin tumor promotion (44,45). PPAR
has a well-defined role as a negative regulator of both native and acquired immune responses (46,47), and its expression among cells of the immune system including macrophages is well known (22,48). In another mouse model of PPAR
haploinsufficiency, PPAR
(+/) mice were reported to have increased arthritis further supporting an anti-inflammatory role for PPAR
(49). PPAR
agonists have also been shown to down-regulate pro-inflammatory mediators such as TNF
and COX-2 in PPAR
-expressing cells (5053). In addition, these pro-inflammatory mediators may up-regulate downstream targets such as prostaglandin E2 and matrix metalloproteinases in a variety of cell types including both macrophages and epidermal cells, and have been shown previously to correlate with the transformation of epithelial cells and skin tumor growth and progression (5456). Thus, decreased PPAR
expression among PPAR
(+/) mice may result in the establishment of a pro-inflammatory environment upon invasion into the skin of immune cells such as macrophages, through induction of paracrine mediators. Ultimately, this altered environment may contribute to the growth and progression of skin tumors following DMBA treatment although this remains speculative. However, the possibility that a spread of tumor cells from lymphatic or blood vessels in the mammary glands or other metastasizing tumor sites contributed in part to the increased incidence of skin tumors among PPAR
haploinsufficient mice cannot be discounted.
After this paper had been accepted, Saez et al. (57) reported that ligand-independent PPAR
signaling promotes mammary tumor development, which may be the result of increased Wnt signaling. Generation of ligand-independent PPAR
target gene expression in the mammary gland was accomplished by placing a construct of a fusion protein of the PPAR
1 isoform with the activation domain of the herpes simplex virus VP16 co-activator protein, under the control of the MMTV promoter. These MMTV-VpPPAR
mice were then bred with a susceptible strain of transgenic mice expressing the polyoma virus middle T antigen in the mammary gland (MMTV-PyV), shown previously to have a rapid spontaneous appearance of multifocal adenocarcinomas (58). While MMTV-VpPPAR
mice were not different from wild-type controls, animals heterozygous for both transgenes had accelerated tumor growth kinetics compared with MMTV-PyV mice, suggesting that following tumor initiation, PPAR
signaling acts as a strong tumor promoter in the mammary gland (57). It was further suggested that the observed PPAR
tumor-promoting effects were the result of enhanced Wnt signaling given the pattern of altered Wnt target gene expression, such as cyclin D1, as well as embryonic defects induced in zebrafish were comparable when either PPAR
or Wnt were activated (57). One explanation for the differences seen between the results of Saez et al. (57) and those here may be the result of the differences in targeting strategies. While the former study focused on constitutive ligand-independent expression of the PPAR
1 in epithelial cells of the mammary gland, the results observed here represent the effects of reduced expression of both PPAR
isoforms in all tissues examined. In addition, as discussed below, the increased expression of genes such as cyclin D1 in tumors from bigenic mice having constitutive PPAR
expression may be independent of Wnt activity and rather reflect a shift in the cellular use of co-regulator proteins such as p300, which would not be necessary for PPAR
activity, although this requires further investigation. Interestingly, crosses between PPAR
heterozygous mice, different from that used in this study, to MMTV-PyV mice were reported to have no significant difference in tumor latency or histology compared with mice with two functional copies of PPAR
(57). However, PPAR
expression is present in not only mammary epithelial, but also stromal cells (11), and RT-PCR has shown that PPAR
mRNA expression was highest in the latter cell type (20). It is possible that since the initiating effects of PyV are restricted to epithelial cells of the mammary gland that the tumors formed may not reflect the role of PPAR
with respect to the whole mammary gland response to tumorigenesis. It is also possible that differences in the genetic backgrounds of the mice used in the two studies contributed to the contrasting observations. Nevertheless, the results here suggest further work is necessary to clarify the role of both PPAR
in the various cell types of the mammary gland to tumorigenesis.
Several in vitro studies have suggested that growth inhibition of human breast cancer cell lines with PPAR
agonists is mediated partly by both reduced activation of epidermal growth factor receptors via inhibition of tyrosine phosphorylation (59), as well as up-regulation of PTEN expression (60). In addition, natural or synthetic ligands for PPAR
have been shown to down-regulate cyclin D1 (61,62). This down-regulation occurs both through inhibition of transcription, via competition with c-fos, which is normally bound to the cyclin D1 promoter, for cellular p300 co-regulator protein, as well as via increased proteasomal degradation through ubiquitin targeting. Similarly, another study reports that cyclin D1 up-regulation can repress PPAR
expression and transactivation (38). Moreover, Qin et al. (62), have shown recently that in MCF-7 cells estrogen receptor alpha (ER
) is similarly down-regulated via ubiquitin-mediated proteasomal degradation. Given that cyclin D1 plays a critical role in G0/G1
S phase cell cycle progression, and a role in early stage breast cancers for over-expression of ER
reported (63), in vivo disruption of one allele of PPAR
in our mouse model may result in increased cyclin D1 and ER
activation leading to the increased DMBA-mediated carcinogenesis seen here. Alternatively, BRCA1 has been implicated as a tumor suppressor in both breast and ovarian carcinomas given its role not only via the repression of estrogen receptor-mediated transcription (64,65), but also through induction of apoptotic pathway genes (66,67). Mutations in BRCA1 have also been associated with familial breast and ovarian cancer, and expression was found to be low to undetectable in sporadic breast cancers, ovarian carcinomas, as well as several breast cancer cell lines (68,69). More recently, PPAR
has been identified as a gene capable of directly regulating expression of BRCA1 through a functional PPAR
responsive element in the BRCA1 promoter, and consequently may play a critical role in BRCA1 regulation (70). In addition, PPAR
expression has been found in ovaries from several species, including humans (71) and mice (20), although primarily located within granulosa cells (72). Thus, decreased PPAR
expression among PPAR
(+/) mice may result in the decreased expression of BRCA1 in mammary glands and ovaries, contributing to the increased carcinomas seen here following DMBA treatment.
Taken as a whole, the results here demonstrate that PPAR
(+/) mice have an increased susceptibility to DMBA-initiated carcinogenesis. These results are the first to show an increased susceptibility in vivo of PPAR
(+/) mice to DMBA-mediated carcinogenesis and thus suggest that PPAR
may act as a tumor modifier. The data also support evidence suggesting a beneficial role for PPAR
-specific ligands in the chemoprevention of mammary, ovarian and skin carcinogenesis.
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Acknowledgments
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We thank Amanda Burns for expert technical assistance.
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Received December 5, 2003;
revised March 19, 2004;
accepted March 31, 2004.