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Cyclooxygenase-2 (COX-2), overexpressed in inflammatory conditions and cancer, regulates angiogenesis and tumorigenesis via the production of biologically active prostanoids. Previously, we showed that COX-2 over-expression in the mammary gland of transgenic mice induces an angiogenic switch and transforms the mammary epithelium into invasive mammary carcinoma. Since COX-2-derived prostanoids can activate the nuclear receptor PPARδ, we crossed Pparδ−/− mice with COX-2 transgenic mice in the FVB/N background. PPARδ was expressed constitutively in the mammary gland of virgin, pregnant and lactating mice. Mammary hyperplasia and tumorigenesis in the COX-2 transgenic mice was markedly reduced in the Pparδ−/− mice compared to their wild type counterparts. Analysis of the mammary tissues indicated that immunoreactive Ki-67, cyclin D1 and phosphorylated histone 3 (Phospho H3) were reduced in Pparδ−/− mice, suggesting that PPARδ activation regulates cell proliferation in the mammary gland. We postulate that activation of the nuclear receptor PPARδ by COX-2-derived prostanoids may be involved in the proliferation of mammary epithelial cells and therefore contribute to mammary cancer development.
Cyclooxygenase (COX)-2 is an inducible enzyme overexpressed in inflammation and cancer. Several lines of evidence support the concept that COX-2 plays a critical role in cancer development and progression [1,2]. Inhibitors of COX-2 and non-steroidal anti-inflammatory drugs which inhibit both COX isoenzymes, suppress cancer development in many animal models [3,4]. Epidemiological studies and clinical trials also indicate that inhibitors of COX-2 reduce cancer incidence and adenoma progression [5,10]. In addition, COX-2 is overexpressed in numerous cancer tissues including breast, colon, prostate, gastric, pancreatic and urinary bladder [2,6–8]. More importantly, exaggerated expression of COX-2 is a negative prognostic factor in breast cancer [9–11]. Genetic studies in which the COX-2 (Ptgs2) gene was overexpressed or deleted in mice resulted in concomitant induction or suppression of neoplasia, respectively [12–14]. These studies strongly suggest that COX-2 expression in the tumor microenvironment is important for the progression of tumors.
We have shown that over-expression of human COX-2 gene in the mammary gland of transgenic mice resulted in the formation of mammary tumors in multiparous mice . Using this mouse model, we have also determined that COX-2 enzyme induces the angiogenic switch and epithelial cell hyperplasia . In previous studies we documented the important role played by the major prostanoid metabolite of COX-2, namely PGE2 in the induction of angiogenic response, and epithelial hyperplasia. Signaling by the cell surface receptor EP2 was shown to be critical for these responses .
The COX-2 enzyme is also known to activate nuclear receptors of the PPAR family. In particular, the PPARδ receptor was shown to be activated prostacyclin and related products. We and others have shown that COX-2 pathway couples to the PPARδ in vitro and in vivo [15–19]. The role of PPARδ in mammary hyperplasia and cancer induced by COX-2 has not been examined. In this report, we assessed the role of the PPARδ in the development of mammary cancer in MMTV-COX-2 transgenic mice. We show that PPARδ activation by COX-2 is an important player in mammary tumorigenesis.
COX-2 transgenic mice was generated as described previously . Pparδ−/− mice in the 129/C57/BL/6 background developed by Peters et al. , was first back-crossed >5 times into the FVB/N background (Taconic Farms, NJ). Pparδ−/− mice obtained in ≥97% FVB/N background were then crossed with the MMTV-COX-2 transgenic mice in FVB/N background to obtain Pparδ−/− COX-2-TG mice.
Mammary glands were dissected and processed for whole mount analysis as described. For histological analysis mammary glands embedded in paraffin were performed as described before . Immnohistochemical staining was conducted with the following antibodies, PPARδ (1:400, Santa Cruz), cyclooxygenase 2 (COX-2) (1:300, Cayman Chemical), phospho H3 (1:1000, Upstate) Ki-67 (1:200, Calbiochem), CyclinD1 (1:1000, Santa Cruz) with a Vectastain ABC kit (Vector) according to the manufacturer’s instructions. The peroxidase staining was visualized with 3,3′-diaminobenzidine (Vector), and the sections were counterstained with methyl green.
In order to observe the expression of PPARδ in Pparδ−/− mice and wild type mice, immunoprecipitated mammary gland extracts were detected with the PPARδ antibody in an immunoblot assay. PPARδK-20 antibody (30 μg; Santa Cruz) was cross-linked with 100 μl of protein A beads. Protein extracts from 12-week-old virgin (5 mg), pregnant and lactating (10 mg) of Pparδ−/− and wild type mice were loaded onto the beads. The PPARδ protein was eluted with 2× sample buffer. Proteins were then loaded into 10% gel and transferred onto nitrocellulose membrane. The membrane was then incubated in 1:1000 PPARδK-20 and 1:10,000 rat anti-goat sequentially and visualized by the ECL reagent (Amersham).
Results are shown as mean ± S.E.M. Statistical significance was calculated by using Student’s t-test.
To establish the role of PPARδ in COX-2 induced mammary tumorigenesis, we obtained Pparδ−/− mice developed by Peters et al.  in the 129/C57BL/6 background and back-crossed to >5 times into the in FVB/N background. The resulting mice were crossed with the MMTV-COX-2 transgenic mice, which is in the FVB/N background. The Pparδ−/− mice in the FVB/N background had small litters, consistent with the role of PPARδ in uterine implantation [19,20].
To verify the expression of PPARδ in different stages of mammary gland development, total protein was extracted from mammary gland of virgin, pregnant and lactating COX-2-TG or COX-2-TG/Pparδ−/− mice and immunoprecipitated using the PPARδ-specific K-20 antibody. Cytosolic extracts of HEK-293T cells transfected with either PPARδ construct was used as positive control and PPARγ construct as negative control. Fig. 1 shows that PPARδ is expressed in virgin (V, 16 weeks), pregnant (P, 18 days pregnant) and lactating (L, 7 days postpartum) COX-2-TG mice but not in the Pparδ−/− mice. The expression was constitutive and did not change during the development and involution of the mammary gland.
We previously showed that multiparous COX-2-TG mice develop mammary hyperplasia and invasive mammary carcinoma . Mammary glands from female COX-2-TG/Pparδ−/− mice or COX-2-TG mice were isolated after three pregnancy/lactation cycles and whole mount preparations were stained and analyzed under a dissecting microscope. Tumor incidence was further confirmed by histological sectioning and H&E staining. As shown in Fig. 2A, marked reduction in tumor incidence was observed in COX-2-TG/Pparδ−/− mice in comparison to the COX-2-TG mice. High tumor incidence was noticed in COX-2-TG mice (10 out of 11, 90%) while it was substantially lower in COX-2-TG/Pparδ−/− mice (1 out of 7, 14%). Estimates of total tumor size per mammary gland was also markedly reduced in COX-2-TG/Pparδ−/− mice, 0.0023 cm3 compared to COX-2-TG mice, 0.28 cm3.
Mammary glands were isolated from multiparous COX-2-TG/Pparδ−/− mice in the weaned stage that have undergone multiple cycles of pregnancy and lactation. Whole mount analysis indicated alveolar development in the COX-2-TG mice whereas COX-2-TG/Pparδ−/− mammary glands showed attenuated lobuloalveolar development. Histological sections indicated that adeno squamous-type, well differentiated mammary tumors were observed in COX-2-TG mice while the occurrence of epithelial hyperplasia and adenocarcinoma were markedly reduced in COX-2-TG/Pparδ−/− mice. These observations suggest a critical role of the PPARδ in mammary tumorigenesis.
The expression of PPARδ, COX-2 and various markers of cell proliferation were examined in mammary tissues isolated from COX-2-TG or COX-2-TG/Pparδ−/− mice. As shown in Fig. 3, PPARδ expression was absent in COX-2-TG/Pparδ−/− mice. As expected, COX-2 was expressed in an equivalent level in both genotypes. Ki-67, a nuclear protein expressed in proliferating cells was used as a standard index of overall cellular proliferation. Because histone 3 is phosphorylated rapidly cycling cells, phospho H3 staining provided a direct indication of mitotic activity. We observed a drastic reduction of Ki-67 and Phospho H3 immunoreactivity in the COX-2-TG/Pparδ−/− mammary tissue sections compared to COX-2-TG mice. Furthermore, expression of CyclinD1, a mammary oncogene was also substantially reduced in the COX-2-TG/Pparδ−/− mammary tissues. These observations suggest that the absence of PPARδ in COX-2-TG mice resulted in reduced cell proliferation of mammary tissues in COX-2-TG mice.
In this report, we show that the PPARδ receptor is critical for mammary tumorigenesis in the COX-2-TG mice. Our data indicate that mammary epithelium in COX-2-TG mice that lack the PPARδ receptor contained less lobuloalveolar differentiation, hyperplasia and tumorigenesis. Markers of mammary epithelial cell differentiation were also down-regulated. These data suggest that PPARδ is a critical mediator in mammary cancer development and is likely to regulate cell proliferation. The expression of PPARδ was observed in mammary epithelial cells as well as stromal cells in the mammary gland. Thus, direct effect on epithelial cells and/or stromal activation, which could indirectly influence epithelial cell proliferation could be involved.
Our data suggest that activation of the PPARδ receptor by prostanoid produced from the COX-2 pathway induces proliferation of mammary epithelial cells and thereby promote hyperplasia and progression of mammary cancer. Previous studies have shown that COX-2 over-expression induces PPARδ activation in colon cancer cells . We recently showed that endocannabinoid metabolism by the vascular endothelial cell COX-2 enzyme results in the activation of the PPARδ receptor selectively . Such a mechanism may be important in mammary tumorigenesis.
Our previous work has shown that COX-2 induced mammary tumor development is also dependent on the presence of the EP2 receptor [6,7]. This receptor is potently activated by PGE2 which is one of the major prostanoids in the mammary gland. EP2 signaling regulated the cAMP-dependent activation of amphiregulin and vascular endothelial cell growth factor, which are potent growth factors for epithelial cells and vascular endothelial cells, respectively. Our results on the PPARδ receptor raise the question about relative importance of EP2 and this nuclear receptor. It is likely that unique as well as overlapping pathways are regulated by both cell surface as well as nuclear receptors for prostanoids and thus contribute to the tumorigenic phenotype.
The role of the PPARδ receptor in tumorigenesis remains unclear [21–24] and somewhat controversial particularly in colon carcinogenesis . Although some studies using the PPARδ knock-out mice and the use of PPARδ agonists have suggested the pro-tumorigenic role, some studies have found that PPARδ receptor knock-out mice have increased tumor development, especially in the intestinal system [16–18,25–30]. The reason for these apparent discrepancies is not well understood [21,31]. It is possible that factors such as genetic background and differences in tumorigenic models contribute to these apparent differences. However, further studies are required to examine the precise role of this pathway in tumor biology. Our results suggest that in the mammary gland, PPARδ receptor has a pro-tumorigenic role.
In summary, we show that PPARδ receptor knock-out mice exhibits an attenuated tumorigenic phenotype induced by COX-2 overexpression. Our data support the pro-tumorigenic role for PPARδ receptor in mammary cancer.