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[Cancer Research 66, 4394-4401, April 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Ligand Activation of Peroxisome Proliferator–Activated Receptor ß Inhibits Colon Carcinogenesis

Holly E. Marin1,2, Marjorie A. Peraza1, Andrew N. Billin3, Timothy M. Willson3, Jerrold M. Ward4, Mary J. Kennett1, Frank J. Gonzalez5 and Jeffrey M. Peters1,2

1 Department of Veterinary and Biomedical Sciences and The Center of Molecular Toxicology and Carcinogenesis, and 2 Graduate Program in Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania; 3 Nuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina; 4 Infectious Disease Pathogenesis Section, Comparative Medicine Branch and SoBran, Inc., National Institute of Allergy and Infectious Diseases; and 5 Laboratory of Metabolism, National Cancer Institute, Bethesda, Maryland

Requests for reprints: Jeffrey M. Peters, Department of Veterinary and Biomedical Sciences and Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, 312 Life Sciences Building, University Park, PA 16802. Phone: 814-863-1387; Fax: 814-863-1696; E-mail: jmp21{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
There is considerable debate whether peroxisome proliferator–activated receptor ß/{delta} (PPARß/{delta}) ligands potentiate or suppress colon carcinogenesis. Whereas administration of a PPARß ligand causes increased small intestinal tumorigenesis in Apcmin/+ mice, PPARß-null (Pparb–/–) mice exhibit increased colon polyp multiplicity in colon cancer bioassays, suggesting that ligand activation of this receptor will inhibit colon carcinogenesis. This hypothesis was examined by treating wild-type (Pparb+/+) and Pparb–/– with azoxymethane, coupled with a highly specific PPARß ligand, GW0742. Ligand activation of PPARß in Pparb+/+ mice caused an increase in the expression of mRNA encoding adipocyte differentiation–related protein, fatty acid–binding protein, and cathepsin E. These findings are indicative of colonocyte differentiation, which was confirmed by immunohistochemical analysis. No PPARß-dependent differences in replicative DNA synthesis or expression of phosphatase and tensin homologue, phosphoinositide-dependent kinase, integrin-linked kinase, or phospho-Akt were detected in ligand-treated mouse colonic epithelial cells although increased apoptosis was found in GW0742-treated Pparb+/+ mice. Consistent with increased colonocyte differentiation and apoptosis, inhibition of colon polyp multiplicity was also found in ligand-treated Pparb+/+ mice, and all of these effects were not found in Pparb–/– mice. In contrast to previous reports suggesting that activation of PPARß potentiates intestinal tumorigenesis, here we show that ligand activation of PPARß attenuates chemically induced colon carcinogenesis and that PPARß-dependent induction of cathepsin E could explain the reported disparity in the literature about the effect of ligand activation of PPARß in the intestine. (Cancer Res 2006; 66(8): 4394-401)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
There are conflicting reports in the literature suggesting that peroxisome proliferator–activated receptor (PPAR)-ß either potentiates or attenuates colon cancer. Overexpression of the adenomatous polyposis coli (APC) gene product in a genetically engineered colorectal HT29-derived tumor cell line leads to a decrease in PPARß mRNA expression (1). The mRNA encoding PPARß has been reported to be increased in colorectal tumors as compared with normal mucosa, consistent with the hypothesis that APC functions to suppress activity of the ß-catenin/Tcf-4 transcription of target genes including PPARß, c-myc, and cyclin D (1, 2). Further, xenografts in nude mice made from PPARß-null HCT116 cells exhibit a decreased ability to form tumors (3). Lastly, treatment of Apc+/– mice with a specific PPARß ligand resulted in an increase in the number and size of small intestinal adenomas but no change in the number of colon tumors (4). Taken together, these studies suggest that the loss of APC expression leads to increased expression of PPARß via the ß-catenin/Tcf-4 transcriptional pathway and potentiation of colon tumorigenesis. In contrast, there are a number of other reports that are inconsistent with this mechanism. For example, normal colonic epithelium and adenomas from Apc+/– mice and human tissue samples showed reduced expression of PPARß in tumors (5). Consistent with these observations, targeted deletion of the APC alleles in mouse intestine results in reduced expression of PPARß mRNA and protein accompanied with the expected increase in the level of mRNA encoding c-myc and accumulation of ß-catenin (6). In the absence of PPARß expression, colon carcinogenesis is exacerbated in both Apc+/– mice and in response to the colon carcinogen, azoxymethane (6, 7). Collectively, these data suggest that reduced expression of PPARß could lead to an increase in colon cancer and that ligand activation of PPARß could inhibit this disease. To definitively test this hypothesis, we examined the effect of ligand activation in two models of intestinal carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
PPARß ligand treatment in Apc+/–Pparb+/+ and Apc+/–Pparb–/– mice. Male Apc+/–Pparb+/+ and Apc+/–Pparb–/– mice on a C57BL/6 genetic background, ages 6 to 8 weeks, were treated with either vehicle control or GW0742 (10 mg/kg) by oral gavage, once per day, five times per week for 6 weeks. At the end of the 6-week experimental period, mice were killed by overexposure to carbon dioxide. The gastrointestinal tract was flushed with saline and lesions were measured and quantified by inspection under a dissecting microscope.

Long-term azoxymethane and PPARß ligand (GW0742) treatment. Male Pparb+/+ and Pparb–/– mice (8), 6 to 8 weeks of age, were injected i.p. with 10 mg azoxymethane/kg body weight, once a week for 10 weeks. A cohort of mice from both genotypes that were treated with azoxymethane were also treated with 2 mg/kg or 10 mg/kg GW0742 by oral gavage thrice per week for 20 weeks or with 5 mg/kg GW0742 five times a week for 22 weeks. At the end of the experiment, mice were euthanized by overexposure to carbon dioxide. The colons were flushed with PBS and lesions were counted and measured by inspection under a dissecting microscope. Representative polyps from each treatment group were fixed in 10% buffered neutral formalin for 24 hours and then placed in 70% ethanol. Fixed tissue was stained with H&E and used for microscopic examination by a pathologist.

Short-term ligand treatments. Male Pparb+/+ or Pparb–/– mice were treated by oral gavage with GW0742 at a concentration of either 5 or 10 mg/kg once per day for 5 days. Two hours before euthanasia by overexposure to carbon dioxide, mice were injected i.p. with bromodeoxyuridine (BrdUrd) at a concentration of 100 mg/kg. Mice were euthanized 8 hours after the last dose of GW0742. The colons were carefully dissected and flushed with saline, cut into 3-mm serial sections, and fixed in 10% buffered neutral formalin. After 24 hours of fixation in formalin, colons were transferred to 70% ethanol and subsequently embedded in paraffin and cut into 3- to 4-µm sections for histologic analyses. To determine whether PPAR{alpha} or PPAR{gamma} transcriptional activity is altered in the absence of PPARß expression, Pparb+/+ and Pparb–/– mice were treated with either 5 mg/kg GW0742 or 100 mg/kg troglitazone, once per day for 5 days, or with 0.1% Wy-14,643 in the diet for 5 days. Mice were euthanized by overexposure to carbon dioxide 8 hours after the last dose of GW0742, Wy-14,643, or troglitazone, and colons were removed and flushed with PBS. Epithelial cells, isolated by scraping the epithelium with a razor blade, were immediately homogenized in Trizol reagent and subsequently frozen until RNA was isolated. To determine the effect of ligand activation on phosphatase and tensin homologue/phosphoinositide-dependent kinase/integrin-linked kinase/phospho-Akt protein expression, Pparb+/+ or Pparb–/– mice were treated by oral gavage with GW0742 at a concentration of 10 mg/kg once per day for 5 days, and Apc+/–Pparb+/+ or Apc+/–Pparb–/– were treated by oral gavage with GW0742 at a concentration of 10 mg/kg for 6 weeks as described above. Mice were euthanized by overexposure to carbon dioxide 8 hours after the last dose of GW0742 and colons were removed and flushed with PBS. Epithelial cells, isolated as above, were homogenized in ice-cold lysis buffer and cytosolic fractions were obtained by differential centrifugation and protein concentration determined using a bicinchoninic acid detection kit (Pierce, Rockford, IL).

RNA analysis. Total RNA was isolated from colon epithelial samples as previously described (7). The mRNAs encoding adipocyte differentiation–related protein (ADRP), fatty acid–binding protein (FABP), cathepsin E, keratin 20, Kruppel-like factor 4 (KLF4), PPAR{alpha}, PPARß, and PPAR{gamma} were quantified using real-time PCR analysis. The cDNA was generated using 2.5 µg total RNA with MultiScribe Reverse Transcriptase kit (Applied Biosystems, Foster City, CA). Primers were designed for real-time PCR using the Primer Express software (Applied Biosystems). The sequence and GenBank accession numbers for the forward and reverse primers used to quantify mRNAs were FABP (NM_017399): forward, 5'-CCATGAACTTCTCCGGCAAGT-3', and reverse, 5'-TCCTTCCCTTTCTGGATGAGGT-3'; ADRP (NM_007408): forward, 5'-CACAAATTGCGGTTGCCAAT-3', and reverse, 5'-ACTGGCAACAATCTCGGACGT-3'; PPARß (NM_011145): forward, 5'-TTGAGCCCAAGTTCGAGTTTGCTG-3', and reverse, 5'-ATTCTAGAGCCCGCAGAATGGTGT-3'; PPAR{alpha} (NM011144): forward, 5'-CGATGCTGTCCTCCTTGATGA-3', and reverse, 5'-CATTGCCGTACGCGATCAG-3'; PPAR{gamma} (NM_011146): forward, 5'-GCTGGCCTCCCTGATGAATAA-3', and reverse, 5'-TCCCTGGTCATGAATCCTTGG-3'; cathepsin E (NM_007799): forward, 5'-TCGACACGATGCCAAACGT-3', and reverse, 5'-CAGCTGGAGGTGGAATGTCAA-3'; keratin 20 (NM_023256): forward, 5'-ATGAAGTCCTGGCCCAGAAGA-3', and reverse, 5'-TTTCAGCTCCTCCGTGTTCACT-3'; and KLF4 (NM_010637): forward, 5'-CCAGACCAGATGCAGTCACAA-3', and reverse, 5'-ACGACCTTCTTCCCCTCTTTG-3'. All mRNAs examined were normalized to the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH; BC083149) using the following primers: forward, 5'-GGTGGAGCCAAAAGGGTCAT-3', and reverse, 5'-GGTTCACACCCATCACAAACAT-3'. Real-time PCR reactions were carried out using SYBR green PCR master mix (Finnzymes, Espoo, Finland) in the PTC-200 DNA Engine Cycler and detected using the CFD-3200 Opticon Detector (MJ Research, Waltham, MA). The following conditions were used for PCR: 95°C for 15 seconds, 94°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and repeated for 45 cycles. The PCR included a no-template control reaction to control for contamination and/or genomic amplification. All reactions had >90% efficiency. Relative expression levels of mRNA were normalized to GAPDH and analyzed for statistical significance using one-way ANOVA (Prism 4.0).

Assessment of cell proliferation, apoptosis, and differentiation. Detection of BrdUrd-labeled cells was done using immunohistochemical methods. Sections were deparaffinized, rehydrated, and endogenous peroxidase was blocked with 3% H2O2 in methanol. Slides were incubated at 37°C for 30 minutes in a 0.08% trypsin solution for antigen retrieval, denatured by incubation in 2N HCl at 37°C for 30 minutes, and neutralized by incubation in 0.1 mol/L Borax for 10 minutes. Sections were blocked with 20% mouse serum for 30 minutes at room temperature and subsequently blocked with M.O.M. blocking reagent (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. Primary mouse monoclonal BrdUrd antibody (Vector Laboratories) was applied to the sections at a 1:200 dilution and incubated at room temperature for 30 minutes. Secondary biotinylated antimouse immunoglobulin G (IgG; Vector Laboratories) was then applied and slides were incubated for an additional 10 minutes at room temperature. Avidin-biotin horseradish peroxidase (ABC kit, Vector Laboratories) was then applied for 5 minutes and diaminobenzidine tetrahydrochloride (DAB) was subsequently applied for detection of positively labeled cells. The sections were counterstained with hematoxylin and visualized under a light microscope. BrdUrd-labeled colonocytes were quantified using light microscopy and labeling indices were quantified as a percentage of labeled cells per total cell number in representative crypts counted. Apoptotic cells were determined in colon sections from long-term and short-term experiments described above using a FragEL DNA Fragmentation Detection Kit (Oncogene Research Products, Boston, MA). Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL)–positive cells were quantified using light microscopy and data are presented as a percentage of average labeled cells per total cell number from representative crypts counted. Relative differentiation of colonocytes was determined using a previously established procedure (9, 10) with colon sections from long-term and short-term experiments that were fixed and sectioned as described above. Sections were deparaffinized, rehydrated, and endogenous peroxidase was blocked with 3% H2O2 in methanol. Sections were then blocked with 20 µg/mL bovine serum albumin and incubated with biotinylated Dolichos biflorus agglutinin (DBA; Vector Laboratories) at a concentration of 10 µg/mL. The lectin DBA was washed with TBS and the sections were then incubated with an avidin-biotin peroxidase complex. Immunodetection was done after treating with DAB and counterstaining with hematoxylin. Staining specificity was determined by adding 0.2 mol/L N-acetylgalactosamine to 10 µg/mL DBA for 30 minutes before the blocking step to inhibit DBA lectin binding. Scoring of the DBA binding was determined as previously described by others (10). Detection of cathepsin E in the colon was done using immunohistochemical methods. Sections were deparaffinized, rehydrated, and endogenous peroxidase was quenched with 1% H2O2 in methanol. Following antigen retrieval, the cathepsin E antibody (R&D Systems, Minneapolis, MN) was applied to the sections and incubated at room temperature for 1 hour. After washing, the sections were then incubated for 30 minutes with biotin-SP-conjugated AffiniPure donkey anti-goat IgG secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Slides were then incubated in a commercial avidin-biotin peroxidase complex (ABC, Vector Laboratories) and immunodetection was achieved by incubation with DAB (Vector Laboratories). The sections were counterstained with hematoxylin and subsequently visualized under a light microscope.

Quantitative Western blot analysis. Cytosolic fractions from colonic epithelium were obtained from control and ligand-treated mice as described above. Cytosol protein (25-50 µg) from each sample of colonic epithelium was resolved using SDS-PAGE. The samples were transferred onto a nitrocellulose membrane using an electroblotting method. After blocking in 5% milk in TBS-Tween 20, the membrane was incubated overnight at 4 °C with primary antibody, followed by incubation with a biotinylated secondary antibody (Jackson ImmunoResearch Laboratories). Immunoreactive proteins were detected after incubation in [125I]-labeled streptavidin (Amersham Biosciences, Piscataway, NJ) using phosphorimaging analysis. Hybridization signals for specific proteins of interest were normalized to the hybridization signal of the housekeeping protein, lactate dehydrogenase (LDH). A minimum of three to four samples from individual mice were used for analysis of each treatment group. The following primary antibodies were used: anti–phosphatase and tensin homologue (Upstate Biotechnology, Lake Placid, NY), anti–phosphoinositide-dependent kinase 1 (BD Transduction Laboratories, San Jose, CA), anti–integrin-linked kinase (Upstate Biotechnology), anti–phospho-Akt (S473; Cell Signaling Technology, Inc., Beverly, MA), anti-Akt (BD Transduction Laboratories), and anti-LDH (Rockland, Inc., Gilbertsville, PA).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
GW0742 is a highly specific PPARß-specific ligand that is very similar in structure and has very similar binding and agonist activity toward PPARß as compared with GW501516 (11). Daily administration of GW0742 had no effect on intestinal tumorigenesis in Apc+/– mice (Table 1 ; Fig. 1A ). In contrast, the average number and size of small intestinal polyps were significantly greater in Apc+/– that also lacked expression of PPARß (Table 1; Fig. 1A). Interestingly, GW0742 treatment did not potentiate intestinal tumorigenesis in Apc+/–Pparb–/– mice but was comparable to that found in control and ligand-treated Apc+/– Pparb+/+ mice (Table 1; Fig. 1A). Whereas these results suggest that ligand activation of PPARß does not potentiate intestinal tumorigenesis in Apc+/– mice, there are a number of limitations to this analysis: (a) these studies were done for only a short duration of ligand treatment (6 weeks); (b) there was significant variation within each experimental group (similar to numerous past studies); (c) the age of mice at the time of examination was 15 ± 2 weeks of age (to allow for comparison with previous studies); and (d) small intestine tumors are more commonly found in the Apc+/– mouse as compared with colon tumors. To examine the effects of PPARß activation in a more specific model of colon carcinogenesis, Pparb+/+ and Pparb–/– mice were treated once per week for 10 weeks with azoxymethane as previously described (7) and also treated with GW0742 (either 2 or 10 mg/kg GW0742, thrice per week) or vehicle control. Decreases of 23% and 51% in colon polyp multiplicity were observed in Pparb+/+ mice treated with 2 and 10 mg/kg GW0742, respectively, compared with control wild-type mice (Fig. 1B), but these differences were only statistically significant at the higher dose. To determine if a more sustained administration of GW0742 could more effectively inhibit colon polyp multiplicity, a similar experiment was done, except the mice were administered GW0742 five times per week at a dose of 5 mg/kg for the duration of the experiment. However, only an 18% decrease in colon polyp multiplicity was observed in Pparb+/+ mice treated with GW0742 five times per week as compared with control, and this was not significantly different than controls (Fig. 1B). Colon polyp multiplicity was significantly greater in all groups of Pparb–/– mice and GW0742 had no effect in these mice (Fig. 1B). The Pparb–/– mice also had a greater percentage of smaller lesions as compared with wild-type mice (Table 1). Histologic examination revealed no differences in colon polyps of either treatment group, independent of genotype, and all lesions observed were benign adenomas (Fig. 1C).


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  		Table 1. Role of PPARß in the size distribution of intestinal polyps in genetic and chemical carcinogenesis models

 

Figure 1
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  		Figure 1. The effect of ligand activation of PPARß on intestinal tumorigenesis. A, effect of administration of GW0742 (10 mg/kg, 5x/wk, for 6 weeks) on colon and small intestinal polyp multiplicity in Apc+/–Pparb+/+ (Apc+/minPparb+/+) and Apc+/–Pparb–/– mice. B, effect of administration of GW0742 (2 mg/kg, 3x/wk, for 20 weeks; 10 mg/kg, 3x/wk, for 20 weeks; or 5 mg/kg, 5x/wk, for 22 weeks) on colon polyp multiplicity in Pparb+/+ mice (+/+) and Pparb–/– mice (–/–). C, representative sections of colon from Pparb+/+ mice (+/+) and Pparb–/– mice (–/–) were fixed in 10% phosphate-buffered formaldehyde and analyzed histologically for microscopic tumor structure. Representative colon polyps stained with H&E. Values with different letters are significantly different at P ≤ 0.05 (ANOVA).

 
Ligand treatment resulted in a marked increase in the mRNA encoding ADRP, FABP, and cathepsin E in wild-type mouse colon and this did not occur in similarly treated Pparb–/– mouse colon (Fig. 2A ). These results show that under the conditions used, ligand treatment caused specific target gene activation by PPARß only in the Pparb+/+ mice. This establishes the efficacy of the dosing regimen and further suggests that the induction of differentiation is one mechanism that could explain how GW0742 inhibits chemically induced colon carcinogenesis because expression of ADRP, FABP, and cathepsin E is associated with terminal differentiation of epithelial tissues including colon epithelium (12–16). To examine this hypothesis, a previously described method (9, 10) was used to quantify relative differentiation of colonic epithelium in response to ligand activation of PPARß. Indeed, GW0742 administration at a dose that specifically activated PPARß (Fig. 2A) caused a significant increase in the level of Dolichos biflorus agglutinin (DBA)–positive cells in Pparb+/+ mouse colon as compared with controls, and this effect was not observed in similarly treated Pparb–/– colons (Figs. 2B and C). Additionally, administration of GW0742 increased cathepsin E protein expression in Pparb+/+ mouse colon as compared with controls, and this effect was not observed in similarly treated Pparb–/– colons (Fig. 2D).


Figure 2
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  		Figure 2. The effect of ligand activation of PPARß on colonocyte differentiation. A, effect of administration of GW0742 (5 mg/kg, 1x day, for 5 days) on mRNA encoding ADRP, FABP, and cathepsin E in colonic epithelium of Pparb+/+ mice and Pparb–/– mice. Columns, mean fold change as compared with respective control; bars, SE. B, effect of administration of GW0742 (5 mg/kg, 1x/d or 10 mg/kg, 1x/d for 5 days) on colonocyte differentiation in Pparb+/+ mice and Pparb–/– mice based on the DBA lectin score. C, representative sections of colon epithelium showing increased DBA lectin–positive cells in Pparb+/+ mice by GW0742 that is not found in similarly treated Pparb–/– mice. D, representative sections of colon epithelium showing increased cathepsin E immunoreactivity in Pparb+/+ mice by GW0742 that is not found in similarly treated Pparb–/– mice. Values with different letters are significantly different at P ≤ 0.05 (ANOVA).

 
Administration of PPAR{gamma} ligands is also known to induce markers of colonic differentiation including mRNA encoding FABP, keratin 20, and KLF4 (12). To determine if PPARß-mediated differentiation of colonocytes is similar to that induced by PPAR{alpha} or PPAR{gamma} ligands, expression of these mRNA markers was examined in Pparb+/+ and Pparb–/– mice treated with either GW0742, Wy-14,643, or troglitazone. GW0742 caused a significant PPARß-dependent increase in mRNA encoding ADRP and FABP but no changes in keratin 20 or KLF4 (Fig. 3A ). Expression of these mRNAs in colon in response to Wy-14,643 was different as compared with changes observed after GW0742 treatment. Increased mRNA encoding FABP was observed in both Pparb+/+ and Pparb–/– mice, and this increase was significantly greater in Wy-14,643-fed Pparb–/– mice as compared with Pparb+/+ mice (Fig. 3A). No increase in the expression level of mRNA encoding keratin 20 was found in either genotype fed Wy-14,643, and the expression of mRNA encoding ADRP and KLF4 was unchanged by Wy-14,643 in Pparb+/+ mice but increased in Pparb–/– mice fed Wy-14,643 (Fig. 3A). In contrast, troglitazone treatment effectively induced all four markers in both genotypes and no differences were found between genotype (Figs. 3A).


Figure 3
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  		Figure 3. Expression of mRNAs in mouse colonic epithelium. A, effect of ligand activation of PPAR{gamma} with troglitazone (100 mg/kg, 1x/d for 5 days), PPAR{alpha} with Wy-14,643 (100 mg/kg, for 5 days), or PPARß with GW0742 (5 mg/kg, 1x/d for 5 days) on mRNA encoding ADRP, FABP, keratin 20 (K20), or KLF4 in Pparb+/+ and Pparb–/– mouse colonic epithelium. B, effect of ligand activation of PPARß with GW0742 (10 mg/kg, 5x/wk, for 6 weeks) on mRNA encoding FABP in Apc+/–Pparb+/+ and Apc+/–Pparb–/– mouse colonic epithelium. C, expression of mRNA encoding PPARß, PPAR{alpha}, and PPAR{gamma} in colon and colon polyps from control (con) and azoxymethane (AOM)-treated Pparb+/+ and Pparb–/– mice and Apc+/–Pparb+/+ mice. Columns, mean fold change as compared with respective control; bars, SE. Values with different letters are significantly different at P ≤ 0.05 (ANOVA).

 
The mRNA encoding FABP was measured to determine whether ligand activation of PPARß resulted in differentiation in Apc+/– mouse colonic epithelium. Results from this analysis show that the mRNA encoding FABP was induced in Apc+/–Pparb+/+ colonic epithelium from mice treated with GW0742 but not in colonic epithelium from similarly treated Apc+/–Pparb–/– (Fig. 3B).

Because there are conflicting reports describing PPARß expression during colon carcinogenesis, expression of mRNA encoding PPARß was measured in colon and colon polyps from both the genetic and chemically induced cancer models. Expression of mRNA encoding PPARß in colonic epithelium was not different between Pparb+/+ and Apc+/– azoxymethane-treated mice as compared with Pparb+/+ control mice (Fig. 3C). In contrast, expression of mRNA encoding PPARß was significantly lower in polyps from both Pparb+/+ and Apc+/– azoxymethane-treated mice as compared with normal colonic epithelium (Fig. 3C), consistent with previous studies (5). Expression of mRNA encoding PPAR{alpha} and PPAR{gamma} in colonic epithelium was similar between genotypes although PPAR{alpha} mRNA was significantly higher in Pparb–/– mice as compared with both Pparb+/+ and Apc+/– mice (Fig. 3C). Expression of mRNA encoding PPAR{alpha} and PPAR{gamma} was significantly lower in polyps from both Pparb+/+ and Apc+/– azoxymethane-treated mice as compared with normal colonic epithelium (Fig. 3C), consistent with previous studies (17–20).

Induction of differentiation can be associated with increased apoptosis and inhibition of cell proliferation. Consistent with this, the average number of TUNEL-positive cells in the colon was significantly greater in GW0742-treated Pparb+/+ mice as compared with GW0742-treated Pparb–/– mice (Fig. 4A ). In skin epithelium, there is evidence that ligand activation of PPARß leads to repression of phosphatase and tensin homologue expression with concomitant increased expression of oncogenic phosphoinositide-dependent kinase 1 and integrin-linked kinase, which leads to phosphorylation of Akt and inhibition of apoptosis (21). Examination of this pathway in colonic epithelium revealed that despite specific activation of the ADRP and FABP genes by PPARß (Figs. 2A and 3A), no changes in phosphatase and tensin homologue, phosphoinositide-dependent kinase, integrin-linked kinase, or phosphorylated Akt were detected between genotypes or in response to GW0742 (Fig. 4B). This shows that the pathway previously described in keratinocytes does not function in colonocytes in vivo. Surprisingly, no statistically significant differences in relative cell proliferation were detected in colonic epithelium after treatment with 5 mg/kg GW0742 (Fig. 4C) but a significant decrease in BrdUrd labeling index was observed after 10 mg/kg GW0742 in both genotypes (Fig. 4C).


Figure 4
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  		Figure 4. Effect of ligand activation of PPARß on cell proliferation in colon. A, effect of administration of GW0742 (10 mg/kg, 3x/wk for 22 weeks) on TUNEL-positive cells in Pparb+/+ and Pparb–/– mice. B, effect of administration of GW0742 (10 mg/kg, 5x/wk, for 1 week) in Pparb+/+ and Pparb–/– mice or GW0742 (10 mg/kg, 5x/wk, for 6 weeks) in Apc+/–Pparb+/+ or Apc+/–Pparb–/– mice on the expression of phosphatase and tensin homologue (PTEN), phosphoinositide-dependent kinase 1 (PDK1), integrin-linked kinase (ILK), phospho-Akt (p-Akt), or Akt in colonic epithelium. Representative Western blots of three to four independent samples from individual mice. Quantified normalized hybridization signals are presented as fold change from control mice. C, effect of GW0742 (5 mg/kg, 5x/wk or 10 mg/kg, 5x/wk for 20 or 22 weeks, respectively) on average relative BrdUrd (BrdU) labeling index of colon crypts in Pparb+/+ and Pparb–/– mice. Values with different letters are significantly different at P ≤ 0.05 (ANOVA).

 
Results from these studies are the first to conclusively show that ligand activation of PPARß inhibits chemically induced colon carcinogenesis in vivo, and that this effect is likely due in part to PPARß-dependent induction of colonocyte differentiation and enhanced apoptosis. These findings are consistent with other reports in other model systems, showing that ligand activation of PPARß induces differentiation and apoptosis in keratinocytes (8, 14, 22–26) and that PPARß expression is associated with differentiation of colon and breast cancer cell lines (27). Indeed, induction of differentiation and apoptosis is typically associated with compounds known to inhibit tumorigenesis. In contrast to results from passaged keratinocytes (21), no changes in the phosphatase and tensin homologue/phosphoinositide-dependent kinase/integrin-linked kinase/phospho-Akt pathway were detected in colonic epithelium after ligand activation of PPARß. The PPARß-dependent induction of cathepsin E represents a novel finding because increased expression of this proteinase is associated with the induction of differentiation of M cells in colonic epithelium, which function to process antigens (28). However, certain pathogens take advantage of this processing (28) and thus increased infection and immune-related responses induced by some bacteria or viruses could be increased by enhanced M-cell differentiation. Thus, it remains a possibility that the increased small intestine tumorigenesis observed by others in response to PPARß ligand administration (4) is secondary to opportunistic pathogenic infections resulting from increased M-cell differentiation. Additionally, similar to PPARß, the role of cathepsin E in carcinogenesis is controversial. For example, increased expression of cathepsin E is found in gastric carcinomas and in cervical intraepithelial neoplasia (29, 30), and there are case reports describing colon adenocarcinomas in association with lymphoid tissue (31–33), which is juxtaposed to M cells that express cathepsin E. However, increased expression of cathepsin E is also associated with an antimetastatic response (34) and decreased expression of cathepsin E correlates well with decreased differentiation, increased cell proliferation, and increased severity of dysplasia in gastric tumors (35, 36). Thus, induction of cathepsin E may be central to some conflicting reports about ligand activation of PPARß during colon carcinogenesis.

The present studies show that administration of the potent PPARß ligand had no effect on colon or small intestinal tumorigenesis in either Apc+/–Pparb–/– or Apc+/–Pparb+/+ mice as compared with controls. However, the average number and size of small intestinal tumors were greater in Apc+/–Pparb–/– mice as compared with Apc+/–Pparb+/+ mice, consistent with past results (6, 7). Surprisingly, the average number and size of intestinal tumors in Apc+/–Pparb–/– mice treated with GW0742 were not statistically different from control Apc+/–Pparb–/– mice or either group of Apc+/–Pparb+/+ mice. It is especially noteworthy that we used a dosing regimen that was essentially identical to one used by others with a very similar PPARß ligand in which increases in the number and size of small intestinal polyps were shown (4). Whereas it cannot be excluded that the difference in polyp number between the two experiments could theoretically be secondary to opportunistic pathogenic infections as described above, it should be noted that different PPARß ligands were used in these studies. In addition, there is the potential for variability in polyp numbers in different colonies of Apc+/– mice. Thus, either there are large differences in target genes activated by the respective PPARß ligand used (e.g., GW0742 versus GW501516) or the variation in polyp numbers found in the Apc+/– mice limits the usefulness of this model to examine the effect of ligand activation. Support for the latter hypothesis is provided from two previous reports from the same group. For example, control Apc+/–Pparb+/+ mice exhibit almost twice as many intestinal polyps between two different experiments (30 ± 2 versus ~50 ± 6; refs. 4 and 37, respectively) and ligand treatment resulted in almost twice as many intestinal polyps as controls (30 ± 2 versus 56 ± 7; ref. 4). Importantly, intestinal tumorigenesis was not potentiated by ligand activation of PPARß in either Apc+/– or Pparb+/+ mice treated with azoxymethane in the present studies, despite specific activation of the PPARß target genes FABP and ADRP in mouse colonic epithelium. This suggests that short-term ligand activation of PPARß is ineffective at inhibiting intestinal tumorigenesis as observed with the chemically induced model, and that more prolonged treatment with ligands could be of benefit. Alternatively, it remains a possibility that ligand activation of PPARß will have no effect in Apc+/– mice, similar to what was found to occur with PPAR{gamma} (38).

The observation that PPARß expression is reduced in both the Apc+/– mouse colon polyps and azoxymethane-treated mouse polyps is in agreement with previous studies (5, 6) but inconsistent with others (1, 2). The reason for this difference is uncertain. However, decreased expression of PPARß is more consistent with the hypothesis that PPARß attenuates colon carcinogenesis as shown from the present study. Whereas induction of the colonocyte differentiation marker FABP occurs in response to ligand activation of PPARß, as shown from the present studies, this also occurs in a PPAR{gamma}-dependent mechanism in colonocytes (12). However, induction of mRNA markers of differentiation by ligand activation of PPARß differs from that associated with PPAR{gamma} and PPAR{alpha} ligands because keratin 20 and KLF4 are not induced by the PPARß ligand GW0742 or the PPAR{alpha} ligand Wy-14,643 but are induced by the PPAR{gamma} ligand troglitazone. Additionally, whereas troglitazone and GW0742 induce expression of mRNA encoding ADRP, no change was observed in response to Wy-14,643. This clearly shows that modulation of target genes is markedly different depending on the PPAR ligand used. Interestingly, administration of Wy-14,643 caused an increase in the expression of mRNA encoding ADRP, FABP, and KLF4 in Pparb–/– mice, which is consistent with the relatively higher level of PPAR{alpha} expression found in the colon of Pparb–/– mice. This observation is similar to in vitro findings (39) but inconsistent with previous studies showing that ligand activation of PPAR{alpha} in the liver of PPARß mice does not lead to enhanced transcriptional activity of PPAR{alpha} in the absence of PPARß expression (40). This may be related to the fact that PPARß expression is considerably higher in colon as compared with liver. In contrast, administration of troglitazone resulted in no significant changes in gene expression in colon between Pparb+/+ and Pparb–/– mice. This is consistent with previous work showing that ligand activation of PPAR{gamma} in white adipose cells of PPARß mice does not lead to enhanced transcriptional activity of PPAR{gamma} in the absence of PPARß expression (41) as suggested by others (39). Combined, these findings do not support the hypothesis that PPARß can inhibit PPAR{gamma} transcriptional activity in colon in vivo but suggest that PPARß may inhibit PPAR{alpha} transcriptional activity in colon in vivo.

In summary, the present studies clearly show that specific ligand activation of PPARß leads to the induction of target gene expression associated with terminal differentiation of colonocytes. No evidence that PPARß ligands potentiate intestinal tumorigenesis was observed in either a genetically predisposed or chemically induced model, and the inclusion of PPARß-null mice clearly shows specificity.


   Acknowledgments
 
Grant support: NIH grants CA97999 and CA89607 (J.M. Peters).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Amanda Burns for providing technical support.


   Footnotes
 
Note: This project was done, in part, using compounds provided by the National Cancer Institute Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute, no. N02-CB-07008.

Received 12/ 1/05. Revised 1/26/06. Accepted 2/ 7/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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