Ligand Activation of Peroxisome Proliferator–Activated Receptor B Inhibits Colon Carcinogenesis Holly E

Research Article

Ligand Activation of Peroxisome Proliferator–Activated Receptor B Inhibits Colon Carcinogenesis Holly E. Marin,1,2 Marjorie A. Peraza,1 Andrew N. Billin,3 Timothy M. Willson,3 Jerrold M. Ward,4 Mary J. Kennett,1 Frank J. Gonzalez,5 and Jeffrey M. Peters1,2

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

Abstract colorectal HT29-derived tumor cell line leads to a decrease in h h There is considerable debate whether peroxisome proliferator– PPAR mRNA expression (1). The mRNA encoding PPAR has been reported to be increased in colorectal tumors as compared activated receptor B/D (PPARB/D) ligands potentiate or suppress colon carcinogenesis. Whereas administration of a with normal mucosa, consistent with the hypothesis that APC B functions to suppress activity of the h-catenin/Tcf-4 transcription PPAR ligand causes increased small intestinal tumorigenesis h Apcmin/+ B PparbÀ/À of target genes including PPAR , c-myc, and cyclin D (1, 2). Further, in mice, PPAR -null ( ) mice exhibit in- h creased colon polyp multiplicity in colon cancer bioassays, xenografts in nude mice made from PPAR -null HCT116 cells suggesting that ligand activation of this receptor will inhibit exhibit a decreased ability to form tumors (3). Lastly, treatment of Apc+/À h colon carcinogenesis. This hypothesis was examined by treating mice with a specific PPAR ligand resulted in an increase in À À wild-type (Pparb+/+) and Pparb / with azoxymethane, cou- the number and size of small intestinal adenomas but no change in pled with a highly specific PPARB ligand, GW0742. Ligand the number of colon tumors (4). Taken together, these studies B Pparb+/+ suggest that the loss of APC expression leads to increased activation of PPAR in mice caused an increase in the h h expression of mRNA encoding adipocyte differentiation– expression of PPAR via the -catenin/Tcf-4 transcriptional related protein, fatty acid–binding protein, and cathepsin E. pathway and potentiation of colon tumorigenesis. In contrast, These findings are indicative of colonocyte differentiation, there are a number of other reports that are inconsistent with this mechanism. For example, normal colonic epithelium and adeno- which was confirmed by immunohistochemical analysis. No +/À B mas from Apc mice and human tissue samples showed reduced PPAR -dependent differences in replicative DNA synthesis or h expression of phosphatase and tensin homologue, phosphoi- expression of PPAR in tumors (5). Consistent with these observations, targeted deletion of the APC alleles in mouse nositide-dependent kinase, integrin-linked kinase, or phospho- h Akt were detected in ligand-treated mouse colonic epithelial intestine results in reduced expression of PPAR mRNA and protein accompanied with the expected increase in the level of cells although increased apoptosis was found in GW0742- h Pparb+/+ mRNA encoding c-myc and accumulation of -catenin (6). In the treated mice. Consistent with increased colonocyte h differentiation and apoptosis, inhibition of colon polyp absence of PPAR expression, colon carcinogenesis is exacerbated Apc+/À multiplicity was also found in ligand-treated Pparb+/+ mice, in both mice and in response to the colon carcinogen, À À and all of these effects were not found in Pparb / mice. In azoxymethane (6, 7). Collectively, these data suggest that reduced expression of PPARh could lead to an increase in colon cancer and contrast to previous reports suggesting that activation of h PPARB potentiates intestinal tumorigenesis, here we show that that ligand activation of PPAR could inhibit this disease. To ligand activation of PPARB attenuates chemically induced definitively test this hypothesis, we examined the effect of ligand activation in two models of intestinal carcinogenesis. colon carcinogenesis and that PPARB-dependent induction of cathepsin E could explain the reported disparity in the B literature about the effect of ligand activation of PPAR in Materials and Methods the intestine. (Cancer Res 2006; 66(8): 4394-401) À À À À PPARB ligand treatment in Apc+/ Pparb+/+ and Apc+/ Pparb / À À À À mice. Male Apc+/ Pparb+/+ and Apc+/ Pparb / mice on a C57BL/6 Introduction genetic background, ages 6 to 8 weeks, were treated with either vehicle There are conflicting reports in the literature suggesting that control or GW0742 (10 mg/kg) by oral gavage, once per day, five times per peroxisome proliferator–activated receptor (PPAR)-h either poten- week for 6 weeks. At the end of the 6-week experimental period, mice were tiates or attenuates colon cancer. Overexpression of the adenoma- killed by overexposure to carbon dioxide. The gastrointestinal tract was tous polyposis coli (APC) gene product in a genetically engineered flushed with saline and lesions were measured and quantified by inspection under a dissecting microscope. Long-term azoxymethane and PPARB ligand (GW0742) treatment. À À Male Pparb+/+ and Pparb / mice (8), 6 to 8 weeks of age, were injected i.p. Note: This project was done, in part, using compounds provided by the National with 10 mg azoxymethane/kg body weight, once a week for 10 weeks. A Cancer Institute Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute, no. N02-CB-07008. cohort of mice from both genotypes that were treated with azoxymethane Requests for reprints: Jeffrey M. Peters, Department of Veterinary and Biomedical were also treated with 2 mg/kg or 10 mg/kg GW0742 by oral gavage thrice Sciences and Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania per week for 20 weeks or with 5 mg/kg GW0742 five times a week for 22 State University, 312 Life Sciences Building, University Park, PA 16802. Phone: 814-863- weeks. At the end of the experiment, mice were euthanized by overexposure 1387; Fax: 814-863-1696; E-mail: [email protected]. I2006 American Association for Cancer Research. to carbon dioxide. The colons were flushed with PBS and lesions were doi:10.1158/0008-5472.CAN-05-4277 counted and measured by inspection under a dissecting microscope.

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Representative polyps from each treatment group were fixed in 10% efficiency. Relative expression levels of mRNA were normalized to GAPDH buffered neutral formalin for 24 hours and then placed in 70% ethanol. and analyzed for statistical significance using one-way ANOVA (Prism 4.0). Fixed tissue was stained with H&E and used for microscopic examination Assessment of cell proliferation, apoptosis, and differentiation. by a pathologist. Detection of BrdUrd-labeled cells was done using immunohistochemical À À Short-term ligand treatments. Male Pparb+/+ or Pparb / mice were methods. Sections were deparaffinized, rehydrated, and endogenous treated by oral gavage with GW0742 at a concentration of either 5 or 10 mg/kg peroxidase was blocked with 3% H2O2 in methanol. Slides were incubated once per day for 5 days. Two hours before euthanasia by overexposure to at 37jC for 30 minutes in a 0.08% trypsin solution for antigen retrieval, carbon dioxide, mice were injected i.p. with bromodeoxyuridine (BrdUrd) at denatured by incubation in 2N HCl at 37jC for 30 minutes, and neutralized a concentration of 100 mg/kg. Mice were euthanized 8 hours after the last by incubation in 0.1 mol/L Borax for 10 minutes. Sections were blocked with dose of GW0742. The colons were carefully dissected and flushed with 20% mouse serum for 30 minutes at room temperature and subsequently saline, cut into 3-mm serial sections, and fixed in 10% buffered neutral blocked with M.O.M. blocking reagent (Vector Laboratories, Burlingame, CA) formalin. After 24 hours of fixation in formalin, colons were transferred to for 1 hour at room temperature. Primary mouse monoclonal BrdUrd 70% ethanol and subsequently embedded in paraffin and cut into 3- to 4-Am antibody (Vector Laboratories) was applied to the sections at a 1:200 dilution sections for histologic analyses. To determine whether PPARa or PPARg and incubated at room temperature for 30 minutes. Secondary biotinylated transcriptional activity is altered in the absence of PPARh expression, antimouse immunoglobulin G (IgG; Vector Laboratories) was then applied À À Pparb+/+ and Pparb / mice were treated with either 5 mg/kg GW0742 or and slides were incubated for an additional 10 minutes at room temperature. 100 mg/kg troglitazone, once per day for 5 days, or with 0.1% Wy-14,643 in Avidin-biotin horseradish peroxidase (ABC kit, Vector Laboratories) was the diet for 5 days. Mice were euthanized by overexposure to carbon dioxide then applied for 5 minutes and diaminobenzidine tetrahydrochloride (DAB) 8 hours after the last dose of GW0742, Wy-14,643, or troglitazone, and was subsequently applied for detection of positively labeled cells. The colons were removed and flushed with PBS. Epithelial cells, isolated by sections were counterstained with hematoxylin and visualized under a light scraping the epithelium with a razor blade, were immediately homogenized microscope. BrdUrd-labeled colonocytes were quantified using light in Trizol reagent and subsequently frozen until RNA was isolated. To microscopy and labeling indices were quantified as a percentage of labeled determine the effect of ligand activation on phosphatase and tensin cells per total cell number in representative crypts counted. Apoptotic cells homologue/phosphoinositide-dependent kinase/integrin-linked kinase/ were determined in colon sections from long-term and short-term experi- À À phospho-Akt protein expression, Pparb+/+ or Pparb / mice were treated ments described above using a FragEL DNA Fragmentation Detection Kit by oral gavage with GW0742 at a concentration of 10 mg/kg once per day (Oncogene Research Products, Boston, MA). Terminal deoxyribonucleotidyl À À À À for 5 days, and Apc+/ Pparb+/+ or Apc+/ Pparb / were treated by oral transferase–mediated dUTP nick end labeling (TUNEL)–positive cells were gavage with GW0742 at a concentration of 10 mg/kg for 6 weeks as quantified using light microscopy and data are presented as a percentage of described above. Mice were euthanized by overexposure to carbon dioxide average labeled cells per total cell number from representative crypts 8 hours after the last dose of GW0742 and colons were removed and flushed counted. Relative differentiation of colonocytes was determined using a with PBS. Epithelial cells, isolated as above, were homogenized in ice-cold previously established procedure (9, 10) with colon sections from long-term lysis buffer and cytosolic fractions were obtained by differential centrifu- and short-term experiments that were fixed and sectioned as described gation and protein concentration determined using a bicinchoninic acid above. Sections were deparaffinized, rehydrated, and endogenous peroxidase detection kit (Pierce, Rockford, IL). was blocked with 3% H2O2 in methanol. Sections were then blocked with RNA analysis. Total RNA was isolated from colon epithelial samples as 20 Ag/mL bovine serum albumin and incubated with biotinylated Dolichos previously described (7). The mRNAs encoding adipocyte differentiation– biflorus agglutinin (DBA; Vector Laboratories) at a concentration of 10 Ag/mL. related protein (ADRP), fatty acid–binding protein (FABP), cathepsin E, The lectin DBA was washed with TBS and the sections were then incubated keratin 20, Kruppel-like factor 4 (KLF4), PPARa,PPARh, and PPARg were with an avidin-biotin peroxidase complex. Immunodetection was done after quantified using real-time PCR analysis. The cDNA was generated using treating with DAB and counterstaining with hematoxylin. Staining specificity 2.5 Ag total RNA with MultiScribe Reverse Transcriptase kit (Applied was determined by adding 0.2 mol/L N-acetylgalactosamine to 10 Ag/mL Biosystems, Foster City, CA). Primers were designed for real-time PCR using DBA for 30 minutes before the blocking step to inhibit DBA lectin binding. the Primer Express software (Applied Biosystems). The sequence and Scoring of the DBA binding was determined as previously described by GenBank accession numbers for the forward and reverse primers used to others (10). Detection of cathepsin E in the colon was done using quantify mRNAs were FABP (NM_017399): forward, 5V-CCATGAACTT- immunohistochemical methods. Sections were deparaffinized, rehydrated, CTCCGGCAAGT-3V, and reverse, 5V-TCCTTCCCTTTCTGGATGAGGT-3V; and endogenous peroxidase was quenched with 1% H2O2 in methanol. ADRP (NM_007408): forward, 5V-CACAAATTGCGGTTGCCAAT-3V,and Following antigen retrieval, the cathepsin E antibody (R&D Systems, reverse, 5V-ACTGGCAACAATCTCGGACGT-3V;PPARh (NM_011145): forward, Minneapolis, MN) was applied to the sections and incubated at room 5V-TTGAGCCCAAGTTCGAGTTTGCTG-3V,andreverse,5V-ATTCTAGA- temperature for 1 hour. After washing, the sections were then incubated for GCCCGCAGAATGGTGT-3V;PPARa (NM011144): forward, 5V-CGATGCTGT- 30 minutes with biotin-SP-conjugated AffiniPure donkey anti-goat IgG CCTCCTTGATGA-3V, and reverse, 5V-CATTGCCGTACGCGATCAG-3V;PPARg secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West (NM_011146): forward, 5V-GCTGGCCTCCCTGATGAATAA-3V, and reverse, Grove, PA). Slides were then incubated in a commercial avidin-biotin 5V-TCCCTGGTCATGAATCCTTGG-3V; cathepsin E (NM_007799): forward, peroxidase complex (ABC, Vector Laboratories) and immunodetection was 5V-TCGACACGATGCCAAACGT-3V, and reverse, 5V-CAGCTGGAGGTGGA- achieved by incubation with DAB (Vector Laboratories). The sections were ATGTCAA-3V; keratin 20 (NM_023256): forward, 5V-ATGAAGTCCTGGCCCA- counterstained with hematoxylin and subsequently visualized under a light GAAGA-3V, and reverse, 5V-TTTCAGCTCCTCCGTGTTCACT-3V; and KLF4 microscope. (NM_010637): forward, 5V-CCAGACCAGATGCAGTCACAA-3V, and reverse, Quantitative Western blot analysis. Cytosolic fractions from colonic 5V-ACGACCTTCTTCCCCTCTTTG-3V. All mRNAs examined were normalized epithelium were obtained from control and ligand-treated mice as to the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH; described above. Cytosol protein (25-50 Ag) from each sample of colonic BC083149) using the following primers: forward, 5V-GGTGGAGCCA- epithelium was resolved using SDS-PAGE. The samples were transferred AAAGGGTCAT-3V, and reverse, 5V-GGTTCACACCCATCACAAACAT-3V. Real- onto a nitrocellulose membrane using an electroblotting method. After time PCR reactions were carried out using SYBR green PCR master mix blocking in 5% milk in TBS-Tween 20, the membrane was incubated (Finnzymes, Espoo, Finland) in the PTC-200 DNA Engine Cycler and detected overnight at 4jC with primary antibody, followed by incubation with a using the CFD-3200 Opticon Detector (MJ Research, Waltham, MA). The biotinylated secondary antibody (Jackson ImmunoResearch Laboratories). following conditions were used for PCR: 95jC for 15 seconds, 94jC for 10 Immunoreactive proteins were detected after incubation in [125I]-labeled seconds, 60jC for 30 seconds, and 72jC for 30 seconds, and repeated for streptavidin (Amersham Biosciences, Piscataway, NJ) using phosphorimag- 45 cycles. The PCR included a no-template control reaction to control ing analysis. Hybridization signals for specific proteins of interest were for contamination and/or genomic amplification. All reactions had >90% normalized to the hybridization signal of the housekeeping protein, lactate www.aacrjournals.org 4395 Cancer Res 2006;66: (8). April 15, 2006

À À À dehydrogenase (LDH). A minimum of three to four samples from individual intestinal tumorigenesis in Apc+/ Pparb / mice but was compa- À mice were used for analysis of each treatment group. The following primary rable to that found in control and ligand-treated Apc+/ Pparb+/+ antibodies were used: anti–phosphatase and tensin homologue (Upstate mice (Table 1; Fig. 1A). Whereas these results suggest that ligand Biotechnology, Lake Placid, NY), anti–phosphoinositide-dependent kinase 1 activation of PPARh does not potentiate intestinal tumorigenesis (BD Transduction Laboratories, San Jose, CA), anti–integrin-linked kinase À in Apc+/ mice, there are a number of limitations to this analysis: (Upstate Biotechnology), anti–phospho-Akt (S473; Cell Signaling Technol- a ogy, Inc., Beverly, MA), anti-Akt (BD Transduction Laboratories), and anti- ( ) these studies were done for only a short duration of ligand b LDH (Rockland, Inc., Gilbertsville, PA). treatment (6 weeks); ( ) 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 F 2 weeks of age (to Results and Discussion allow for comparison with previous studies); and (d) small intestine À GW0742 is a highly specific PPARh-specific ligand that is very tumors are more commonly found in the Apc+/ mouse as similar in structure and has very similar binding and agonist compared with colon tumors. To examine the effects of PPARh activity toward PPARh as compared with GW501516 (11). Daily activation in a more specific model of colon carcinogenesis, À À administration of GW0742 had no effect on intestinal tumorigen- Pparb+/+ and Pparb / mice were treated once per week for 10 À esis in Apc+/ mice (Table 1; Fig. 1A). In contrast, the average weeks with azoxymethane as previously described (7) and also number and size of small intestinal polyps were significantly treated with GW0742 (either 2 or 10 mg/kg GW0742, thrice per À greater in Apc+/ that also lacked expression of PPARh (Table 1; week) or vehicle control. Decreases of 23% and 51% in colon polyp Fig. 1A). Interestingly, GW0742 treatment did not potentiate multiplicity were observed in Pparb+/+ mice treated with 2 and

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

Genetic model Colon

Genotype Treatment n <1 mm 1 mm 1-2 mm 2-3 mm >3 mm Mean size (mm)

À Apc+/ Pparb+/+ Control 11 0.5 F 0.9a 0.5 F 0.5a 0.6 F 1.0a 0.4 F 0.7a 0.7 F 0.8a 2.1 F 1.4a À Apc+/ Pparb+/+ GW0742 10 0.3 F 0.7a 0.0 F 0.0a 0.5 F 0.8a 0.8 F 1.1a 0.5 F 0.7a 3.2 F 1.6a À À À Apc+/ Pparb / Control 10 0.2 F 0.4a 0.3 F 0.7a 0.2 F 0.4a 0.0 F 0.0a 0.5 F 0.8a 2.0 F 1.4a À À À Apc+/ Pparb / GW0742 11 0.2 F 0.6a 0.3 F 0.5a 0.3 F 0.5a 0.5 F 0.7a 0.3 F 0.5a 2.1 F 0.6a P 0.5940 0.2148 0.5094 0.1202 0.5329 0.3354

Genetic model Small intestine

Genotype Treatment n <1 mm 1 mm 1-2 mm 2-3 mm >3 mm Mean size (mm)

À Apc+/ Pparb+/+ Control 11 2.0 F 2.9a 3.2 F 3.3a 9.9 F 4.9a 1.7 F 1.4a 0.3 F 0.5a 1.5 F 0.3a À Apc+/ Pparb+/+ GW0742 10 3.7 F 5.9a 2.0 F 2.9a 12.5 F 4.8a 7.3 F 5.1a,b 1.1 F 1.5a,b 1.8 F 0.5a À À À Apc+/ Pparb / Control 10 3.8 F 6.5a 3.9 F 4.0a 24.6 F 16.0b 10.1 F 9.8b 3.9 F 5.6b 1.8 F 0.4a À À À Apc+/ Pparb / GW0742 11 2.4 F 2.3a 2.6 F 2.8a 15.2 F 5.8a,b 4.5 F 4.0a,b 0.9 F 1.4a,b 1.7 F 0.4a P 0.7503 0.6107 0.0041 0.0140 0.0363 0.4974

Chemical model Colon

Genotype Treatment n <1 mm 1 mm 1-2 mm 2-3 mm >3 mm Mean size (mm)

Apc+/+Pparb+/+ Control 8 1.3 F 1.3a 1.0 F 1.0a 1.4 F 1.2a 0.5 F 0.8a 0.4 F 0.7a 1.3 F 0.4a,b Apc+/+Pparb+/+ GW0742 (5 mg/kg) 8 0.5 F 0.8a 1.1 F 0.6a 1.4 F 1.1a 1.0 F 1.4a 0.3 F 0.5a 1.6 F 0.6a À À Apc+/+Pparb / Control 10 3.5 F 2.0b 2.5 F 1.4b 2.0 F 1.6a 0.2 F 0.4a 0.1 F 0.3a 1.0 F 0.1b À À Apc+/+Pparb / GW0742 (5 mg/kg) 8 3.0 F 2.0a,b 1.4 F 1.5a,b 2.4 F 1.7a 0.6 F 0.7a 0.5 F 1.0a 1.4 F 0.6a,b P 0.0016 0.0443 0.4230 0.3131 0.6526 0.0414

À À À À NOTE: Apc+/ Pparb+/+ or Apc+/ Pparb / mice on a C57BL/6 genetic background, ages 6 to 8 weeks, were treated with either vehicle control or 10 mg/kg GW0742 by oral gavage once per day, 5Â/wk for 6 weeks. At the end of the 6-week experimental period, mice were killed by overexposure to carbon dioxide and lesions were quantified by inspection under a dissecting microscope. The size distribution of intestinal polyps are presented as the mean number of lesions F SD for each representative size. Within a column section, values with different superscripts are significantly different from each other (P V 0.05, ANOVA and post hoc testing). P values are provided for data sets in each column section and were determined by ANOVA. For À À azoxymethane treatment, Apc+/+Pparb+/+ or Apc+/+Pparb / mice on a C57BL/6 genetic background were injected with azoxymethane (10 mg/kg) once per week for 10 weeks. Mice were also treated once per day, 5Â per week with GW0742 (5 mg/kg) by oral gavage during azoxymethane and for 12 weeks after the last injection. At the end of the 22-week experimental period, mice were killed and intestinal tracts were examined for lesions as described above. The size distribution of intestinal polyps is presented as the mean number of lesions F SD for each representative size. Within a column section, values with different superscripts are significantly different from each other (P V 0.05 by ANOVA and post hoc testing). P values are provided for data sets in each column section and were determined by ANOVA.

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Figure 1. The effect of ligand activation of PPARh on intestinal tumorigenesis. A, effect of administration of GW0742 (10 mg/kg, 5Â/wk, for 6 weeks) on colon and small intestinal polyp multiplicity in Apc +/ÀPparb +/+ (Apc+/minPparb+/+ ) and Apc +/ÀPparb À/À mice. B, effect of administration of GW0742 (2 mg/kg, 3Â/wk, for 20 weeks; 10 mg/kg, 3Â/wk, for 20 weeks; or 5 mg/kg, 5Â/wk, for 22 weeks) on colon polyp multiplicity in Pparb +/+ mice (+/+) and Pparb À/À mice (À/À). C, representative sections of colon from Pparb +/+ mice (+/+) and Pparb À/À mice (À/À) 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 V 0.05 (ANOVA).

10 mg/kg GW0742, respectively, compared with control wild-type dose that specifically activated PPARh (Fig. 2A) caused a significant mice (Fig. 1B), but these differences were only statistically increase in the level of Dolichos biflorus agglutinin (DBA)–positive significant at the higher dose. To determine if a more sustained cells in Pparb+/+ mouse colon as compared with controls, and this À À administration of GW0742 could more effectively inhibit colon effect was not observed in similarly treated Pparb / colons (Figs. polyp multiplicity, a similar experiment was done, except the mice 2B and C). Additionally, administration of GW0742 increased were administered GW0742 five times per week at a dose of 5 mg/ cathepsin E protein expression in Pparb+/+ mouse colon as kg for the duration of the experiment. However, only an 18% compared with controls, and this effect was not observed in À À decrease in colon polyp multiplicity was observed in Pparb+/+ mice similarly treated Pparb / colons (Fig. 2D). treated with GW0742 five times per week as compared with Administration of PPARg ligands is also known to induce control, and this was not significantly different than controls markers of colonic differentiation including mRNA encoding FABP, (Fig. 1B). Colon polyp multiplicity was significantly greater in all keratin 20, and KLF4 (12). To determine if PPARh-mediated À À groups of Pparb / mice and GW0742 had no effect in these mice differentiation of colonocytes is similar to that induced by PPARa À À (Fig. 1B). The Pparb / mice also had a greater percentage of or PPARg ligands, expression of these mRNA markers was À À smaller lesions as compared with wild-type mice (Table 1). examined in Pparb+/+ and Pparb / mice treated with either Histologic examination revealed no differences in colon polyps of GW0742, Wy-14,643, or troglitazone. GW0742 caused a significant either treatment group, independent of genotype, and all lesions PPARh-dependent increase in mRNA encoding ADRP and FABP observed were benign adenomas (Fig. 1C). but no changes in keratin 20 or KLF4 (Fig. 3A). Expression of these Ligand treatment resulted in a marked increase in the mRNA mRNAs in colon in response to Wy-14,643 was different as encoding ADRP, FABP, and cathepsin E in wild-type mouse colon compared with changes observed after GW0742 treatment. À À and this did not occur in similarly treated Pparb / mouse colon Increased mRNA encoding FABP was observed in both Pparb+/+ À À (Fig. 2A). These results show that under the conditions used, ligand and Pparb / mice, and this increase was significantly greater in À À treatment caused specific target gene activation by PPARh only in Wy-14,643-fed Pparb / mice as compared with Pparb+/+ mice the Pparb+/+ mice. This establishes the efficacy of the dosing regimen (Fig. 3A). No increase in the expression level of mRNA encoding and further suggests that the induction of differentiation is one keratin 20 was found in either genotype fed Wy-14,643, and the mechanism that could explain how GW0742 inhibits chemically expression of mRNA encoding ADRP and KLF4 was unchanged by À À induced colon carcinogenesis because expression of ADRP, FABP, Wy-14,643 in Pparb+/+ mice but increased in Pparb / mice fed and cathepsin E is associated with terminal differentiation of Wy-14,643 (Fig. 3A). In contrast, troglitazone treatment effectively epithelial tissues including colon epithelium (12–16). To examine induced all four markers in both genotypes and no differences were this hypothesis, a previously described method (9, 10) was used to found between genotype (Figs. 3A). quantify relative differentiation of colonic epithelium in response to The mRNA encoding FABP was measured to determine whether À ligand activation of PPARh. Indeed, GW0742 administration at a ligand activation of PPARh resulted in differentiation in Apc+/ www.aacrjournals.org 4397 Cancer Res 2006;66: (8). April 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research mouse colonic epithelium. Results from this analysis show that the PPARa and PPARg was significantly lower in polyps from both À À mRNA encoding FABP was induced in Apc+/ Pparb+/+ colonic Pparb+/+ and Apc+/ azoxymethane-treated mice as compared with epithelium from mice treated with GW0742 but not in colonic normal colonic epithelium (Fig. 3C), consistent with previous studies À À À epithelium from similarly treated Apc+/ Pparb / (Fig. 3B). (17–20). Because there are conflicting reports describing PPARh expres- Induction of differentiation can be associated with increased sion during colon carcinogenesis, expression of mRNA encoding apoptosis and inhibition of cell proliferation. Consistent with this, PPARh was measured in colon and colon polyps from both the the average number of TUNEL-positive cells in the colon was genetic and chemically induced cancer models. Expression of mRNA significantly greater in GW0742-treated Pparb+/+ mice as compared À À encoding PPARh in colonic epithelium was not different between with GW0742-treated Pparb / mice (Fig. 4A). In skin epithelium, À Pparb+/+ and Apc+/ azoxymethane-treated mice as compared with there is evidence that ligand activation of PPARh leads to Pparb+/+ control mice (Fig. 3C). In contrast, expression of mRNA repression of phosphatase and tensin homologue expression with encoding PPARh was significantly lower in polyps from both concomitant increased expression of oncogenic phosphoinositide- À Pparb+/+ and Apc+/ azoxymethane-treated mice as compared with dependent kinase 1 and integrin-linked kinase, which leads to normal colonic epithelium (Fig. 3C), consistent with previous studies phosphorylation of Akt and inhibition of apoptosis (21). Examina- (5). Expression of mRNA encoding PPARa and PPARg in colonic tion of this pathway in colonic epithelium revealed that despite epithelium was similar between genotypes although PPARa mRNA specific activation of the ADRP and FABP genes by PPARh (Figs. 2A À À was significantly higher in Pparb / mice as compared with both and 3A), no changes in phosphatase and tensin homologue, À Pparb+/+ and Apc+/ mice (Fig. 3C). Expression of mRNA encoding phosphoinositide-dependent kinase, integrin-linked kinase, or

Figure 2. The effect of ligand activation of PPARh on colonocyte differentiation. A, effect of administration of GW0742 (5 mg/kg, 1Â day, for 5 days) on mRNA encoding ADRP, FABP, and cathepsin E in colonic epithelium of Pparb +/+ mice and Pparb À/À mice. Columns, mean fold change as compared with respective control; bars, SE. B, effect of administration of GW0742 (5 mg/kg, 1Â/d or 10 mg/kg, 1Â/d for 5 days) on colonocyte differentiation in Pparb +/+ mice and Pparb À/À 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 À/À 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 À/À mice. Values with different letters are significantly different at P V 0.05 (ANOVA).

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Figure 3. Expression of mRNAs in mouse colonic epithelium. A, effect of ligand activation of PPARg with troglitazone (100 mg/kg, 1Â/d for 5 days), PPARa with Wy-14,643 (100 mg/kg, for 5 days), or PPARh with GW0742 (5 mg/kg, 1Â/d for 5 days) on mRNA encoding ADRP, FABP, keratin 20 (K20), or KLF4 in Pparb +/+ and Pparb À/À mouse colonic epithelium. B, effect of ligand activation of PPARh with GW0742 (10 mg/kg, 5Â/wk, for 6 weeks) on mRNA encoding FABP in Apc +/ÀPparb +/+ and Apc +/ÀPparb À/À mouse colonic epithelium. C, expression of mRNA encoding PPARh, PPARa, and PPARg in colon and colon polyps from control (con) and azoxymethane (AOM)-treated Pparb +/+ and Pparb À/À mice and Apc +/ÀPparb +/+ mice. Columns, mean fold change as compared with respective control; bars, SE. Values with different letters are significantly different at P V 0.05 (ANOVA). phosphorylated Akt were detected between genotypes or in small intestine tumorigenesis observed by others in response to response to GW0742 (Fig. 4B). This shows that the pathway PPARh ligand administration (4) is secondary to opportunistic previously described in keratinocytes does not function in pathogenic infections resulting from increased M-cell differentia- colonocytes in vivo. Surprisingly, no statistically significant differ- tion. Additionally, similar to PPARh, the role of cathepsin E in ences in relative cell proliferation were detected in colonic carcinogenesis is controversial. For example, increased expression epithelium after treatment with 5 mg/kg GW0742 (Fig. 4C)buta of cathepsin E is found in gastric carcinomas and in cervical significant decrease in BrdUrd labeling index was observed after intraepithelial neoplasia (29, 30), and there are case reports 10 mg/kg GW0742 in both genotypes (Fig. 4C). describing colon adenocarcinomas in association with lymphoid Results from these studies are the first to conclusively show that tissue (31–33), which is juxtaposed to M cells that express ligand activation of PPARh inhibits chemically induced colon cathepsin E. However, increased expression of cathepsin E is also carcinogenesis in vivo, and that this effect is likely due in part to associated with an antimetastatic response (34) and decreased PPARh-dependent induction of colonocyte differentiation and expression of cathepsin E correlates well with decreased differen- enhanced apoptosis. These findings are consistent with other tiation, increased cell proliferation, and increased severity of reports in other model systems, showing that ligand activation of dysplasia in gastric tumors (35, 36). Thus, induction of cathepsin E PPARh induces differentiation and apoptosis in keratinocytes may be central to some conflicting reports about ligand activation (8, 14, 22–26) and that PPARh expression is associated with of PPARh during colon carcinogenesis. differentiation of colon and breast cancer cell lines (27). Indeed, The present studies show that administration of the potent induction of differentiation and apoptosis is typically associated PPARh ligand had no effect on colon or small intestinal À À À À with compounds known to inhibit tumorigenesis. In contrast to tumorigenesis in either Apc+/ Pparb / or Apc+/ Pparb+/+ mice results from passaged keratinocytes (21), no changes in the as compared with controls. However, the average number and size À À À phosphatase and tensin homologue/phosphoinositide-dependent of small intestinal tumors were greater in Apc+/ Pparb / mice as À kinase/integrin-linked kinase/phospho-Akt pathway were detected compared with Apc+/ Pparb+/+ mice, consistent with past results in colonic epithelium after ligand activation of PPARh. The PPARh- (6, 7). Surprisingly, the average number and size of intestinal À À À dependent induction of cathepsin E represents a novel finding tumors in Apc+/ Pparb / mice treated with GW0742 were not À À À because increased expression of this proteinase is associated with statistically different from control Apc+/ Pparb / mice or either À the induction of differentiation of M cells in colonic epithelium, group of Apc+/ Pparb+/+ mice. It is especially noteworthy that we which function to process antigens (28). However, certain used a dosing regimen that was essentially identical to one used by pathogens take advantage of this processing (28) and thus others with a very similar PPARh ligand in which increases in the increased infection and immune-related responses induced by number and size of small intestinal polyps were shown (4). some bacteria or viruses could be increased by enhanced M-cell Whereas it cannot be excluded that the difference in polyp number differentiation. Thus, it remains a possibility that the increased between the two experiments could theoretically be secondary to www.aacrjournals.org 4399 Cancer Res 2006;66: (8). April 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research opportunistic pathogenic infections as described above, it should polyps is in agreement with previous studies (5, 6) but inconsistent be noted that different PPARh ligands were used in these studies. with others (1, 2). The reason for this difference is uncertain. In addition, there is the potential for variability in polyp numbers However, decreased expression of PPARh is more consistent with À in different colonies of Apc+/ mice. Thus, either there are large the hypothesis that PPARh attenuates colon carcinogenesis as differences in target genes activated by the respective PPARh shown from the present study. Whereas induction of the ligand used (e.g., GW0742 versus GW501516) or the variation in colonocyte differentiation marker FABP occurs in response to À polyp numbers found in the Apc+/ mice limits the usefulness of ligand activation of PPARh, as shown from the present studies, this this model to examine the effect of ligand activation. Support for also occurs in a PPARg-dependent mechanism in colonocytes (12). the latter hypothesis is provided from two previous reports from However, induction of mRNA markers of differentiation by ligand À the same group. For example, control Apc+/ Pparb+/+ mice exhibit activation of PPARh differs from that associated with PPARg and almost twice as many intestinal polyps between two different PPARa ligands because keratin 20 and KLF4 are not induced by the experiments (30 F 2 versus f50 F 6; refs. 4 and 37, respectively) PPARh ligand GW0742 or the PPARa ligand Wy-14,643 but are and ligand treatment resulted in almost twice as many intestinal induced by the PPARg ligand troglitazone. Additionally, whereas polyps as controls (30 F 2 versus 56 F 7; ref. 4). Importantly, troglitazone and GW0742 induce expression of mRNA encoding intestinal tumorigenesis was not potentiated by ligand activation of ADRP, no change was observed in response to Wy-14,643. This À PPARh in either Apc+/ or Pparb+/+ mice treated with azoxy- clearly shows that modulation of target genes is markedly different methane in the present studies, despite specific activation of the depending on the PPAR ligand used. Interestingly, administration PPARh target genes FABP and ADRP in mouse colonic epithelium. of Wy-14,643 caused an increase in the expression of mRNA À À This suggests that short-term ligand activation of PPARh is encoding ADRP, FABP, and KLF4 in Pparb / mice, which is ineffective at inhibiting intestinal tumorigenesis as observed with consistent with the relatively higher level of PPARa expression À À the chemically induced model, and that more prolonged treatment found in the colon of Pparb / mice. This observation is similar to with ligands could be of benefit. Alternatively, it remains a in vitro findings (39) but inconsistent with previous studies possibility that ligand activation of PPARh will have no effect in showing that ligand activation of PPARa in the liver of PPARh À Apc+/ mice, similar to what was found to occur with PPARg (38). mice does not lead to enhanced transcriptional activity of PPARa The observation that PPARh expression is reduced in both the in the absence of PPARh expression (40). This may be related to the À Apc+/ mouse colon polyps and azoxymethane-treated mouse fact that PPARh expression is considerably higher in colon as

Figure 4. Effect of ligand activation of PPARh on cell proliferation in colon. A, effect of administration of GW0742 (10 mg/kg, 3Â/wk for 22 weeks) on TUNEL-positive cells in Pparb +/+ and Pparb À/À mice. B, effect of administration of GW0742 (10 mg/kg, 5Â/wk, for 1 week) in Pparb +/+ and Pparb À/À mice or GW0742 (10 mg/kg, 5Â/wk, for 6 weeks) in Apc +/ÀPparb +/+ or Apc +/ÀPparb À/À 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, 5Â/wk or 10 mg/kg, 5Â/wk for 20 or 22 weeks, respectively) on average relative BrdUrd (BrdU) labeling index of colon crypts in Pparb +/+ and Pparb À/À mice. Values with different letters are significantly different at P V 0.05 (ANOVA).

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Ligand Activation of PPARb Inhibits Colon Cancer compared with liver. In contrast, administration of troglitazone expression associated with terminal differentiation of colonocytes. resulted in no significant changes in gene expression in colon No evidence that PPARh ligands potentiate intestinal tumorigen- À À between Pparb+/+ and Pparb / mice. This is consistent with esis was observed in either a genetically predisposed or chemically previous work showing that ligand activation of PPARg in white induced model, and the inclusion of PPARh-null mice clearly shows adipose cells of PPARh mice does not lead to enhanced specificity. transcriptional activity of PPARg in the absence of PPARh expression (41) as suggested by others (39). Combined, these Acknowledgments findings do not support the hypothesis that PPARh can inhibit Received 12/1/2005; revised 1/26/2006; accepted 2/7/2006. PPARg transcriptional activity in colon in vivo but suggest that Grant support: NIH grants CA97999 and CA89607 (J.M. Peters). PPARh may inhibit PPARa transcriptional activity in colon in vivo. 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 In summary, the present studies clearly show that specific ligand with 18 U.S.C. Section 1734 solely to indicate this fact. activation of PPARh leads to the induction of target gene We thank Amanda Burns for providing technical support.

References selectively induces differentiation and inhibits cell differentiation and function. Annu Rev Cell Dev Biol proliferation. Cell Death Differ 2006;13:53–60. 2000;16:301–32. 1. He TC, Chan TA, Vogelstein B, Kinzler KW. PPARy is an 15. Lawrie LC, Dundas SR, Curran S, Murray GI. Liver 29. Matsuo K, Kobayashi I, Tsukuba T, et al. Immuno- APC-regulated target of nonsteroidal anti-inflammatory fatty acid binding protein expression in colorectal histochemical localization of cathepsins D and E in drugs. Cell 1999;99:335–45. neoplasia. Br J Cancer 2004;90:1955–60. human gastric cancer: a possible correlation with local 2. Gupta RA, Tan J, Krause WF, et al. Prostacyclin- 16. Wong NA, Herriot M, Rae F. An immunohistochem- invasive and metastatic activities of carcinoma cells. mediated activation of peroxisome proliferator-activated ical study and review of potential markers of human Hum Pathol 1996;27:184–90. receptor y in colorectal cancer. Proc Natl Acad Sci U S A intestinal M cells. Eur J Histochem 2003;47:143–50. 30. Mota F, Kanan JH, Rayment N, et al. Cathepsin E 2000;97:13275–80. 17. Bogazzi F, Ultimieri F, Raggi F, et al. Peroxisome expression by normal and premalignant cervical 3. Park BH, Vogelstein B, Kinzler KW. Genetic disrup- proliferator activated receptor g expression is reduced epithelium. Am J Pathol 1997;150:1223–9. tion of PPARy decreases the tumorigenicity of human in the colonic mucosa of acromegalic patients. J Clin 31. Clouston AD, Clouston DR, Jass JR. Adenocarcinoma colon cancer cells. Proc Natl Acad Sci U S A 2001;98: Endocrinol Metab2002;87:2403–6. of colon differentiating as dome epithelium of gut- 2598–603. 18. Bogazzi F, Ultimieri F, Raggi F, et al. Colonic polyps of associated lymphoid tissue. Histopathology 2000;37:567. 4. Gupta RA, Wang D, Katkuri S, et al. Activation of acromegalic patients are not associated with mutations 32. De Petris G, Lev R, Quirk DM, et al. Lymphoepithe- nuclear hormone receptor peroxisome proliferator- of the peroxisome proliferator activated receptor g gene. lioma-like carcinoma of the colon in a patient with activated receptor-y accelerates intestinal adenoma J Endocrinol Invest 2003;26:1054–8. hereditary nonpolyposis colorectal cancer. Arch Pathol growth. Nat Med 2004;10:245–7. 19. Bogazzi F, Ultimieri F, Raggi F, et al. Changes in the LabMed 1999;123:720–4. 5. Chen LC, Hao CY, Chiu YS, et al. Alteration of gene expression of the peroxisome proliferator-activated 33. Jass JR, Constable L, Sutherland R, et al. Adenocar- expression in normal-appearing colon mucosa of receptor g gene in the colonic polyps and colonic cinoma of colon differentiating as dome epithelium of APC(min) mice and human cancer patients. Cancer mucosa of acromegalic patients. J Clin Endocrinol gut-associated lymphoid tissue. Histopathology 2000; Res 2004;64:3694–700. Metab2003;88:3938–42. 36:116–20. 6. Reed KR, Sansom OJ, Hayes AJ, et al. PPARy status and 20. Jackson L, Wahli W, Michalik L, et al. Potential role 34. Sakakura C, Hasegawa K, Miyagawa K, et al. Possible Apc-mediated tumourigenesis in the mouse intestine. for peroxisome proliferator activated receptor (PPAR) in involvement of RUNX3 silencing in the peritoneal Oncogene 2004;23:8992–6. preventing colon cancer. Gut 2003;52:1317–22. metastases of gastric cancers. Clin Cancer Res 2005; 7. Harman FS, Nicol CJ, Marin HE, et al. Peroxisome 21. Di-Poi N, Tan NS, Michalik L, Wahli W, Desvergne B. 11:6479–88. proliferator-activated receptor-y attenuates colon carci- Antiapoptotic role of PPARbin keratinocytes via 35. Kakei N, Ichinose M, Tatematsu M, et al. Effects of nogenesis. Nat Med 2004;10:481–3. transcriptional control of the Akt1 signaling pathway. long-term omeprazole treatment on adult rat gastric 8. Peters JM, Lee SST, Li W, et al. Growth, adipose, brain Mol Cell 2002;10:721–33. mucosa—enhancement of the epithelial cell prolifera- and skin alterations resulting from targeted disruption 22. Kim DJ, Akiyama TE, Harman FS, et al. Peroxisome tion and suppression of its differentiation. Biochem of the mouse peroxisome proliferator-activated receptor proliferator-activated receptor h (y)-dependent regula- Biophys Res Commun 1995;214:861–8. h(y). Mol Cell Biol 2000;20:5119–28. tion of ubiquitin C expression contributes to attenuation 36. Lin CK, Lai KH, Lo GH, et al. Cathepsin E and 9. Boland CR, Montgomery CK, Kim YS. Alterations in of skin carcinogenesis. J Biol Chem 2004;279:23719–27. subtypes of intestinal metaplasia in carcinogenesis of human colonic mucin occurring with cellular differen- 23. Kim DJ, Murray IA, Burns AM, et al. Peroxisome the human stomach. Zhonghua Yi Xue Za Zhi (Taipei) tiation and malignant transformation. Proc Natl Acad proliferator-activated receptor-h/y (PPARh/y) inhibits 2001;64:331–6. Sci U S A 1982;79:2051–5. epidermal cell proliferation by down-regulation of 37. Wang D, Wang H, Shi Q, et al. Prostaglandin E(2) 10. Chang WC, Chapki R, Lupton JR. Predictive value of kinase activity. J Biol Chem 2005;280:9519–27. promotes colorectal adenoma growth via transactiva- proliferation, differentiation and apoptosis as interme- 24. Schmuth M, Haqq CM, Cairns WJ, et al. Peroxisome tion of the nuclear peroxisome proliferator-activated diate markers for colon tumorigenesis. Carcinogenesis proliferator-activated receptor (PPAR)-h/y stimulates receptor y. Cancer Cell 2004;6:285–95. 1997;18:721–30. differentiation and lipid accumulation in keratinocytes. 38. Girnun GD, Smith WM, Drori S, et al. APC-dependent 11. Sznaidman ML, Haffner CD, Maloney PR, et al. Novel J Invest Dermatol 2004;122:971–83. suppression of colon carcinogenesis by PPARg. Proc selective small molecule agonists for peroxisome 25. Tan NS, Michalik L, Noy N, et al. Critical roles of Natl Acad Sci U S A 2002;99:13771–6. proliferator-activated receptor y (PPARy)—synthesis PPARh/y in keratinocyte response to inflammation. 39. Shi Y, Hon M, Evans RM. The peroxisome proliferator- and biological activity. Bioorg Med Chem Lett 2003;13: Genes Dev 2001;15:3263–77. activated receptor y, an integrator of transcriptional 1517–21. 26. Westergaard M, Henningsen J, Svendsen ML, et al. repression and nuclear receptor signaling. Proc Natl Acad 12. Drori S, Girnun GD, Tou L, et al. Hic-5 regulates an Modulation of keratinocyte gene expression and differ- Sci U S A 2002;99:2613–8. epithelial program mediated by PPARg. Genes Dev entiation by PPAR-selective ligands and tetradecylthio- 40. PetersJM,AoyamaT,BurnsAM,GonzalezFJ. 2005;19:362–75. acetic acid. J Invest Dermatol 2001;116:702–12. Bezafibrate is a dual ligand for PPARa and PPARh: 13. Gupta RA, Brockman JA, Sarraf P, Willson TM, 27. Aung CS, Faddy HM, Lister EJ, Monteith GR, Roberts- studies using null mice. Biochim Biophys Acta 2003;1632: DuBois RN. Target genes of peroxisome proliferator- Thomson SJ. Isoform specific changes in PPARa and h 80–9. activated receptor g in colorectal cancer cells. J Biol in colon and breast cancer with differentiation. Biochem 41. Matsusue K, Peters JM, Gonzalez FJ. PPARh/y Chem 2001;276:29681–7. Biophys Res Commun 2006;340:656–60. potentiates PPARg-stimulated adipocyte differentiation. 14. Kim DJ, Bility MT, Billin AN, et al. PPARb/d 28. Kraehenbuhl JP, Neutra MR. Epithelial M cells: FASEB J 2004;18:1477–9.

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