Activators of the nuclear receptor PPARγ enhance colon polyp formation

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1058 NATURE MEDICINE VOLUME 4 NUMBER 9 SEPTEMBER 1998 ARTICLES A high-fat diet increases the risk of colon, breast and prostate cancer. The molecular mechanism by which dietary lipids pro- mote tumorigenesis is unknown. Their effects may be mediated at least in part by the peroxisome proliferator-activated recep- tors (PPARs). These ligand-activated nuclear receptors modulate gene expression in response to fatty acids, lipid-derived metabolites and antidiabetic drugs. To explore the role of the PPARs in diet-induced carcinogenesis, we treated mice predis- posed to intestinal neoplasia with a synthetic PPARγ ligand. Reflecting the pattern of expression of PPARγ in the gastroin- testinal tract, treated mice developed a considerably greater number of polyps in the colon but not in the small intestine, in- dicating that PPARγ activation may provide a molecular link be- tween a high-fat diet and increased risk of colorectal cancer. An estimated 510,000 people worldwide died of colorectal can- cer in 1996 (ref. 1). Epidemiological studies have suggested that a diet rich in fat, particularly fat derived from red meat, can aug- ment the risk of colon cancer 2,3 . Animal experiments have also shown that diets high in saturated and ϖ-6 polyunsaturated fatty acids (PUFAs) can promote colon tumor development 4–8 . However, little is known regarding the mechanism whereby di- etary lipids influence the process of colon carcinogenesis. The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors that may serve as sensors of dietary fats, translating nutritional stimuli into changes in gene expression. PPARs are members of the nuclear hormone receptor superfamily, ligand-responsive transcription factors that participate in many processes important for cell and tis- sue homeostasis 9 . Three different PPARs (α, δ and γ29 have been identified in mammals 10 . These receptors are believed to be chief regulators of fatty acid catabolism and storage 11,12 . Diverse polyunsaturated fatty acids can bind the PPARs and stimulate their transcriptional function. The activity of the PPARs can also be modulated by arachidonic acid derivatives, such as prostaglandins and eicosanoids 13–16 . More than 85% of human colon tumors and premalignant adenomas overexpress cyclooxygenase 2 (COX-2)(refs. 17,18), an enzyme that con- verts arachidonic acid to its downstream metabolites. Treatment with specific inhibitors of COX-2 activity can pre- vent polyp formation and cause regression of existing colorec- tal tumors 19 . COX-2 levels are also induced by high-fat diets rich in ϖ-6 polyunsaturated fatty acids 20 . Because the PPARs can alter gene expression in response to fatty acids, eicosanoids and prostaglandins, the PPARs may be associated with the neoplastic reaction to high-fat diets. We have used synthetic PPARγ ligands to test directly the role of this receptor in colon carcinogenesis. Although PPARγ was originally characterized as a regulator of adipocyte differentiation and lipid metabolism 12 , this recep- tor is also found in a variety of other specialized cell types. We have examined the expression of this gene in the gastrointesti- nal system of wild-type mice. Northern blot analysis indicates that PPARγ is expressed at high levels in the cecum and the colon, whereas barely detectable amounts can be found in the jejunum and the ileum (Fig. 1). The quantity of PPARγ mRNA present in the large bowel is substantial, approaching the level found in white fat. The Min mouse is a widely used cancer model in which loss of a single copy of the tumor suppressor gene APC predisposes animals to the development of a large number of intestinal polyps 21 . The pattern of PPARγ expression in the gastrointesti- nal tract of Min mice is similar to that of wild-type mice (Fig. 1a). To evaluate the role of PPARγ in colon carcinogenesis, we treated Min mice with the highly specific PPARγ ligand troglita- zone, a representative of the thiazolidinedione class of antidia- betic drugs 22,23 . Min mice seven weeks of age were fed pharmacological doses of a bioavailable form of troglitazone for five weeks. After completing this treatment, mice were killed and the number of tumors in the small and large intes- tine was evaluated. Troglitazone administration did not no- tably influence tumorigenesis in the small intestine (Fig. 2). In Activators of the nuclear receptor PPARγ enhance colon polyp formation ENRIQUE SAEZ 1 , PETER TONTONOZ 1,2 , MICHAEL C. NELSON 1 , JACQUELINE G.A. ALVAREZ 1 , TZE MING U 1 , STEPHEN M. BAIRD 2,3 , VILMOS A. THOMAZY 4 & RONALD M. EVANS 1 1 The Salk Institute for Biological Studies, Howard Hughes Medical Institute, La Jolla, California 92037 USA 2 Department of Pathology, University of California, San Diego, California 92093 USA 3 Pathology and Laboratory Medicine, VA Medical Center, San Diego, CA 92161 USA 4 Departments of Pathology and Integrative Biology, University of Texas at Houston Medical School, Houston, Texas 77225 USA Correspondence should be addressed to R.M.E.; e-mail: [email protected] WT MIN WT MIN Ifeum Ifeum Jejunum Jejunum Cecum Cecum Colon Colon Tro Tro Cecum Colon Fig. 1 Expression of PPARγ mRNA in the gastrointestinal tracts of wild- type and Apc Min mice. Total RNA was analyzed by northern blot. a, PPARγ is expressed at its highest levels in the cecum and colon of wild type and Apc Min mice. b, Troglitazone treatment has no effect on expression of PPARγ mRNA in the cecum or colon of wild-type or Apc Min mice.

Transcript of Activators of the nuclear receptor PPARγ enhance colon polyp formation

1058 NATURE MEDICINE • VOLUME 4 • NUMBER 9 • SEPTEMBER 1998

ARTICLES

A high-fat diet increases the risk of colon, breast and prostatecancer. The molecular mechanism by which dietary lipids pro-mote tumorigenesis is unknown. Their effects may be mediatedat least in part by the peroxisome proliferator-activated recep-tors (PPARs). These ligand-activated nuclear receptors modulategene expression in response to fatty acids, lipid-derivedmetabolites and antidiabetic drugs. To explore the role of thePPARs in diet-induced carcinogenesis, we treated mice predis-posed to intestinal neoplasia with a synthetic PPARγ ligand.Reflecting the pattern of expression of PPARγ in the gastroin-testinal tract, treated mice developed a considerably greaternumber of polyps in the colon but not in the small intestine, in-dicating that PPARγ activation may provide a molecular link be-tween a high-fat diet and increased risk of colorectal cancer.

An estimated 510,000 people worldwide died of colorectal can-cer in 1996 (ref. 1). Epidemiological studies have suggested thata diet rich in fat, particularly fat derived from red meat, can aug-ment the risk of colon cancer2,3. Animal experiments have alsoshown that diets high in saturated and ω-6 polyunsaturatedfatty acids (PUFAs) can promote colon tumor development4–8.However, little is known regarding the mechanism whereby di-etary lipids influence the process of colon carcinogenesis.

The peroxisome proliferator-activated receptors (PPARs) area family of transcription factors that may serve as sensors ofdietary fats, translating nutritional stimuli into changes ingene expression. PPARs are members of the nuclear hormonereceptor superfamily, ligand-responsive transcription factorsthat participate in many processes important for cell and tis-sue homeostasis9. Three different PPARs (α, δ and γ) have beenidentified in mammals10. These receptors are believed to bechief regulators of fatty acid catabolism and storage11,12.Diverse polyunsaturated fatty acids can bind the PPARs andstimulate their transcriptional function. The activity of thePPARs can also be modulated by arachidonic acid derivatives,such as prostaglandins and eicosanoids13–16. More than 85% ofhuman colon tumors and premalignant adenomas overexpresscyclooxygenase 2 (COX-2)(refs. 17,18), an enzyme that con-verts arachidonic acid to its downstream metabolites.Treatment with specific inhibitors of COX-2 activity can pre-vent polyp formation and cause regression of existing colorec-tal tumors19. COX-2 levels are also induced by high-fat dietsrich in ω-6 polyunsaturated fatty acids20. Because the PPARscan alter gene expression in response to fatty acids,

eicosanoids and prostaglandins, the PPARs may be associatedwith the neoplastic reaction to high-fat diets. We have usedsynthetic PPARγ ligands to test directly the role of this receptorin colon carcinogenesis.

Although PPARγ was originally characterized as a regulatorof adipocyte differentiation and lipid metabolism12, this recep-tor is also found in a variety of other specialized cell types. Wehave examined the expression of this gene in the gastrointesti-nal system of wild-type mice. Northern blot analysis indicatesthat PPARγ is expressed at high levels in the cecum and thecolon, whereas barely detectable amounts can be found in thejejunum and the ileum (Fig. 1). The quantity of PPARγ mRNApresent in the large bowel is substantial, approaching the levelfound in white fat.

The Min mouse is a widely used cancer model in which lossof a single copy of the tumor suppressor gene APC predisposesanimals to the development of a large number of intestinalpolyps21. The pattern of PPARγ expression in the gastrointesti-nal tract of Min mice is similar to that of wild-type mice (Fig.1a). To evaluate the role of PPARγ in colon carcinogenesis, wetreated Min mice with the highly specific PPARγ ligand troglita-zone, a representative of the thiazolidinedione class of antidia-betic drugs22,23. Min mice seven weeks of age were fedpharmacological doses of a bioavailable form of troglitazonefor five weeks. After completing this treatment, mice werekilled and the number of tumors in the small and large intes-tine was evaluated. Troglitazone administration did not no-tably influence tumorigenesis in the small intestine (Fig. 2). In

Activators of the nuclear receptor PPARγ enhancecolon polyp formation

ENRIQUE SAEZ1, PETER TONTONOZ1,2, MICHAEL C. NELSON1, JACQUELINE G.A. ALVAREZ1,TZE MING U1, STEPHEN M. BAIRD2,3, VILMOS A. THOMAZY4 & RONALD M. EVANS1

1The Salk Institute for Biological Studies, Howard Hughes Medical Institute, La Jolla, California 92037 USA2Department of Pathology, University of California, San Diego, California 92093 USA3Pathology and Laboratory Medicine, VA Medical Center, San Diego, CA 92161 USA

4Departments of Pathology and Integrative Biology, University of Texas at Houston Medical School,Houston, Texas 77225 USA

Correspondence should be addressed to R.M.E.; e-mail: [email protected]

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Fig. 1 Expression of PPARγ mRNA in the gastrointestinal tracts of wild-type and ApcMin mice. Total RNA was analyzed by northern blot. a, PPARγ isexpressed at its highest levels in the cecum and colon of wild type andApcMin mice. b, Troglitazone treatment has no effect on expression of PPARγmRNA in the cecum or colon of wild-type or ApcMin mice.

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Fig. 2 Troglitazone enhances polyp for-mation in the colon but not small intestineof ApcMin mice. The number of polyps in thejejunum and ileum (a) or cecum and colon(b) of each animal is shown.

contrast, the number of colon polyps signifi-cantly increased in Min mice treated with thePPARγ ligand (3 tumors per mouse, comparedwith 1 tumor per mouse in untreated mice; P< 0.02). Furthermore, whereas no control ani-mal had more than two polyps, mice fedtroglitazone had as many as seven colonpolyps (Fig. 2). Mice receiving sulindac, aninhibitor of intestinal tumorigenesis19, hadfewer and smaller tumors. The threefold en-hancement in colon polyp formation in-duced by troglitazone was independent ofgender. The weight of mice on troglitazonewas somewhat reduced, but as their food intake was similar tothat of untreated mice, the slight difference is probably be-cause of the increased tumor burden (data not shown).

To examine at which stage of colorectal tumor developmentPPARγ ligands may be acting, the results of troglitazone treat-ment in these 7-week-old Min mice (an age at which an averageof 20 small-bowel and 0.4 large-bowel polyps are already appar-ent) were compared with those from experiments using 9-week-old mutants (with an average of 29 small-bowel and 0.6large-bowel preexisting tumors). Older animals were still sus-ceptible to the deleterious effects of PPARγ activation, but themagnitude of polyposis induced by troglitazone was smaller(an increase of 1.4 to 2.83 tumors per mouse in 9-week-olds,compared with 1 to 3 tumors per mouse in 7-week-olds; Table),indicating that PPARγ function maybe most relevant duringthe promotion phase of colorectal tumor development.

Colon polyps from troglitazone-treated mice were similar inmorphology to tumors from Min mice fed a standard diet; nomeaningful polyp size differences were detected. Two patholo-gists, blinded to the categories of the samples, analyzed histo-logic sections of polyps from treated and untreated animals.Although tumors in both groups had a range of size, mitoticrate and degree of dysplasia, there was no meaningful histo-logic difference (Fig. 3). Of those examined, 10 of 14 polyps intreated animals and 8 of 12 tumors in untreated mice wereclassified as carcinoma in situ, documenting a similar degree ofdysplasia in both groups. No invasive tumors were found in ei-ther group. The enormous tumor burden of Min mice results inintestinal bleeding and the development of a fatal anemia thatprecludes thorough study of the late stages of colon tumorige-nesis. Nonetheless, comparison of the pathology of controland troglitazone-induced polyps seems to obviate a substan-

tial role for PPARγ activators in tumor progression.To elucidate the molecular mechanism whereby PPARγ acti-

vators influence colorectal tumorigenesis, we examined theexpression of PPARγ in the colon before and after drug treat-ment. Troglitazone administration did not affect the globallevels of PPARγ message detectable by northern blot analysis(Fig. 1b). Immunohistochemical staining showed that in nor-mal mucosa, PPARγ expression is mostly confined to the pro-liferating compartment of the epithelium, that is, the crypts;little or no PPARγ protein could be detected in the terminallydifferentiated surface epithelium (Fig. 4). In contrast, polypsfrom Min mice showed diffuse staining of the epithelium, bothin the glands and in the surface. Although the level of PPARγexpression varied between individual polyps, expression wasusually stronger in the tumor than in normal epithelium. Thepattern of PPARγ expression in normal and neoplastic tissueswas not altered by troglitazone treatment. These results showthat PPARγ expression seems to correlate with proliferation,not differentiation, of colonic cells, and that ligand activationof the receptor does not alter its pattern of expression.

To determine whether PPARγ activation in mice not predis-posed to intestinal neoplasia can result in colon tumor devel-opment in the absence of cooperating genetic alterations, wetreated an additional set of 7-week-old Min and wild-type micewith troglitazone for 5 weeks. Consistent with our previousfindings, we documented a twofold to threefold increase incolon polyp number in Min mice fed the PPARγ activator (2.9tumors per mouse, compared with 1.3 tumors per mouse inuntreated controls; P < 0.02). In contrast, no colon tumorswere found in 17 troglitazone-treated wild-type mice (Table).Troglitazone had no notable effect on the weight or food in-take of wild-type animals.

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Age Genotype n Treatment Total tumors Tumors/mouse

First trial 7 weeks APCMin 17 none 17 17 weeks APCMin 10 troglitazone 30 37 weeks APCMin 6 sulindac 8 1.3

9 weeks APCMin 5 none 7 1.49 weeks APCMin 6 troglitazone 17 2.8

Second trial 7 weeks APCMin 6 none 8 1.37 weeks APCMin 7 troglitazone 20 2.97 weeks wild-type 17 troglitazone 0 0

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Fig. 4 Expression of PPARγ protein in normal colonicmucosa and polyps from ApcMin mice. PPARγ protein waslocalized by immunohistochemistry using affinity-purifiedanti-PPARγ antisera. a and b, PPARγ protein is expressedprimarily in crypts in normal colonic mucosa. d, Colonicpolyp from an ApcMin mouse stained with preimmuneserum. c, e and f, Expression of PPARγ protein in colonicpolyps from ApcMin mice. Objective magnification: 20× fora and 40× for b,c,d,e,f.

Fig. 3 Histology of tumors from control (a–d)and troglitazone-treated (e–h) ApcMin mice. Polypswere fixed in formalin and stained with hema-toxylin and eosin. All show glandular hyperplasiaand dysplasia. Objective magnification: 20× for aand e, and 40× for b,c,d,f,g,h.

This study explored the hypothesis that PPARγ activationmay contribute to diet-induced colorectal cancer. We found adistinct correlation between PPARγ expression and increasedtumorigenesis in the colons of Min mice treated with a highlyspecific PPARγ ligand. Similar results have been obtained in anindependent study in which Min mice were treated with a dif-ferent high-affinity PPARγ ligand24. PPARγ expression also mayincrease during colorectal carcinogenesis25. These findings in-dicate that PPARγ can influence colon tumor development.

Our observations are in agreement with studies designed toassess the impact of high-fat diets on intestinal neoplasia. Inmany studies4–7,26–29, animals fed a diet rich in ω-6 PUFAs de-veloped twofold to threefold more colon tumors than con-trols fed a normal diet; a considerably smaller effect was seenin the small intestine. In a particularly relevant study withMin mice, the number of colon tumors per mouse increasedfrom 1.83 in controls to 4.12 in the high-fat group29.Moreover, the size and degree of malignancy of the colorectaltumors was not different in the two groups. The magnitudeand specificity of this tumor number increase mirrors ourdata obtained with PPARγ activators, indicating that PPARγmay act as a molecular link between a high-fat diet and in-creased risk of colon cancer.

High-fat diets may stimulate formation of colon polyps byproviding an unusual abundance of PPARγ activators. Colonicepithelial cells may procure lipids and fatty acids from the in-testinal lumen or the bloodstream that can directly serve asPPARγ agonists. Colonic cells may also convert these lipidsinto more specific PPARγ ligands, such as prostaglandinmetabolites. Induction of COX-2 overexpression by high-fatdiets and during neoplasia could generate local PPARγ activa-

tors. Because PPARα and PPARδ are also expressed in areas ofthe intestine10, perhaps an interplay between the PPARs canexplain why large amounts of dietary saturated fatty acidsand ω-6 PUFAs promote colon cancer, whereas ω-3 PUFAsoffer a protective benefit7,30.

The mechanism whereby PPARγ activation leads to increasedpolyp formation is unclear. PPARγ can prompt differentiationof monocytes and adipocyte precursors12,31; thus, PPARγ expres-sion might promote colonic cell differentiation, and chronictreatment with PPARγ ligands might select cells that retain pro-liferative potential because of a functional loss of the receptor.This scenario seems unlikely, as PPARγ expression is main-tained in proliferating cells in both normal mucosa and polyps.Furthermore, PPARγ protein can be easily detected in colon tu-mors, indicating that neoplastic cell growth is neither becauseof PPARγ loss nor inhibited by its presence. PPARγ activationcan induce terminal differentiation of human liposarcoma andbreast cancer cells cultured in vitro32,33. Given the increasedcolon tumor multiplicity produced by troglitazone in the Minmouse, the therapeutic significance of cell culture studies withPPARγ ligands remains to be determined.

Although additional work is needed to determine how PPARγactivation promotes colon polyp formation, our findings mayhave implications for type II diabetes patients treated withtroglitazone and other thiazolidinediones. The dose of troglita-zone in our studies is that routinely used to elicit an insulin-sensitizing response in diabetic mice22,34. The effect we havenoted may be species-specific, and humans may not respond inthe same way to PPARγ activators. It is also evident from our re-sults with wild-type mice that troglitazone may be innocuousfor patients without a history of colorectal cancer. However, as

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6–15% of humans are estimated to be at risk for colon cancer34,it would be prudent to evaluate the effects of troglitazone andother PPARγ activators on the pathogenesis of human colorec-tal carcinoma. In a preliminary experiment, simultaneous ad-ministration of sulindac was not sufficient to abrogate thetumor-promoting effect of troglitazone, although other colontumor inhibitors may be more effective.

A high-fat diet is a considerable risk factor for several com-mon human neoplasms other than colon cancer, includingbreast and prostate cancer2. A feature all these tumors share isthat they express PPARγ (ref. 33 and P.T. and R.M.E., unpub-lished). Our studies with the Min mouse indicate that PPARγmay mediate the deleterious effects of dietary lipids on col-orectal tumorigenesis. It will be important to investigatewhether PPARγ can act as a molecular link between a high-fatdiet and increased cancer risk in these other nutritionally-sensitive tumors.

MethodsAnimal treatments. C57BL/6J-ApcMin mice (called Min mice here) werefed ad libitum a powdered form of standard chow (Teklad 7001M, 4%fat; Harlan Teklad, Madison, Wisconsin). Troglitazone was delivered bymixing Troglitazone Solid Dispersion (a formulation with better bioavail-ability than troglitazone in crystal form) with powdered food to achievean effective 0.2% troglitazone admixture. Sulindac (Sigma) was addedto the water at a dose of 160 p.p.m. Supplemented food and water werereplaced on a weekly basis. Animal weights and food intake were moni-tored bi-weekly.

Tumor scoring and histology. After mice were killed, the intestine wasligated beneath the stomach with dental floss and the gut was filled fromthe anal end with 10% neutral formalin to distend and fix the epithelium.The intestine was opened longitudinally and stained with Diff-Quik II(Dade Diagnostics, Aguada, Puerto Rico) to facilitate polyp detection.Polyp counts were done by the same investigator, blinded to the identityof the samples; all colon samples were scored at least twice.

PPARγ expression. For PPARγ northern blot analysis, the gastrointestinaltract was divided into its morphological boundaries and total RNA wasprepared from each section. Polyps for immunohistochemistry were em-bedded in O.C.T. (Tissue-Tek; Miles, Naperville, Illinois). The specificity ofthe antibody and the protocol used to assess PPARγ expression in tissueshave been described31.

Statistical analysis. Calculations were done by the Cancer Prevention-Biostatistics department at the UCSD Cancer Center. Significance oftumor counts was evaluated by unpaired t-tests and Wilcoxon’s nonpara-metric 2-sample test. Raw tumor numbers were also transformed usingthe transformation lvar = log10(var+1) to make the group variances morehomogeneous. Tumor multiplicity differences were evaluated using bothtests on raw and transformed data, and considered significant if all ap-proaches yielded a P < 0.05.

AcknowledgmentsTroglitazone was a gift from Sankyo Co.LTD. We thank R. Boland, R.Kucherlapati, R. Kolodner, G. Wahl, R. Heyman and J. Auwerx for discussions,and B. Gilpin for statistical analysis. R.M.E. is an Investigator of the HowardHughes Medical Institute at the Salk Institute for Biological Studies. E.S. is afellow of the Susan G. Komen Breast Cancer Foundation.

RECEIVED 22 MAY; ACCEPTED 4 AUGUST 1998

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