Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/؉ mice with natural ligands Kaname Kawajiria,1, Yasuhito Kobayashib, Fumiaki Ohtakec,d, Togo Ikutaa, Yoshibumi Matsushimaa, Junsei Mimurae, Sven Petterssonf, Richard S. Pollenzg, Toshiyuki Sakakih, Takatsugu Hirokawai, Tetsu Akiyamad, Masafumi Kurosumib, Lorenz Poellingerj, Shigeaki Katoc,d, and Yoshiaki Fujii-Kuriyamac,e aResearch Institute for Clinical Oncology and bHospital, Saitama Cancer Center, 818 Komuro, Ina, Saitama, 362-0806, Japan; cExploratory Research for Advanced Technology and Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan; dInstitute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan; eTsukuba Advanced Research Alliance Center, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, 305-8577, Japan; fGerm-Free Facility and jDepartment of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden; gDepartment of Biology, University of South Florida, Tampa, FL 33620; hDepartment of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan; and iComputational Biology Research Center, National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koto-ku, Tokyo, 135-0064, Japan Edited by Tadatsugu Taniguchi, University of Tokyo, Tokyo, Japan, and approved June 23, 2009 (received for review February 26, 2009) Intestinal cancer is one of the most common human cancers. reported for some tumor suppressor gene products, such as VHL Aberrant activation of the canonical Wnt signaling cascade, for (11) and adenomatous polyposis coli (APC) (12). Also, the example, caused by adenomatous polyposis coli (APC) gene mu- natural AhR ligands of indole derivatives (13, 14), such as tations, leads to increased stabilization and accumulation of indole-3-acetic acid (IAA, so-called plant auxin), indole-3- ␤-catenin, resulting in initiation of intestinal carcinogenesis. The carbinol (I3C) and 3,3Ј-diindolylmethane (DIM), are natural aryl hydrocarbon receptor (AhR) has dual roles in regulating AhR ligands and generated through conversion from dietary intracellular protein levels both as a ligand-activated transcription tryptophan (Trp) and glucosinolates, respectively, by commensal factor and as a ligand-dependent E3 ubiquitin ligase. Here, we intestinal microbes (15). Notably, glucosinolates have been show that the AhR E3 ubiquitin ligase has a role in suppression of reported to exert the chemopreventive effects on colorectal intestinal carcinogenesis by a previously undescribed ligand-de- cancers in humans by cruciferous vegetables (16–18). Together, pendent ␤-catenin degradation pathway that is independent of these lines of evidence suggest that AhR has some functional and parallel to the APC system. This function of AhR is activated by association with intestinal carcinogenesis. both xenobiotics and natural AhR ligands, such as indole deriva- tives that are converted from dietary tryptophan and glucosino- Results -lates by intestinal microbes, and suppresses intestinal tumor Cecal Tumor Development in AhR؊/؊ Mice. After thoroughly exam development in ApcMin/؉ mice. These findings suggest that che- ining the digestive tracts of AhRϪ/Ϫ mice, we found that AhRϪ/Ϫ moprevention with naturally-occurring and chemically-designed mice, but not heterozygous AhRϩ/Ϫ or wild-type AhRϩ/ϩ mice, AhR ligands can be used to successfully prevent intestinal cancers. frequently developed colonic tumors, mostly in the cecum near the ileocecal junction (Fig. 1 A and B). AhRϪ/Ϫ mice bred at 2 cecal cancer ͉ ubiquitin ligase ͉ ␤-catenin ͉ tumor chemoprevention independent animal houses showed a similar time course of macroscopic tumor incidence (Fig. S1B), and the tumor size Ϸ he aryl hydrocarbon receptor (AhR, also known as dioxin increased gradually by age, reached a plateau at 30 to 40 weeks Ϫ/Ϫ Treceptor) is a member of a transcription factor superfamily (Fig. 1B). To date, 3 independent AhR mice lines have been that is characterized by structural motifs of basic helix–loop– reported (4, 19, 20). Although one report described frequent helix (bHLH)/Per-AhR nuclear translocator (Arnt)-Sim (PAS) rectal prolapse (Fig. S1A) and marked colonic hyperplasia with Ϫ/Ϫ domains, and also includes hypoxia-inducible factors (HIFs). severe inflammation in AhR mice (19), there have been no Over the past decade, many studies have been focused on systematic studies on intestinal carcinogenesis, which may ex- elucidating the functions of AhR as a mediator of multiple plain why the tumor suppressor function of AhR has been pharmacological and toxicological effects such as the induction unreported to date. Colorectal cancer is one of the most common of drug-metabolizing enzymes, teratogenesis, tumor promotion, human cancers, 5–10% of which originates in the cecum. There- Ϫ/Ϫ and immunosuppression caused by environmental contaminants fore, we were interested in investigating how AhR mice such as 3-methylcholanthrene (MC) and 2,3,7,8-tetrachloro- develop spontaneous cecal tumors. dibenzo-p-dioxin (TCDD) (1, 2). On ligand binding, AhR trans- Randomly selected mice were examined histologically for locates from the cytoplasm into the nucleus where it het- atypia classified according to the standards as shown in Fig. S2. ϩ/ϩ ϩ/Ϫ erodimerizes with the Arnt and activates the transcription of Although AhR and AhR mice of all ages had normal Ϫ/Ϫ target genes such as Cyp1a1. Induction of the Cyp1a1 gene leads (Grade 1) to mild hyperplasia (Grade 2) at worst, AhR mice to the biotransformation of polycyclic aromatic hydrocarbons older than 11 weeks had abnormal histology with atypia ranging into active genotoxic metabolites, resulting in the initiation of from mild malignancy of polyps to severe carcinomas that were chemical carcinogenesis (3). AhR-deficient (AhRϪ/Ϫ) mice are exacerbated with age (Fig. 1C). Close microscopic examination Ϫ/Ϫ resistant to most, if not all, of these toxicological adverse effects, revealed that the AhR mice bore cecal lesions with a mod- indicating that AhR is a key factor in the development of these chemical-induced diseases (4, 5). Also, we recently found that MEDICAL SCIENCES Author contributions: K.K., S.K., and Y.F.-K. designed research; K.K., Y.K., F.O., T.I., Y.M., AhR functions as a ligand-dependent E3 ubiquitin ligase of certain J.M., S.P., T.S., T.H., M.K., and L.P. performed research; R.S.P. and T.A. contributed new nuclear receptors (6), such as the estrogen (ER) and androgen reagents/analytic tools; K.K., L.P., S.K., and Y.F.-K. analyzed data; and K.K., L.P., and Y.F.-K. receptors (AR). Most recently, AhR has been reported to have a wrote the paper. crucial role in the differentiation of regulatory T cells (7–9). The authors declare no conflict of interest. AhR is a nucleocytoplasmic shuttling protein, the intracellular This article is a PNAS Direct Submission. localization of which is changed depending on cell density in the 1To whom correspondence should be addressed. E-mail: [email protected]. absence of exogenous ligands (10). Such cell density-dependent This article contains supporting information online at www.pnas.org/cgi/content/full/ movements between the cytoplasm and nucleus have also been 0902132106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902132106 PNAS ͉ August 11, 2009 ͉ vol. 106 ͉ no. 32 ͉ 13481–13486 Downloaded by guest on September 29, 2021 +/+ +/+ A AhR-/- (21W) AhR+/- (21W) AhR+/+ (21W) A AhR AhR-/- C AhR AhR-/- HE HE β-catenin β-catenin Paneth cells Paneth cells AhR AhR 4 3 P<0.05 B 100 100 β β 2 ) -catenin -catenin 2 80 1 -catenin (Relative) 0 60 β 50 c-myc c-myc Anti-β-catenin 40 AhR-/- Anti-AhR AhR+/- Anti-α-actin AhR+/+ 20 AhR+/+ AhR-/- Tumor-bearing (%) 0 0 ,mm Tumor size ( AhR+/+ AhR-/- 0 10 20 30 40 50 60 70 B β-catenin β-catenin β-catenin D Age in weeks c-myc GAPDH C 5 AhR+/+ AhR-/- 4 Fig. 2. Abnormal ␤-catenin accumulation in the intestines of AhRϪ/Ϫ mice. (A) H&E staining and immunohistochemical staining of mouse small intes- 3 tines. Paneth cells were observed at the bottom of the crypts in the small intestine in both genotypes. Expression of AhR, ␤-catenin, and c-myc are ␤ 2 shown. Nuclear accumulation of -catenin in Paneth cells of the small intestine and cecum is noted by red arrowheads. Immunohistochemical staining of Grade of atypia ␤-catenin in the colons (B) or cecum (C)ofAhRϩ/ϩ or AhRϪ/Ϫ mice. (C) Levels of 1 ␤-catenin, AhR and ␣-actin in the cecum were detected by Western blotting. ␤ Ͻ 96 10 1611 21 4025 55 >70 The amount of -catenin was quantified using the ImageJ software (NIH). (P 0.05; AhRϩ/ϩ versus AhRϪ/Ϫ group). (D) RT-PCR was performed to detect mRNA Age in weeks ϩ ϩ Ϫ Ϫ levels for ␤-catenin, c-myc (P Ͻ 0.05; AhR / versus AhR / group), and GAPDH in the cecal epithelium of AhRϩ/ϩ or AhRϪ/Ϫ mice. Data are representative of D 3 independent experiments. comitant overexpression of ␤-catenin and c-myc, a target gene of ␤-catenin/TCF4 (21). It remains uninvestigated whether there β-catenin c-myc Ϫ Ϫ HE should occur any further genetic alterations in AhR / mice Fig. 1. Cecal tumor development in AhRϪ/Ϫ mice. (A) Representative profiles leading to carcinogenesis. In human cecal cancers, markedly of colon tumors at the cecum in AhRϪ/Ϫ mice. (B) Relationship between the reduced expression of AhR was also found concomitantly with time course of macroscopic tumor incidence and tumor growth by age. Tumor an abnormal accumulation of ␤-catenin in all of 12 cancer size was estimated based on NIH images as shown by beige circles. Error bars, specimens from our hospital (Fig. S4). means Ϯ SD. (C) Summary of histological atypia grades of tumors in AhRϪ/Ϫ ϩ/ϩ ϩ/Ϫ Ϫ/Ϫ -mice by age.