Cooperation Between the Thyroid Hormone Receptor TR 1 and the WNT Pathway in the Induction of Intestinal Tumorigenesis

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1 GASTROENTEROLOGY 2010;138: Cooperation Between the Thyroid Hormone Receptor TR 1 and the WNT Pathway in the Induction of Intestinal Tumorigenesis ELSA KRESS,* SEHAM SKAH,* MARIA SIRAKOV,* JULIEN NADJAR,* NICOLAS GADOT, JEAN YVES SCOAZEC, JACQUES SAMARUT,* and MICHELINA PLATEROTI* *Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon, France; ANIPATH, Lyon, France; INSERM U865,; # and Hospices Civils de Lyon, Lyon, France BACKGROUND & AIMS: Colorectal tumorigenesis is a multistep process involving the alteration of oncogenes and tumor suppressor genes, leading to the deregulation of molecular pathways that govern intestinal homeostasis. We have previously shown that the thyroid hormone receptor 1 (TR 1) controls intestinal development and homeostasis through the WNT pathway. More precisely, TR 1 directly enhances the transcription of several components of this pathway, allowing increased expression of -catenin/tcf4 target genes and stimulation of cell proliferation. Because the WNT pathway is a major player in controlling intestinal homeostasis, we addressed whether the TR 1 receptor has tumor-inducing potential. Methods: We generated mice overexpressing TR 1 specifically in the intestinal epithelium in a wild-type (vil-tr 1) or a WNT-activated (vil-tr 1/Apc /1638N ) genetic background. RESULTS: The intestine of vil-tr 1 mice presents aberrant intestinal mucosal architecture and increased cell proliferation and develops adenoma at a low rate. However, TR 1 overexpression is unable to induce cancer development. On the contrary, we observed accelerated tumorigenesis in vil-tr 1/Apc /1638N mice compared with the Apc /1638N mutants. CONCLUSION: Our results suggest that this phenotype is due to cooperation between the activated TR 1 and WNT pathways. This is the first report describing the tumor-inducing function of TR 1 in the intestine. Keywords: c-fos; Intestinal Tumor; TR 1; WNT. The continuous cell renewal of the mammalian intestinal epithelium is fueled by stem cells and committed progenitors located in the crypts of Lieberkün. The balance between cell proliferation and differentiation assures the homeostasis of this highly organized tissue. 1 The physiology of the crypt compartment is tightly regulated by several signaling pathways comprising the canonical WNT. 2,3 Mutations to or altered expression of genes in this pathway are associated with very early gut tumorigenesis. 2 4 Intestinal cancer is known to develop through a defined series of histological changes, the so-called adenoma carcinoma sequence. Each step is characterized by genetic alteration of a specific oncogene or tumor suppressor gene. 4,5 Interestingly, these genes are components of the molecular pathways that control the homeostasis of the intestinal epithelium. Thus, symmetry exists between their roles in gut physiology and in tumor initiation and/or progression. This is particularly well-characterized in the case of the canonical WNT pathway, for which the mammalian intestine is one of the most-studied paradigms. 6,7 Hormones and growth factors are also important for proper gut development and homeostasis. In particular, we have shown that the thyroid hormone (TH) nuclear receptor TR 1 controls intestinal homeostasis. 8 The mechanism underlying this effect involves direct transcriptional regulation and stabilization of -catenin. 9,10 Due to the direct control of WNT activation by TH and TR 1, we hypothesized that the TR 1 receptor might have tumor-inducing properties and further investigated the integration of WNT and TR 1 signals in intestinal tumorigenesis. Here we show for the first time that the TR 1 receptor acts as a tumor inducer in the gut. Specifically, overexpression of TR 1 in the mouse intestinal epithelium accelerates the adenoma carcinoma development induced by mutations in the WNT pathway. The overactivation of WNT in conjunction with a TR 1- dependent increased expression of the oncogene c-fos accounts for the phenotype of this new model of intestinal carcinogenesis. Materials and Methods Transgenesis, Animal Breeding, and Sample Preparation The mouse TR 1 complementary DNA followed by SV40 polyadenylation signal was cloned into a pgemt-easy vector (Promega, Charbonnières, France). After verification by DNA sequencing, the fragment was inserted into the vector pvill-aatii, excised by Xho1 digestion, and purified with the QIAex II extraction kit (Qiagen, Courtaboeuf Cedex, France). The microinjection Abbreviations used in this paper: mrna, messenger RNA; RTqPCR, real-time quantitative polymerase chain reaction; TH, thyroid hormone; TR, TH receptor; WB, Western blot; WT, wild-type by the AGA Institute /10/$36.00 doi: /j.gastro

2 1864 KRESS ET AL GASTROENTEROLOGY Vol. 138, No. 5 was conducted at the Plateau de Biologie Expérimentale de la Souris, Ecole Normale Supérieure de Lyon (Lyon, France) and 8 founders were obtained. The transgenic lines were backcrossed into the C57BL6 genetic background. For the genotyping of the vil-tr 1 mice, we used a nested polymerase chain reaction (PCR) approach (Supplementary Figure 1D). Apc /1638N mice were obtained from Professor R. Fodde (Rotterdam, The Netherlands). Animals received standard mouse chow and water ad libitum. They were euthanized at the indicated ages and the intestine was quickly removed, flushed with cold phosphate-buffered saline and fixed in 4% paraformaldehyde for paraffin embedding, or frozen in liquid nitrogen for DNA, RNA, and protein extraction. The transgenesis protocol, mouse housing, and experimentation were approved from the animal experimental committee of the Ecole Normale Supérieure de Lyon (Lyon, France), and are in accordance with European legislation on animal care and experimentation. Induction of Hypo- and Hyperthyroidism in Mice; Histological Analysis, Semi-Thin Preparation and Scanning Electron Microscopy; RNA Extraction and Real-Time Quantitative PCR; Immunolabeling and Western Blot Details are reported in Supplementary Materials and Methods. Results Generation and Characterization of vil-tr 1 Transgenic Mice A 9-kb regulatory region of the mouse Villin gene was used to drive the expression of the murine TR 1 complementary DNA in the epithelium of both small intestine and colon 11 (Supplementary Figure 1A). As expected, the transgene was expressed at the messenger RNA (mrna) level specifically in these of several independent transgenic lines (Supplementary Figure 1B). We also confirmed the overexpression of TR 1 by real-time quantitative PCR (RTqPCR) and Western blot (WB) on whole intestinal extracts in transgenic compared with wild-type (WT) animals (Supplementary Figure 1C and D). Finally, immunostaining of intestinal sections showed that in WT intestine, TR 1 is mainly present in the nuclei of small intestinal crypt cells and in the lower part of the colonic crypts (Figure 1A), which are associated with cell proliferation. 2 This pattern of expression explains the very faint band visualized by WB in WT animals (Supplementary Figure 1D). In transgenic mice, TR 1 was highly and homogenously expressed in all epithelial units (Figure 1A). The viability, growth, and lifespan of vil-tr 1 mice were not changed compared to the WT mice. The expression of endogenous TR 1 mrna (analyzed by amplification of a 3=UTR region by RTqPCR) and of TR 2 and TR 1 mrnas was not affected. Additionally, the levels of circulating T3 and T4 were not altered (not shown). We have analyzed the morphology of small intestine and colon of vil-tr 1 animals in H&E-stained paraffin sections (Figure 1B). Several intestinal abnormalities together with histologically normal parts were present in mice aged 1 month compared with WT animals (Figure 1B). The abnormal structures include an extension of the crypt compartment (Figure 1B, middle panels), shorter and larger villi than in WT or normal counterparts (Supplementary Figure 2C). Distribution of the abnormal structures was equal in the different regions of the small intestine and colon and was also highly frequent. In fact, we observed such structures in every histological section analyzed. Intestinal mucosa whole-mounts from mice aged 1 month were observed under the binocular microscope, and normal and abnormal parts from the vil-tr 1 mice were removed and used for semi-thin analysis and scanning electron microscopy. The abnormal samples clearly displayed misshapen structures growing in the wrong orientation, or forming branched structures compared with the normal parts (Supplementary Figure 2B, C, and E vs A and D). Ectopic crypts were also observed (Supplementary Figure 2F). More importantly, with a frequency of 1 of 6 mice, some abnormal portions represent microadenoma lesions (Figure 1B, right panels). We also wanted to assess whether the microadenomas degenerate into adenoma during adulthood. A systematic histopathological analysis revealed the presence of adenomatous lesions in both small intestine and colon with a frequency of approximately 5% in 3-month-old mice and 13% in 6-month-old mice (Table 1). These data clearly indicate that vil-tr 1 animals suffer from abnormal intestinal architecture, microadenoma, and adenoma development starting from a young age. To address the etiology of the abnormalities observed in vil-tr 1 mice, we analyzed the gut proliferating compartments in both transgenic and nontransgenic litters (Figure 1C). Ki-67 immunolabeling showed that the increased thickness of the crypts in histologically normal intestinal portions of the vil-tr 1 mice corresponds to an increased proliferation unit size (Figure 1C). This result has also been confirmed by counting the number of Ki-67 positive nuclei per proliferating units (Figure 1D). In microadenomatous lesions, the proliferation marker confirmed the extended hyperproliferation in those structures (Figure 1C, lower panels). The presence of different epithelial cytotypes was not affected by TR 1 overexpression (not shown). We also analyzed the responsiveness of these mice to TH-alterations with regard to cell proliferation. Supplementary Figure 3A clearly shows that vil-tr 1 animals are more sensitive to changes in TH status. However, we did not observe any macroscopic long-term consequences of transient alteration of the hormones (not shown).

3 May 2010 THYROID HORMONE RECEPTOR AND INTESTINAL CANCER 1865 Figure 1. Generation of vil-tr 1 animals. (A) TR 1 protein immunolabeling in paraffin sections from wild-type (WT) or vil-tr 1 mice. Images show merged TR 1 immunolabeling (red) and nuclear staining (blue) in small intestine and colon. (B) Morphological analysis of small intestine and colon from WT and vil-tr 1 mice. c, crypts; v, villi; sm, smooth muscle; se, surface epithelium. (C) Ki-67 immunolabeling of proliferating cells in intestinal sections from WT or vil-tr 1 mice. Images show merged Ki-67 immunolabeling (red) and nuclear staining (blue) in small intestine and colon. (D) Quantification of the proliferating cells along the proximo-distal intestinal axis. The Ki-67 positive cells have been counted under a Zeiss Axioplan microscope on well-oriented sections from 4 different animals per genotype. Thirty crypts per condition have been evaluated under the microscope. Numeric values illustrate mean standard deviation. Dotted bars in (A) and (C) define the limit between the crypts and the villi. The vil-tr 1 Mice Display High Levels of -Catenin and Overexpress TR 1 and WNT Target Genes We have previously shown that TR 1 regulates intestinal epithelial progenitor proliferation through the activation of WNT and induction of proliferation-controlling genes. 9,10 Supplementary Figure 3 illustrates the results of RT-qPCR analysis of the 2 direct targets of TR 1, Ctnnb1, and Sfrp2, together with c-fos, some cyclins, and the WNT targets Ccnd1, c-myc, and c-jun (Supplementary Figure 3B and C). Because all of these genes are implicated in cell proliferation, 9,12 their deregulation is directly linked to the hyperproliferative phenotype observed in the intestine of the vil-tr 1 animals. We also examined the pattern of -catenin expression by immunolabeling on sections. Figure 2 shows that in WT, -catenin is present mainly at cell cell junctions of the intestinal epithelial cells (Figure 2A, left panels). Detection of nuclei positive for -catenin is very rare in those animals, in agreement with published observations. 13 In vil-tr 1 mice, we could observe a clear-cut increase in -catenin in the epithelial cells. Moreover, cell clusters with strong -catenin expression could be clearly observed (Figure 2A), and nuclear -catenin labeling is present within these structures (magnifications of Figure 2A). These foci of high -catenin expression resemble the very early events of the normal-adenoma sequence. 13 WB analysis (Figure 2B) quantitatively confirmed that expression of -catenin is higher in the intestine of the vil-tr 1 mice than that of the WT litters. We also used a specific antibody to determine the level of activated -catenin. 14 Based on the results, we concluded that TR 1 overexpression in the intestine allows increased stabilization of -catenin, as also confirmed by increased levels of the

4 1866 KRESS ET AL GASTROENTEROLOGY Vol. 138, No. 5 Table 1. Analysis of the Lesions in Animals of Different Age and Genotype Genotype Mice, n Incidence, n (%) Adenoma/animal, mean SD Adenocarcinoma/ animal, mean SD Tumor range, absolute no. of tumors Tumor localization, small intestine/colon, % Age: 3 months WT 8 0/0 (0) /0 vil-tr /20 (5) /50 Apc 1638N/ 10 0/10 (0) /0 vil-tr 1/Apc 1638N/ 10 10/10 (100) a,b,c /36 Age: 6 months WT 6 0/6 (0) /0 vil-tr /15 (13) /33 Apc 1638N/ 10 10/10 (100) a,b a,b /0 vil-tr 1/Apc 1638N/ 8 8/8 (100) a,b a,b,c /32 SD, standard deviation; WT, wild-type. a Significantly different compared with WT mice. b Significantly different compared with the vil-tr 1 mice. c Significantly different compared with the Apc mice, by Student t test. -catenin targets Cyclin D1 and c-myc at both mrna (Supplementary Figure 3B) and protein level (Figures 2B and 6B). Altogether, overexpression of TR 1 in the intestinal epithelium allows increased cell proliferation and induces adenoma development, likely through activation of the WNT pathway and proliferation control genes. However, TR 1 overexpression per se is unable to trigger intestinal malignancy; thus we speculated that it may cause susceptibility to malignancy in an appropriate genetic background. Generation and Analysis of Double Transgenic vil-tr 1/Apc /1638N Mice To assess whether TR 1 overexpression in the intestine can interact with WNT signaling in tumorigenesis, we crossed the vil-tr 1 mice with mutant Apc /1638N mice, which are characterized by a homogenous low rate of tumor development. 15 We performed comparative histological analyses of both the small intestine and colon of Apc /1638N (referred to as Apc) and double transgenic vil-tr 1/Apc /1638N (referred to as vil-tr 1/Apc) mice at different ages (Figure 3A and C; complete data sets in Supplementary Figures 4 and 5). The 3-month-old Apc mice have normal small intestine and colon features (Figure 3A), resembling those of the age-matched WT mice (Supplementary Figure 4A and B). On the contrary, the double transgenic vil-tr 1/Apc mice present adenomatous lesions in both small intestine and colon (Figure 3A). Systematic histopathological analysis indicated the presence of approximately 2 adenomas per animal in the vil-tr 1/Apc background (Table 1). Aberrant hyperproliferation in the intestinal lesions of those animals was confirmed by immunolabeling (Figure 3B and Supplementary Figure 6). Next, we analyzed the intestinal morphology of 6-month-old animals (Figure 3C and Supplementary Figure 5) and showed that the Apc mice already present adenoma and adenocarcinoma lesions only in the small intestine (Figure 3C). In contrast, vil-tr 1/Apc mice had adenomas and adenocarcinomas in both small intestine and colon (Figure 3C). The number of lesions per animal was comparable between the single Apc and double vil- TR 1/Apc mutants (Table 1). However, all the lesions in the double transgenic mice could be histopathologically classified as adenocarcinoma, while most lesions observed in the Apc mice were still at the adenoma stage (Table 1). All the lesions were hyperproliferative, independent of genotype (Figure 3D and Supplementary Figure 7). These data show that TR 1 overexpression in an Apc-mutated background accelerates carcinoma development. We then asked whether the metastatic process was also accelerated in these animals. However, no metastases were observed by histopathologic analysis of livers from 6-month-old animals. Instead, we detected micrometastasis consisting of disseminated intestinal tumor cells, strongly villin-positive, in livers from vil-tr 1/Apc mice (Figure 3E). Indeed, small groups of villin-expressing cells, distinct from hepatocytes or bile duct cells were visualized. This ectopic presence of villin-expressing cells clearly indicates their intestinal epithelium origin, consistent with other reports. 16,17 This last result provides supplementary evidence that the entire tumorigenesis process taking place in the double transgenic mice is accelerated. -Catenin Stabilization and WNT Target Gene Expression in vil-tr 1/Apc Mice To clarify the molecular mechanisms underlying the acceleration of the adenoma-carcinoma sequence in the vil-tr 1/Apc mice, we analyzed the pattern of -catenin expression as well as its targets in the different mutant mice. -catenin immunolocalization in normal mucosa of Apc and double transgenic vil-tr 1/Apc mice showed that the protein is mainly present at cell cell junctions of the epithelial cells in both small intestine and colon (Figure 4A). Compared with the healthy mucosa, in the

5 May 2010 THYROID HORMONE RECEPTOR AND INTESTINAL CANCER 1867 Figure 2. The vil-tr 1 mice display a high level of -catenin and WNT activation. (A) -catenin immunolabeling in intestinal paraffin sections from WT or vil-tr 1 mice. Images show merged -catenin immunolabeling (red) and nuclear staining (blue) in small intestine and colon. Bars 15 m; enlargements 5 m. (B) Western blot analysis (50 g protein/ lane) confirming the increased levels of total and activated -catenin in vil-tr 1 mice compared with WT. The activation of WNT is confirmed by increased amounts of c-myc. The image is representative of 3 independent experiments. Actin has been used as loading control. adenomas of small intestine and colon of 3-month-old vil-tr 1/Apc mice, there was an obvious increase of -catenin labeling. Clusters of cells strongly expressing -catenin with a clear nuclear localization were evident (Figure 4B). Moreover, in comparing the small-intestine lesions in the 6-month-old Apc and vil-tr 1/Apc mice, it was clear that cell clusters of high-expressing -catenin were present in both genotypes (Figure 4C). However, differences in -catenin staining were evident in the adenomas developed in the colon of vil-tr 1/Apc mice at this age, compared with the healthy mucosa (Figure 4C vs A). WB analysis allowed us to quantify the differences in the levels of total and activated -catenin (Figure 5). As expected, the levels of -catenin were higher in adenoma

6 1868 KRESS ET AL GASTROENTEROLOGY Vol. 138, No. 5 Figure 3. Analysis of vil-tr 1/Apc mice. (A) H&E-stained sections of small intestine and colon from 3-month-old Apc and vil-tr 1/Apc. (B) Analysis of cell proliferation by Ki-67 immunolabeling in small intestine and colon of 3-month-old Apc and vil-tr 1/Apc mice. Images show merged Ki-67 immunolabeling (red) and nuclear staining (blue). (C) H&E-stained sections of small intestine and colon from 6-month-old Apc and vil-tr 1/Apc as indicated. (D) Analysis of cell proliferation by Ki-67 immunolabeling in small intestine and colon of 6-month-old Apc and vil-tr 1/Apc mice. Images show merged Ki-67 immunolabeling (red) and nuclear staining (blue). (E) Presence of micrometastasis in the liver of vil-tr 1/Apc mice. Images show merged villin (red) and nuclear staining (blue). We analyzed 3 animals per genotype and the micrometastasis were present in the liver of all the double transgenic animals.

7 May 2010 THYROID HORMONE RECEPTOR AND INTESTINAL CANCER 1869 Figure 4. Activation of -catenin in the intestine of vil-tr 1/Apc. (A C) -catenin immunolabeling in paraffin sections from Apc or vil-tr 1/Apc mice in normal mucosa (A), in tumors from 3-month-old vil-tr 1/Apc mice (B) or 6-months-old (C) animals of indicated genotype. Images show merged -catenin immunolabeling (red) and nuclear staining (blue) in small intestine and colon as indicated. Bars 30 m; enlargements 7 m. and adenocarcinoma than in normal mucosa in both vil-tr 1/Apc and Apc mice (Figure 5). However, these levels were generally higher in lesions from the double vil-tr 1/Apc than in those from Apc mice (Figure 5B). It is worth noting that in the vil-tr 1 and in healthy mucosa of vil-tr 1/Apc mice, the levels of activated -catenin were similar. Moreover, they were higher than in WT or in the normal mucosa of Apc mice (Figure 5B). Overactivation of -catenin signaling in vil-tr 1 and vil-tr 1/Apc animals was also confirmed by analyzing the -catenin targets Cyclin D1 and c-myc (Figure 6). No changes were observed in the mrna expression of these genes in normal mucosa of Apc mice. Interestingly, Ccnd1 mrna expression in the vil-tr 1 mice and in the normal mucosa of the vil-tr 1/Apc mice was significantly increased relative to WT or the normal mucosa of Apc mice at both ages (Figure 6A). The trend for c-myc expression was similar to that of Ccnd1 at 3 months, while in older mice there was no strong difference in the expression of c-myc when compared to the healthy mucosa in all genotypes. In the lesions of vil-tr 1/Apc animals, we observed a further increase in the expression of both mrnas compared with the healthy mucosa. This trend was different from that observed for other targets of WNT such as Axin2, Mmp7, and c-jun (Supplementary Figure 8A F). Finally, the overall differences in expression of Ccnd1 and c-myc mrnas were confirmed at the protein level by WB (Figure 6B). Exploration of Other Molecular Players The results described here strongly suggested the existence of additional player(s) accounting for the accelerated adenoma carcinoma progression in vil-tr 1 animals. We have previously described a positive regulation of the oncogene c-fos by TH and TR 1 in intestinal progenitor cells. 9 Here we observed an increase in c-fos mrna in the intestine of vil-tr 1 mice (Supplementary Figure 3B) and decided to focus on this oncogene. c-fos

8 1870 KRESS ET AL GASTROENTEROLOGY Vol. 138, No. 5 intestine and colon. Both cytoplasmic and nuclear staining can be observed, consistent with its nuclear-cytoplasm shuttling. 19 We also performed WB analysis (Figure 7C) and confirmed the increased expression of c-fos protein in vil-tr 1 and in the normal mucosa of vil- TR 1/Apc compared with the WT mice, independent of animal age. Moreover, the protein level was further increased, specifically in the lesions of vil-tr 1/Apc animals. Notably, the expression profiles of both Cyclin D1 and c-myc mrna and protein perfectly overlapped with those of c-fos (Figure 6). Both of these genes are described targets of both WNT and c-fos/c-jun complex. 20 Altogether, our data strongly suggest that the overexpression of c-fos together with the unique expression pattern of cyclin D1 and c-myc is the most evident molecular feature of the vil-tr 1 genetic background. Figure 5. Western blot analysis of total and activated -catenin. (A, B) The study was performed in the intestine (50 g protein/lane) of3-(a) and 6-month (B) animals. The picture is representative of 3 independent experiments. Actin has been used as loading control. N, normal mucosa; A, adenoma; AC, adenocarcinoma. mrna was significantly up-regulated in vil-tr 1 and in normal mucosa of vil-tr 1/Apc mice compared with WT and Apc mice at 3 months of age (Figure 7A). Interestingly, at 6 months, the level of c-fos expression in the healthy mucosa of vil-tr 1/Apc mice was higher compared with vil-tr 1 mice. Finally, c-fos mrna expression was further increased in the lesions of vil-tr 1/Apc mice compared to their normal counterparts at both ages (Figure 7A). The 3-month-old Apc animals showed no difference in the expression of c-fos mrna compared with WT animals (Figure 7A), while c-fos expression in normal mucosa was increased in older mice. However, no further difference was observed in the lesions compared with the normal intestinal parts in those animals (Figure 7A). We also examined the localization of c-fos in intestinal sections of vil-tr 1/Apc and Apc mice. As shown in Figure 7B, the expression of c-fos in the normal portions of Apc mutant small intestine is almost undetectable by immunolabeling, similar to what is observed in WT mice (Supplementary Figure 9A). A few c-fos positive cells are present in the lamina propria, as described previously 18 ; several positive cells were also present at the bottom of the crypts in the colon of Apc (Figure 7B and Supplementary Figure 9H) or WT animals (Supplementary Figure 9B). Staining for c-fos remained very low in lesions of Apc mutants (Figure 7B and Supplementary Figure 9I and J). In contrast, the vil-tr 1/Apc mice displayed high expression of c-fos in both normal mucosa and in lesions (Figure 7B, Supplementary Figure 9K P) from the small Figure 6. Analysis of WNT target genes in vil-tr 1/Apc mice. (A) Real-time quantitative polymerase chain reaction analysis of Ccnd1 and c-myc in the intestine of 3- and 6-month-old mice. Values represent fold change standard deviation, after normalization to wild-type (WT). A 2-tailed Student t test was used for statistical analysis. *P.05 and **P.001, in comparison with WT. $ P.05 and $$ P.001, in comparison with healthy tissue of the same genotype; n 4. (B) Western blot analysis (50 g protein/lane) of cyclin D1 and c-myc expression in the intestine of 3- and 6-month animals. The picture is representative of 3 independent experiments. Actin has been used as loading control. N, normal mucosa; A, adenoma; AC, adenocarcinoma.

9 May 2010 THYROID HORMONE RECEPTOR AND INTESTINAL CANCER 1871 Figure 7. Analysis of c-fos in vil-tr 1/Apc mice. (A) Realtime quantitative polymerase chain reaction analysis of c- Fos in the intestine of 3- and 6-month-old mice. Values represent fold change standard deviation after normalization to wild-type (WT). A 2-tailed Student t test was used for statistical analysis. *P.05 and **P.001, in comparison with WT. $ P.05 and $$ P.001, in comparison with the normal mucosa of the same genotype; n 4. (B) c-fos immunolabeling in paraffin sections from Apc or vil-tr 1/Apc mice. Images show merged c-fos immunolabeling (red) and nuclear staining (blue) in healthy small intestine, healthy colon, lesions from small intestine, or lesions from colon as labeled. c, crypt; v, villus; sm, smooth muscle; se, surface epithelium; lp, lamina propria. Bars 15 m. (C) Western blot analysis (50 g protein/lane) confirming the differential expression of c-fos in 3- and 6-month-old animals of different genotypes. The image is representative of 3 independent experiments. Actin has been used as loading control. N, normal mucosa; A, adenoma; AC, adenocarcinoma. Discussion Our previous works pointed to a key role of the thyroid hormone receptor TR 1 in intestinal cell proliferation, due in part to the activation of WNT. 9,10 Because this is one of the best-characterized pathways in intestinal tumorigenesis, 2,3 we tested the hypothesis that TR 1 might have tumor-inducing properties. Indeed, we demonstrate here that the intestinal epithelium-targeted overexpression of TR 1 leads to hyperproliferation and induces adenoma development in both the small intestine and colon. This overexpression per se is unable to trigger a complete adenoma carcinoma sequence. However, we observed that in Apc-mutated mice, which are genetically predisposed to develop intestinal cancer, 15 the additional overexpression of TR 1 accelerates the rate of tumor appearance and progression. This is the first re-

10 1872 KRESS ET AL GASTROENTEROLOGY Vol. 138, No. 5 port describing an oncogenic role for TR 1 in vivo in the intestine. Most animal models develop tumors preferentially in the small intestine. 21 This represents a major problem in translational research because human tumors arise mostly in the colon. 4 On the other hand, our new model of intestinal tumorigenesis also displays tumors in the colon (Table 1). It is thus a valuable tool in comparing and studying these events in both mice and humans. During the past decade, mouse models of intestinal cancer has proved to be an excellent system for understanding the key events involved in the development and progression of this highly frequent human cancer. Although heterogeneity is evident on a case-by-case basis, genetically speaking it can be considered a relatively homogeneous disease because a vast majority of sporadic cases are known to be triggered by somatic mutation of the Apc, Axin, or Ctnnb1 genes, leading to the constitutive activation of the canonical WNT pathway. 3 A current view is that intestinal cancer results, at least in part, from the accumulation of multiple mutations in oncogenes and tumor suppressor genes in an affected cell. 4 This means that interactions between different pathways can play an important role in triggering the cancer as well as in its development. Consistent with this assumption, we show here the integration between the WNT and TR 1 signals, and propose that this interaction could have important implications for the initiation of intestinal tumorigenesis. It is well known that signaling pathways can work synergistically to affect cell fate. This is a fundamental issue in developmental biology and has been already reported in the context of intestinal cancer for the WNT and Notch pathways. 22 We had already shown that TR 1 regulates expression of WNT components, such as -catenin and sfrp2, and that it activates the WNT cascade and the epithelial proliferation via this regulation. 9,10 The expression of these mrnas is clearly enhanced in the vil-tr 1 mice. Moreover, we also showed that levels of activated -catenin and its molecular targets cyclin D1 and c-myc in the vil-tr 1/Apc animals were generally higher than in Apc mutant mice. This result strongly suggests cooperation between TR 1 and WNT signals. As regarding sfrp2, the down-regulation of its expression has been related to epigenetic modifications during carcinogenesis. 23 The results obtained in our double transgenic vil-tr 1/Apc mice (Supplementary Figure 10) gave molecular confirmation of the histopathological classification of the lesions reported in Table 1. In fact, the lesions from the vil-tr 1/Apc mice represent more advanced tumor stages than those observed in the Apc mice. The decreased expression of sfrp2 mrna in the healthy mucosa of vil-tr 1/Apc compared with the vil-tr 1 mice remains difficult to explain. Indeed, more in-depth analysis is necessary to fully describe the molecular features of the intestine in animals of different genotypes and in relation to healthy/ unhealthy mucosa. Taken together, these results strongly suggest that the mechanisms of -catenin stabilization by TR 1 might evolve differently throughout the diverse stages of tumor progression. These intriguing results point to the existence of other players involved in the accelerated rate of the adenoma carcinoma sequence in the vil-tr 1/Apc mice. Our data strongly suggest that the genetic programs triggering intestinal tumorigenesis in Apc or in vil-tr 1/ Apc mice are different. Indeed, we demonstrated here that the oncogene c-fos is specifically and highly upregulated in animals overexpressing TR 1, and its levels increase further, specifically in the lesions of those animals. More importantly, we also show that TR 1-stimulated-expression of c-fos is integrated and synergistic with the WNT-dependent tumoral program. In fact, cyclin D1 and c-myc, targets of both WNT and c-fos/c- Jun, 20 presented an expression profile that strongly overlaps with the expression of c-fos. We conclude that this feature represents the most evident peculiarity of the lesions that occur in the context of TR 1 overexpression. Moreover, according to data in the literature, the overexpression of c-fos, cyclin D1, and c-myc directly correlates with the phenotype of the intestinal lesions of vil-tr 1/ Apc mice. In particular, increased expression of c-fos and cyclin D1 are considered markers of tumor aggressiveness in different organs. 24,25 More in-depth studies will be necessary, however, to define the molecular signature characterizing these tumors in detail, given that the role of the c-fos/c-jun (AP-1) transcriptional complex in intestinal tumorigenesis is still unsolved. 18,26 29 Several reports have indicated that mutant TRs and altered TH status are involved in various cancers Data have shown a correlation between altered levels of TH and development of human breast and colon cancers. 33,35 However, discrepancies in the literature exist, and both increased and decreased levels of hormones have been reported. As for TRs, it has been shown that the mutation 34,36,37 or aberrant expression 30 of TRs is associated with gastrointestinal tumors. Some of these articles described a TR 1-dominant negative activity, but did not approach the function of these mutant receptors in tumorigenesis. However, our unpublished observations of a TR 1-dominant negative mouse model 38 did not allow for the detection of intestinal abnormalities. Finally, a recent article by Modica and colleagues 39 describes the pattern of expression of nuclear receptors, including the TRs, in normal intestine and tumors. In that article, they reported a decrease in both TR and TR mrna expression in tumors, without reference to a specific TR isoform. Their work appears to be in contradiction to our assumptions, but this contradiction is only apparent. When we compared TR 1 mrna expression in normal intestinal mucosa and tumors from Apc mutant mice, we did not identify differential expression of this

11 May 2010 THYROID HORMONE RECEPTOR AND INTESTINAL CANCER 1873 receptor (not shown). This result strongly suggests that we need a careful investigation that takes into consideration the diverse isoforms encoded by the TR gene. Until now, there has been no conclusive evidence to link the pro-proliferative action of TRs to tumorigenesis. Here we have reported for the first time that the specific overexpression of a nonmutated TR receptor in vivo triggers the transformation of the intestinal epithelium and leads to tumorigenesis. Our next challenge will be to confirm that TR 1 overexpression might be related to human colorectal cancer, which could lead to translational research in which specific antagonists of TR 1 are used to down-modulate its activity in human tumors. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Stappenbeck TS, Wong MH, Saam JR, et al. 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12 1874 KRESS ET AL GASTROENTEROLOGY Vol. 138, No. 5 hepatocellular carcinoma. Endocrinology 1997;138: Lin KH, Zhu XG, Shieh HY, et al. Identification of naturally occurring dominant negative mutants of thyroid hormone alpha 1 and beta 1 receptors in a human hepatocellular carcinoma cell line. Endocrinology 1996;137: Quignodon L, Vincent S, Winter H, et al. A point mutation in the activation function 2 domain of thyroid hormone receptor alpha1 expressed after CRE-mediated recombination partially recapitulates hypothyroidism. Mol Endocrinol 2007;21: Modica S, Gofflot F, Murzilli S, et al. The intestinal nuclear receptor signature with epithelial localization patterns and expression modulation in tumors. Gastroenterology 2009 Oct 8. [Epub ahead of print]. Received October 9, Accepted January 21, Reprint requests Address requests for reprints to: Prof Jacques Samarut or Dr Michelina Plateroti, Institut de Génomique Fonctionnelle de Lyon, Ecole Normale Supérieure de Lyon, 46 allée d Italie, Lyon Cedex 07, France. Michela.Plateroti@ens-lyon.fr; Jacques. Samarut@ens-lyon.fr; fax: (33) Acknowledgments We gratefully acknowledge Nadine Aguilera for animal handling, Denise Aubert for transgenesis, and Simone Peyrol for the scanning electron microscopy and semi-thin. We are indebted to Dr Sylvie Robine for providing us the villin promoter and antibody and to Professor Riccardo Fodde for the Apc /1638N mice. We are especially grateful to Professor Nicholas O. Davidson for helpful discussion and the critical reading of the manuscript. E. Kress s present address is Department of Genetic Medicine and Development, University of Geneva, Switzerland. Conflicts of interest The authors disclose no conflicts. Funding This work was supported by the Agence Nationale pour la Recherche (grant ANR-06-BLAN ) and the Institut pour le Cancer (grant INCA-2007-Col6) and the European Network of excellence CRESCENDO. E.K. was supported by the Ligue Nationale contre le Cancer; M.S. by INCA.

13 May 2010 THYROID HORMONE RECEPTOR AND INTESTINAL CANCER 1874.e1 Supplementary Materials and Methods Induction of Hypo- and Hyperthyroidism in Mice TH deficiency was induced by feeding the vil- TR 1 and WT mice with a low-iodine diet supplemented with 0.15% of propylthiouracil (Harlan/Teklad) and by adding 0.005% methimazole (Sigma) in the drinking water. Hyperthyroidism was induced by adding M T3 and M T4 in the drinking water. Animals in each experimental condition were sacrificed after 2 weeks of treatment. TH status was confirmed by measuring the circulating levels of free T3 and T4 (Biomerieux). Histological Analysis, Semi-Thin Preparation, and Scanning Electron Microscopy We recovered abnormal and normal portions of the mucosa upon observation under a binocular microscope (Olympus) and fixed the sections in 4% paraformaldehyde for histology and immunohistochemical studies according to Kress and colleagues. 1 For semi-thin sections, we fixed the samples for 1 hour at 4 C in 0.1 M cacodylate (0.1 M cacodylate, ph 6.9, 10 mm MgCl 2,10 mm CaCl 2, 90 mm sucrose)-buffered 2% glutaraldehyde, ph 7.4. They were then postfixed in cacodylate-buffered 1% osmium tetroxide, ph 7.4 for 30 minutes at 4 C; dehydrated; and embedded in araldite. For scanning electron microscopy observations, the fragments of intestinal mucosa were fixed for 1 hour in cacodylate buffer containing 3% glutaraldehyde and 5% formaldehyde, dehydrated in increasing concentrations of acetone, and dried with liquid CO 2. We observed the samples with an Hitachi 5800 scanning electron microscope. RNA Extraction and RT-qPCR Samples were lysed using a Precellys 24 homogenizer (Bertin). Total RNA was extracted with the QIAGEN RNeasy kit (Qiagen). To avoid the presence of contaminating DNA, DNase digestion (Qiagen) was performed in all preparations. Reverse transcription was performed using MMLV reverse transcriptase (Promega) on 1 g total RNA according to manufacturer s instructions, using random hexanucleotide primers (Invitrogen). Detailed protocols and primers sequences are described in Kress and colleagues. 1 Immunolabeling and WB Paraffin sections (5- m thickness) were used for indirect immunostaining; whole-protein extracts were obtained by homogenizing intestinal samples in RIPA buffer, as described in Plateroti and colleagues. 2 Wholeprotein extracts (50 g/lane) from animals of different genotype were analyzed. We used the following antibodies for immunofluorescence (IF) and WB: anti-tr 1 2 (dilution WB 1:500; IF 1/50); anti Ki-67 (Labvision: dilution 1:200); anti -catenin (Santa Cruz; dilution WB 1:1000; IF 1:50); anti c-myc (Santa Cruz; WB 1:2500); anti c-fos (Santa Cruz; dilution WB 1:200; IF 1:50; anti-cyclind1 (Labvision; dilution WB 1:500); anti-activated -catenin (Upstate; dilution WB 1:250); anti-villin (gift from Dr S. Robine, dilution IF 1:500). For immunolabeling experiments, we used fluorescent anti-mouse or anti-rabbit secondary antibodies (Jackson Laboratories). All nuclei were stained with Hoechst (Sigma). For WB analysis, we used secondary anti-rabbit or anti-mouse immunoglobulin G horseradish peroxidase conjugated antibodies (Promega). The signal was then analyzed using the enzymatic chemiluminescence detection kit (LumiLight, Roche). References 1. Kress E, Rezza A, Nadjar J, et al. The frizzled-related sfrp2 gene is a target of thyroid hormone receptor alpha1 and activates beta-catenin signaling in mouse intestine. J Biol Chem 2009;284: Plateroti M, Kress E, Mori JI, et al. Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells. Mol Cell Biol 2006;26: