Reduced Level of Smoothened Suppresses Intestinal Tumorigenesis by Down-Regulation of Wnt Signaling

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1 GASTROENTEROLOGY 2009;137: Reduced Level of Smoothened Suppresses Intestinal Tumorigenesis by Down-Regulation of Wnt Signaling SUMIMASA ARIMURA, AKIHIRO MATSUNAGA, TAKANORI KITAMURA, KOJI AOKI, MASAHIRO AOKI, and MAKOTO M. TAKETO Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan See editorial on page 422. BACKGROUND & AIMS: Although the Hedgehog (Hh) pathway regulates development and progression of several types of cancer, its involvement in colon cancer remains unclear. We aimed to clarify the roles of Hh signaling in intestinal tumorigenesis. METHODS: We studied expression of the Hh signaling components in the intestinal tumors of Apc / 716 mouse, a model for familial adenomatous polyposis. We used small interfering RNAs against Smoothened (SMO), which encodes the major signal transducer of the Hh pathway, to knockdown SMO expression and explore its function in human colon cancer cell lines. We also compared the intestinal tumor phenotypes of Apc / 716 Smo / mice with those of Apc / 716 mice. RESULTS: Expression of Smo was markedly increased in the intestinal adenoma epithelium of Apc / 716 mice. Importantly, SMO knockdown in human colon cancer cell lines suppressed proliferation in culture; cells arrested at the G1/S phase. Furthermore, Apc / 716 Smo / mice had decreased numbers of polyps in the large size class ( 1 2 mm) and recessed polyp morphology, accompanied by reduced proliferation of the tumor epithelial cells. Unexpectedly, reduced expression of Smo suppressed -catenin-dependent transcription, rather than Hhresponsive Gli-dependent transcription. Interestingly, SMO knockdown reduced protein levels of active -catenin and induced its nuclear exclusion. CONCLUSIONS: Smo contributes to intestinal tumorigenesis by increasing Wnt signaling. SMO might be a good therapeutic target for patients with colorectal polyps and carcinomas, even in the absence of Hh signal activation. The Hedgehog (Hh) signaling pathway plays essential roles in cell growth and differentiation, as well as in morphogenesis of many organs including the mammalian gastrointestinal tracts. 1,2 There are 3 mammalian Hh homologs: Sonic hedgehog, Indian hedgehog, and Desert hedgehog. In the absence of these Hh ligands, Patched 1 (Ptc1), a 12-pass transmembrane receptor, represses the activity of Smoothened (Smo), a G-protein-coupled receptor-like protein. The binding of the Hh ligands to Ptc relieves it from this repression of the Smo activity, causing Smo to activate the downstream signaling pathway and control the glioma-associated oncogene homologue (Gli) proteins, namely Gli1, Gli2, and Gli3. These Gli proteins regulate transcription of downstream target genes, including Ptc1 and Gli1, forming negative and positive feedback loops of the Hh signaling pathway, respectively. 3 Aberrations of the Hh-signaling components have been implicated in human cancers. Loss-of-function mutations of PTCH1 and gain-of-function mutations of SMO can be found in basal cell carcinomas, medulloblastomas, and rhabdomyosarcomas. 4 Autocrine activation of the Hh signaling also plays essential roles in a wide range of tumors, such as carcinomas of the breast, lung, liver, pancreas, esophagus, stomach, biliary tract, and prostate. 4 Accumulating evidence indicates that efficacious inhibitors of Hh signaling can prevent formation or progression of various types of cancer, suggesting the essential roles of the Hh signaling in these tumors. However, the contribution of the Hh signaling to colorectal cancer has been controversial. Some reports show that expression of the Hh signaling components is increased in hyperplastic polyps, adenomas, and adenocarcinomas of the human colon. 5 8 These Hh signaling components are also shown to be expressed in many cell lines derived from human colonic adenomas and adenocarcinomas. 9,10 In addition, treatment with the Hh inhibitor cyclopamine induces apoptosis in a colon cancer cell line, which can be rescued partially by further stimulation of the Hh signaling. 9 Although these reports suggest that activation of the Hh signaling may contribute to colon carcinogenesis, it has been reported that Indian hedgehog antagonizes Wnt signaling in a colon cancer cell line and that loss of Indian hedgehog may help intestinal tumor formation. 11 Furthermore, transgenic expression of hedgehog interacting protein, a pan-hedgehog inhibitor, in the small intestine has also been shown to enhance activation Abbreviations used in this paper: Apc, adenomatous polyposis coli; Gli, glioma-associated oncogene homologue; Hh, Hedgehog; Smo, Smoothened by the AGA Institute /09/$36.00 doi: /j.gastro

2 630 ARIMURA ET AL GASTROENTEROLOGY Vol. 137, No. 2 of the Wnt signaling. 12 In addition, treatment with cyclopamine does not alter expression of the Hh target genes PTCH1 and GLI1 in some human colon caner cell lines. 13 These reports suggest that activation of the Hh signaling may suppress the intestinal tumorigenesis. Here, we show that Smo is expressed at high levels in the intestinal polyps of Apc / 716 mice, a mouse model for familial adenomatous polyposis), 14 as well as in some human colon cancer cell lines. We provide evidence that Smo can contribute to intestinal tumorigenesis by enhancing the Wnt signaling. Materials and Methods Knockout Mice The Apc 716, cis-apcsmad4, and Smo / knockout mouse strains have been described previously Smo / knockout mice were from Jackson Laboratory (Bar Harbor, ME). We used Apc 716, Smad4 /, and Smo / mice at N 24 29, N 15 20, and N 8 9 generations, respectively. All animal experiments were carried out according to the protocols approved by the Animal Care and Use Committee of Kyoto University. Reverse-Transcription Polymerase Chain Reaction and Quantitative Reverse-Transcription Polymerase Chain Reaction Analysis Levels of messenger RNA (mrna) were determined by reverse-transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qrt-pcr) as described. 18 Primers for RT-PCR and qrt-pcr are shown in Supplementary Table 1. Transfection of RNA Oligonucleotides Two different small interfering RNA (sirna) against SMO and CTNNB1 were designed to prevent the off-target effects (Dharmacon, Lafayette, CO). Sequences of specific sirnas are shown in Supplementary Table 1. Nonsilencing RNA without homology sequences were used as negative controls (Hokkaido System Science Co, Sapporo, Hokkaido, Japan). Transfection of the sirnas was performed using RNAi MAX (Invitrogen, Carlsbad, CA) to adjust final RNA concentration to 40 nmol/l. Cell Proliferation Assay Cell proliferation was determined using Beckman Coulter Z Series (Beckman Coulter, Fullerton, CA) and CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega, Madison, WI). Reporter Gene Assays Reporter plasmids 8 3=Gli-BS-Luc and 8 mut3= Gli-BS-Luc were kindly provided by H. Sasaki (Riken, Kobe, Japan). 19 -Catenin-dependent Wnt transcriptional activity was determined using TOPFLASH and FOPFLASH reporters (Upstate Biotechnology, Charlottesville, VA). Transfection was performed using Lipofectamine 2000 (Invitrogen), and luciferase activity was determined. Tumor Analyses Polyp scoring, histopathologic analysis, 5-bromo- 2=-deoxyuridine (BrdU)-labeling index determination, and TdT-mediated dutp-biotin nick-end labeling (TUNEL) analysis were performed as described. 18,20 Full Materials and Methods and references are included in Supplementary Information. Results Expression of Smo Is Increased in Intestinal Polyps of Apc / 716 Mice To determine the possible roles of the Hh-signaling components in intestinal tumorigenesis, we first investigated their expression in the polyps of Apc / 716 (Apc 716 ) mice, a mouse model for familial adenomatous polyposis. 15 These mice develop numerous adenomatous polyps in the small intestine as well as some in the colon. As shown in Figure 1A and Supplementary Figure 1A, we found that expression of Smo mrna estimated by qrt- PCR and semiquantitative RT-PCR was higher ( 3 times) in the small intestinal and colonic polyps of Apc 716 mice compared with the normal epithelium, whereas expression of other components of the pathway was not affected significantly. Consistently, a Western blot analysis also confirmed that expression of Smo protein was increased by 3 times in the polyps of both intestines in the Apc 716 mice (Figure 1B). Similar results were obtained with the cis-apc / 716 Smad4 / (cis-apcsmad4) mice, a model for invasive intestinal adenocarinoma (Supplementary Figure 1B). 16 We next determined the localization of Smo mrna in the polyps of Apc 716 mice. In situ hybridization showed abundant Smo mrna in the polyp adenoma epithelial cells of both intestines in Apc 716 mice (Figure 1D), whereas little expression was found in the tumor stroma. Expression of the Smo mrna in adenoma epithelium was further confirmed by an RT-PCR analysis of the laser-microdissected samples (Figure 1C). On the other hand, Smo mrna was found only at low levels at the base of the normal colonic crypts and was hardly detectable in the small intestine (Supplementary Figure 1C). These results indicate that expression of Smo is significantly increased in the intestinal polyps of Apc 716 mice as compared with the normal epithelium. SMO Knockdown in SMO-High Human Colon Cancer Cell Lines Induces G 1 /S Arrest Overexpression of Smo in the intestinal polyp epithelium of Apc 716 and cis-apcsmad4 mice prompted us to determine the roles of SMO in human colon cancer

3 August 2009 SMOOTHENED AND INTESTINAL TUMORIGENESIS 631 Figure 1. Expression of Smo in intestinal polyps of Apc 716 mice. (A) Expression of Smo and Hh target genes analyzed by quantitative RT-PCR in the small intestinal (left) and colonic (right) polyps of the Apc 716 mice. Error bars indicate SD (n 4). *P.05; **P.01. (B) Expression of Smo and -actin (control) analyzed by Western blotting in the intestinal polyps of the Apc 716 mice. (C) Expression of Smo analyzed by RT-PCR in cdna prepared from laser-microdissected adenoma cells of the Apc 716 mouse intestinal polyps. Whole, whole adenoma tissues; Epithel, laser-microdissected adenoma epithelial cells. (D) Expression of Smo analyzed by in situ hybridization in the small intestinal (a, b, e, f) and colonic (c, d, g, h) polyps of the Apc 716 mice. Scale bars, a and e: 50 m; c and g:30 m; b, d, f, h: 5 m. Letters A, adenoma; E, normal epithelium; S, stroma. cell lines. A semiquantitative RT-PCR analysis of 9 human colon cancer cell lines revealed expression of SMO in 5 of them (Supplementary Figure 1D), with the highest levels in SW480 and HCT116. We thus investigated the roles of SMO in colon cancer cell lines by knocking down its expression using sirnas. Based on the RT-PCR results, we chose colon cancer cell lines SW480 and HCT116 (SMOhigh) and RKO (SMO-low) as test cells. Cell lines SW480 and HCT116 carry mutations in APC and CTNNB1 (for -catenin), respectively, 21,22 whereas no mutation has been found in the RKO cells for genes in the Wntsignaling pathway. Two independent sirnas targeted to the SMO gene (sismo-1 and sismo-2) both decreased SMO expression dramatically in SW480 and HCT116 cells, although a nonsilencing sirna (sin.s.) did not (Figure 2A). Because it was reported that suppression of the SMO gene by antisense RNA inhibited proliferation of 2 cancer cell lines, 23,24 we then tested the effects of SMO knockdown on cell proliferation. We found that proliferation of the SMO-high cells was significantly reduced by SMO sirnas but not by sin.s. (Figure 2B). Importantly, flow cytometric analyses showed that SMO knockdown by sirnas blocked the G 1 /S progression of the SMO-high cells (Figure 2C and Supplementary Figure 2). Following the treatment with SMO sirnas, approximately 63% and 59% of SW480 and HCT116 cells accumulated in the G 1 phase, respectively, whereas only 45% and 38% of the same cell lines were found in G 1 when treated with sin.s., respectively. On the other hand, SMO sirnas did not significantly affect the sub-g 1 population in these cells, suggesting that apoptosis was not induced as a result of SMO knockdown (Figure 2D and Supplementary Figure 2). Consistently, SMO knockdown in these cells markedly reduced the levels of cyclin D, cyclin E, and phosphorylated Rb (Ser795 and Ser807/811) (Figure 2E). In the SMO-low RKO cells, treatment with SMO sirnas did not affect cell proliferation or expression of the G 1 /S regulators (Supplementary Figures 2 and 3). Taken together, these results suggest that SMO knockdown in the SMOhigh human colon cancer cells caused strong growth suppression through cell cycle arrest at the G 1 /S boundary.

4 632 ARIMURA ET AL GASTROENTEROLOGY Vol. 137, No. 2 Figure 2. Effects of SMO down-regulation by sirnas on cell proliferation in human colon cancer cell lines. (A) Expression of SMO and GAPDH (control) analyzed by RT-PCR and Western blotting in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours. (B) Growth curves of the SW480 and HCT116 cells treated with SMO sirnas for 96 hours, determined in a Coulter counter. (C and D) Cell cycle distribution (C) and percentages of the sub-g 1 populations (D) in flow-cytometric analyses of the SW480 and HCT116 cells treated with SMO sirnas for 72 hours. (E) Expression of cell cycle regulators involved in the G 1 /S progression analyzed by Western blotting in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours. Error bars indicate SD. *P.05; **P.01. N.T., no treatment with sirna; sin.s., 40 nmol/l nonsilencing sirna; sismo, 40 nmol/l sirnas directed against SMO gene. Smo Heterozygosity Inhibits Proliferation of Tumor Cells in Apc 716 Mice We next investigated the role of Smo in intestinal tumorigenesis of Apc 716 mice. Because the Smo homozygous null mice had an embryonic lethal phenotype, 17 we crossed Apc 716 mice with Smo / mice and tested the effects of the Smo heterozygosity on the polyp formation. As shown in Figure 3A and Supplementary Figure 4A, the expression levels of Smo protein in the small intestinal and colonic polyps of Apc / 716 Smo / (ApcSmo) mice showed an approximately 70% and 50% decrease, respectively, as compared with those of the polyps of littermate Apc 716 mice. The control littermate Apc 716 mice developed (mean SD) polyps in the small intestine at weeks of age. In the small intestine of ApcSmo mice of the same age, the total polyp number was reduced to , showing a 26% decrease compared with the littermate Apc 716 mice (Figure 3B). Most importantly, the number of polyps larger than 1.0 mm in diameter was significantly reduced in the ApcSmo mice compared with Apc 716 mice (Figure 3C). On the other hand, the total numbers of colonic polyps were not significantly different between the Apc 716 and ApcSmo mice (Supplementary Figure 4B). Interestingly, however, the number of colonic polyps larger than 2.0 mm in diameter was significantly decreased in the ApcSmo mice (Supplementary Figure 4C). As shown in Figure 3D, large polyps ( 1 2 mm) were not found in the ApcSmo mice as seen in the ileum or colon of Apc 716 mice. In addition, the well-developed small intestinal polyps in the ApcSmo mice appeared recessed at the top, whereas the polyps of Apc 716 mice were dome shaped, as observed under a dissection microscope (Figure 3E, top) and by histopathologic analysis of the H&E-stained sections (Figure 3E, bottom). Consistent with these tumor phenotypes, 7 of the 19 ApcSmo mice survived longer than 45 weeks, whereas all of the littermate Apc 716 mice (n 18) became moribund by 35 weeks of age (data not shown). These results suggest that heterozygous Smo mutation affects tumor multiplicity and size and that Smo plays an important role in the expansion of intestinal polyps in the Apc 716 mice. To investigate the mechanism by which Smo heterozygosity suppresses the expansion of the intestinal polyps,

5 August 2009 SMOOTHENED AND INTESTINAL TUMORIGENESIS 633 Figure 3. Effects of Smo heterozygosity on intestinal tumor growth in Apc mutants. (A) Expression of Smo and -actin analyzed by Western blotting in the small intestinal polyps of the Apc 716 and ApcSmo mice. (B and C) Total polyp number per mouse (B) and size distribution of the intestinal polyps (C) in the small intestine of the ApcSmo and littermate Apc 716 mice. (D) Dissection micrographs of the intestines from the Apc 716 and ApcSmo mice. Polyps visible here are in the large size class ( 1 2 mm). Duo, duodenum; Jej, jejunum; Ile, ileum. Scale bars, 1 cm. (E) Dissection micrographs and H&E-stained sections of the intestinal mucosa containing well-developed ileal polyps in the Apc 716 and ApcSmo mice. Scale bars, 200 m (red) and 100 m (black). Arrowheads indicate polyps. (F) Staining of the adenoma cells with BrdU in the small intestinal polyps of Apc 716 and ApcSmo mice (top). Quantified data for BrdU-positive adenoma cells (bottom). Scale bars, 50 m. Error bars indicate SDs (n 9 for B and C, and 6 for F). *P.05; **P.01. we next determined cell proliferation and apoptotic rates in the polyps by BrdU labeling and TUNEL assay, respectively. The BrdU labeling index of the normal epithelial cells was not different between the Apc 716 and ApcSmo mice. However, the index was significantly lower in the intestinal adenoma cells of the ApcSmo mice than in those of the Apc 716 mice (Figure 3F and Supplementary Figure 4E). In contrast, the apoptotic rate of both intestinal polyps was not significantly different between them (Supplementary Figure 5A and B). These results collectively indicate that reduced level of Smo causes inhibition of cell proliferation in the intestinal polyps of Apc 716 mice. Reduced Level of Smo Does Not Suppress Gli-Dependent Hh Signaling Because Smo is a major transducer of the Hhsignaling pathway, we hypothesized that the observed growth suppression by reduced level of Smo was mediated by inhibition of the Hh signaling. To assess the Hh signal activation, we performed qrt-pcr to determine the levels of the Hh target genes and luciferase assays using 8 3=Gli-BS-Luc and 8 mut3=gli-bs-luc reporter plasmids that carried a minimal promoter with 8 GLI1 binding sites and mutated binding sites, respectively. 19 As shown in Supplementary Figure 6B and C, the Hh inhibitor cyclopamine affected neither the luciferase activity of GLI-responsive reporter plasmids nor expression of the Hh target genes PTCH1 or GLI1 in SW480 or HCT116 cells. These data are in sharp contrast to those in human gastric cancer cell line AGS, which is highly sensitive to cyclopamine, and are consistent with reports by others that showed the lack of the Hh signal activation in colon cancer cells. 13,25 Thus, inhibition of the Hh signaling may not account for growth suppression induced by reduced level of Smo in intestinal tumor cells. To determine whether SMO knockdown affected the Hh-signaling activity in intestinal tumor cells, we next performed luciferase assays using GLI-responsive reporter plasmids, as well as qrt-pcr analyses of some known Hh target genes in SW480 and HCT116 cells upon transfection of SMO sirnas. Suppression of SMO by sirnas in these cell lines did not significantly alter the activity of the GLI-responsive reporter (Figure 4A) nor the expression of PTCH1 or GLI1 (Figure 4B and Supplementary Figure 7A). Furthermore, mrna levels for Ptc1 and Gli1 were not different between

6 634 ARIMURA ET AL GASTROENTEROLOGY Vol. 137, No. 2 Figure 4. Effects of SMO down-regulation on GLI-dependent transcription in SMO-high human colon cancer cell lines and in the polyps of Apc 716 mice. (A) Luciferase reporter assays using 8 3=Gli-BS-Luc and 8 mut3=gli-bs-luc reporter plasmid in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours. (B and C) Expression of SMO and the Hh target genes analyzed by quantitative RT-PCR in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours (B) and in the small intestinal and colonic polyps of Apc 716 and ApcSmo mice (C). Error bars indicate SD. **P.01. N.T., no treatment with sirna; sin.s., 40 nmol/l nonsilencing sirna; sismo, 40 nmol/l sirnas directed against SMO gene; ND, not detectable. the polyps of the Apc 716 mice and those of the ApcSmo mice (Figure 4C and Supplementary Figure 7B). These results suggest that Smo can regulate cell proliferation through a mechanism independent of Gli-mediated transcription in intestinal tumor cells. Reduced Expression of Smo Inhibits -Catenin/Tcf-Dependent Wnt Signaling Colon cancer cells with activating mutations in the CTNNB1 (for -catenin) gene or inactivating mutations in APC suffer accumulation of -catenin that subsequently translocates from the cytoplasm into the nucleus where it binds to the TCF/LEF family of transcription factors and turns on transcription of the downstream target genes. 14,26 Because both SW480 and HCT116 cells, as well as the intestinal tumor cells of the Apc 716 mice, carry such mutations, we next investigated the possibility that down-regulation of the SMO gene may inhibit the Wnt-signaling pathway. We first measured -catenin-dependent transcription by luciferase assays using TOPFLASH and FOPFLASH reporter plasmids that have been used widely to monitor the -catenin-dependent Wnt signaling, containing a minimal promoter with 6 TCF/LEF binding sites and mutated binding sites, respectively. As shown in Figure 5A, SMO sirnas, but not sin.s., reduced the TOPFLASH activity to 50% in the SMO-high SW480 and HCT116 cells. To determine whether SMO sirnas suppress expression of the endogenous Wnt target genes in these cells, we next performed qrt-pcr and semiquantitative RT-PCR analyses. Knockdown of SMO in the SW480 or HCT116 cells significantly reduced expression of the Wnt target genes CCND1 (for cyclin D1) and tended to reduce the levels of MYC, STRA6, and T (encoding Brachyury protein) (Figure 5B and Supplementary Figure 7C) Expression of another Wnt target gene, PROX1, was also decreased by SMO knockdown in the SW480 cells, although its expression was not detectable in the HCT116 cells. Knockdown of CTNNB1 by sirna in these cell lines suppressed the TOPFLASH activity as well as expression of these target genes, confirming that their expression depended on -catenin (Supplementary Figure 8A and B). Accordingly, these results suggest that SMO can regulate the Wnt signaling in colon cancer cell lines that express SMO at high levels. To determine whether Smo heterozygosity also suppressed the Wnt signaling in Apc 716 mouse polyps, we next analyzed expression of the Wnt target genes in the polyps. As shown in Figure 5C and Supplementary Figure 7D, the expression levels of Wnt target genes Ccnd1, Prox1, Stra6, and T were lower in the polyps of the ApcSmo mouse than in those of the Apc 716 mouse.

7 August 2009 SMOOTHENED AND INTESTINAL TUMORIGENESIS 635 Figure 5. Effects of SMO down-regulation on -catenin-dependent transcription in SMO-high human colon cancer cell lines and in the polyps of Apc 716 mice. (A) Luciferase reporter assays using TOPFLASH and FOPFLASH reporter plasmids in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours. (B and C) Expression of SMO and the Wnt target genes analyzed by quantitative RT-PCR in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours (B) and in the small intestinal and colonic polyps of Apc 716 and ApcSmo mice (C). Error bars indicate SD. *P.05; **P.01. N.T., no treatment with sirna; sin.s., 40 nmol/l nonsilencing sirna; sismo, 40 nmol/l sirnas directed against SMO gene; ND, not detectable. SMO Knockdown Reduces Protein Level of Active -Catenin and Induces Its Nuclear Exclusion We then investigated the molecular mechanisms by which SMO knockdown reduced the Wnt-dependent transcriptional activity in the SW480 and HCT116 cells. It has been reported that the mrna level of -catenin is up-regulated in rhabdomyosarcomas and basal cell carcinomas of Smo transgenic mice, as well as in medulloblastomas of Ptc1 / mice. 31,32 However, SMO knockdowns did not significantly reduce the level of CTNNB1 (for -catenin) mrna in the SW480 or HCT116 cells (Supplementary Figure 9A). Likewise, Smo heterozygosity did not alter expression of Catnb (for -catenin) mrna in the intestinal polyps of Apc 716 mice (Supplementary Figure 9B). Notably, SMO knockdowns modestly reduced the protein level of total -catenin (by 50%) and strongly reduced that of the active (ie, unphosphorylated) form (by 80%) in these cells (Figure 6A). Furthermore, the levels of the active -catenin were reduced by 70% in the intestinal polyps of ApcSmo mice compared with the Apc 716 mice polyps (Figure 6B). To investigate whether SMO knockdown in SMO-high colon cancer cell lines affected the subcellular localization of the active -catenin, we then immunostained the active -catenin. As shown in Figure 6C, active -catenin was found mainly in the nucleus in SW480 and HCT116 cells. Importantly, SMO knockdown caused strong nuclear exclusion of the active -catenin in these SMO-high cells. These results suggest that suppression of Smo can inhibit the -catenin/tcf-dependent Wnt signaling by down-regulation and nuclear exclusion of active -catenin in the intestinal tumor cells. On the other hand, treatment with cyclopamine or the Hh-neutralizing antibody 5E1, or forced expression of Ptc1 in SW480 and HCT116 colon cancer cells, did not alter the TOPFLASH activity, protein level, or nuclear localization of the active -catenin (Supplementary Figure 10A F, and data not shown). Taken together, these data suggest that reduction of Smo expression inhibits the Wnt signaling by a mechanism independent of the Gli-mediated canonical Hh signaling. Discussion In the present study, we have identified a novel role for Smo in intestinal tumorigenesis. Our results demonstrate that Smo can stimulate intestinal tumor cell proliferation both in culture and in mice and provide the first direct genetic evidence that Smo plays an essential role in the formation of intestinal tumors (Figures 1 3). Consistent with our findings, expression of SMO at high levels has been reported in human colonic tumors, 5 and Smo transgenic mice develop hamartomatous polyps in

8 636 ARIMURA ET AL GASTROENTEROLOGY Vol. 137, No. 2 Figure 6. Effects of SMO knockdown on active -catenin in SMO-high human colon cancer cell lines and in the polyps of Apc 716 mice. (A and B) Expression of total and active -catenin analyzed by Western blotting in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours (A) and in the intestinal polyps of Apc 716 and ApcSmo mice (B). (C) Immunofluorescence analysis for subcellular localization of active -catenin in the SW480 and HCT116 cells treated with SMO sirnas for 72 hours. N.T., no treatment with sirna; sin.s., 40 nmol/l nonsilencing sirna; sismo, 40 nmol/l sirnas directed against SMO gene. the small intestine. 31 Our preliminary results also show that expression of SMO is increased in 9 out of 20 clinical samples of the primary colorectal adenocarcinomas compared with the normal epithelium of the same patients (data not shown). Smo-mediated activation of the Hh signaling is implicated in tumorigenesis. 4 However, our results suggest that the Hh-signaling activity is rather low in the adenoma epithelial cells of the Apc 716 mice that abundantly express Smo or in the SMO-high colon cancer cell lines (Figure 1A and Supplementary Figure 6). Furthermore, reduced expression of Smo does not suppress Gli-dependent Hh signaling in intestinal tumor cells (Figure 4A C, Supplementary Figure 7A and B). Our results are consistent with a previous report that found no mutations in the SMO gene in human colon cancer cell lines 33 and with reports showing that the Hh inhibitor cyclopamine does not affect the signaling activity of colon cancer cell lines in culture. 13,25 Although it was reported that the Hh ligand SHH was overexpressed in tumor tissues of colon cancer patients, a recent report suggests that Hh ligands produced by the tumor cells serve to activate the Hh signaling in the surrounding tumor stromal cells, 8 which also suggests that autocrine activation of the Hh signaling is rather rare in colon cancer. Although we cannot rule out some effects of Smo heterozygosity on the polyp size mediated by the tumor stroma of Apc 716 mice, expression of Smo was specifically detected in the polyp adenoma epithelial cells but not in the tumor stroma (Figure 1C and D). Taken together, these results indicate that Smo overexpression can contribute to the proliferation of intestinal tumor epithelial cells by a mechanism independent of Gli-mediated transcription. We have provided evidence that SMO can modulate the Wnt signaling in intestinal tumor cells. Namely, reduction of Smo suppressed TOPFLASH activity and expression of the Wnt target genes (Figure 5A C, Supplementary Figure 7C and D). Furthermore, SMO knockdown caused the reduced protein level and induced nuclear exclusion of the active -catenin (Figure 6). In addition, suppression of the Wnt signaling by Smo knockdown may be independent of the presence of Hh ligands (Supplementary Figure 10). These results are not likely to be secondary effects of the G1/S arrest caused by SMO sirnas because G1/S arrest induced by thymidine block did not affect the TOPFLASH reporter activity, expression of the Wnt target genes, or subcellular localization of the active -catenin significantly (data not shown). Cross talks between the Wnt signaling and Hh signaling are not unprecedented. The mrna level of -catenin is increased in tumors developed in the Smo transgenic mice and in the Ptc1 heterozygous knockout mice. 31,32 It has been reported that the level of active -catenin is raised in the skin tumors of the Smo mice, accompanied by increased transcription of the Wnt ligands by Gli1. 34 Although these reports suggest that activation of the Hh signaling can stimulate Wnt signaling through transcriptional induction of the Wnt ligands by Gli1, our results show that Smo can contribute to the Wnt signaling in a manner independent of Gli activation (Supplementary Figures 6 and 10). On the other hand, some papers report that the Wnt pathway is negatively regulated by the Hh signaling in colon cancer cell lines. Although 1 study shows that overexpression of IHH inhibits the Wnt signaling in DLD1 cells, 11 SMO expression in DLD1 cells appears to be silenced by methylation of the SMO promoter, 10 and

9 August 2009 SMOOTHENED AND INTESTINAL TUMORIGENESIS 637 we have confirmed an undetectably low level of SMO mrna in DLD1 cells (Supplementary Figure 1D). It is therefore not clear how Indian hedgehog can affect the Wnt signaling through a Smo-independent mechanism in DLD1 cells. Another report shows that overexpression of GLI1 may suppress the Wnt signaling in SW480 and HCT116 cells. 35 However, we found no alterations in the Gli1 level in SW480 and HCT116 cells treated with SMO sirna or in the polyps of ApcSmo mice as compared with that in the Apc 716 mice (Figure 4A C, Supplementary Figure 7A and B). Surprisingly, we have found the decreased protein level and strong nuclear exclusion of the active -catenin by SMO knockdown, which provides a novel type of interaction between Smo and Wnt signaling. With respect to the decreased level of the active -catenin, SMO sirnas did not affect the half-life of -catenin or the kinase activity of GSK3 in SW480 and HCT116 cells (data not shown), suggesting that reduced level of Smo does not affect degradation of -catenin. It is currently understood that Wnt signaling is mediated by the active -catenin that is unphosphorylated at serine 37 and threonine ,37 Although it remains to be investigated how the nuclear localization of this active -catenin is regulated, a recent report showed that Ran binding protein 3 (RanBP3) directly binds to the active -catenin and enhances its export from the nucleus in a chromosome region maintenance 1 (CRM1)-independent manner. 38 Further studies are required to elucidate the precise molecular mechanisms by which Smo modulates the protein level and subcellular localization of the active -catenin. In summary, we have demonstrated significant roles of Smo overexpression in the growth of mouse intestinal tumors and some human colon cancer cell lines. Importantly, we have found that Smo can regulate -catenindependent Wnt signaling in intestinal tumors through a mechanism that is independent of the canonical Hh pathway. These results suggest that Smo can be a novel therapeutic target for treating colorectal polyposis and cancer, even in the absence of Hh signal activation. Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev 2001;15: van den Brink GR. Hedgehog signaling in development and homeostasis of the gastrointestinal tract. Physiol Rev 2007;87: Lum L, Beachy PA. The Hedgehog response network: sensors, switches, and routers. Science 2004;304: Rubin LL, de Sauvage FJ. Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 2006;5: Oniscu A, James RM, Morris RG, et al. Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia. J Pathol 2004;203: Douard R, Moutereau S, Pernet P, et al. Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer. Surgery 2006;139: Monzo M, Moreno I, Artells R, et al. Sonic hedgehog mrna expression by real-time quantitative PCR in normal and tumor tissues from colorectal cancer patients. Cancer Lett 2006;233: Yauch RL, Gould SE, Scales SJ, et al. Paracrine requirement for hedgehog signalling in cancer. Nature 2008;18: Qualtrough D, Buda A, Gaffield W, et al. Hedgehog signalling in colorectal tumour cells: induction of apoptosis with cyclopamine treatment. Int J Cancer 2004;110: Zhu Y, James RM, Peter A, et al. Functional Smoothened is required for expression of GLI3 in colorectal carcinoma cells. 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Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node. Cell 2001;106: Fujishita T, Aoki K, Lane HA, et al. Inhibition of the mtorc1 pathway suppresses intestinal polyp formation and reduces mortality in Apc 716 mice. Proc Natl Acad Sci U S A 2008;105: Sasaki H, Hui C, Nakafuku M, et al. A binding site for Gli proteins is essential for HNF-3 floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 1997;124: Oshima M, Oshima H, Matsunaga A, et al. Hyperplastic gastric tumors with spasmolytic peptide-expressing metaplasia (SPEM) caused by TNF- -dependent inflammation in COX-2/mPGES-1 transgenic mice. Cancer Res 2005;65: Gayet J, Zhou XP, Duval A, et al. Extensive characterization of genetic alterations in a series of human colorectal cancer cell lines. Oncogene 2001;20: Ilyas M, Tomlinson IPM, Rowan A, et al. -Catenin mutations in cell lines established from human colorectal cancers. 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