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Research Article

Transcriptional Cooperation between the Transforming Growth Factor-B and Wnt Pathways in Mammary and Intestinal Tumorigenesis

Etienne Labbe´,1 Lisa Lock,2 Ainhoa Letamendia,2 Agnieszka E. Gorska,4 Robert Gryfe,3 Steven Gallinger,3 Harold L. Moses,4 and Liliana Attisano1,2

Departments of 1Medical Biophysics and 2Biochemistry, University of Toronto; 3Department of Surgery, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; and 4Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee

Abstract initiate signal transduction via cell surface serine/threonine kinase Transforming growth factor-B (TGF-B)andWntligands receptors that directly phosphorylate receptor-regulated Smad function in numerous developmental processes, and alter- proteins. Phosphorylated receptor-regulated Smads complex with ations of both signaling pathways are associated with Smad4 and then translocate to the nucleus and interact with DNA- common pathologic conditions, including cancer. To obtain bound transcription factors and other transcriptional regulators to insight into the extent of interdependence of the two signaling control gene expression (3). Signaling downstream of Wnt ligands h cascades in regulating biological responses, we used an in the canonical Wnt/ -catenin pathway involves stabilization of h oligonucleotide microarray approach to identify Wnt and cytoplasmic -catenin, a protein that is normally targeted for TGF-B target genes using normal murine mammary gland ubiquitin-mediated degradation via the action of a destruction epithelial cells as a model. Combination treatment of TGF-B complex composed of proteins including adenomatous polyposis h and Wnt revealed a novel transcriptional program that could coli (APC), Axin, and glycogen synthase kinase 3 . Wnt binding to h not have been predicted from single ligand treatments and the Frizzled and LRP5/6 receptors inhibits -catenin degradation, h included a cohort of genes that were cooperatively induced by leading to -catenin nuclear accumulation and activation of gene both pathways. These included both novel and known transcription in association with the DNA-binding lymphoid components or modulators of TGF-B and Wnt pathways, enhancer factor (LEF)/T-cell factor (TCF) proteins and other suggesting that mutual feedback is a feature of the coordi- cofactors (2). h nated activities of the ligands. The majority of the cooperative TGF- and Wnt ligands regulate a number of common processes targets display increased expression in tumors derived from during embryonic development, such as patterning of imaginal either Min (many intestinal neoplasia) or mouse mammary discs in Drosophila and tissue specification and organogenesis in tumor virus (MMTV)–Wnt1 mice, two models of Wnt-induced vertebrate embryos (2). These pathways can coordinate develop- tumors, with nine of these genes (Ankrd1, Ccnd1, Ctgf, Gpc1, mental events by regulating the expression of target genes such as Hs6st2, IL11, Inhba, Mmp14, and Robo1) showing increases in cerberus, chordin, crescent, goosecoid, noggin, siamois, and Xtwn in both. Reduction of TGF-B signaling by expression of a the Xenopus Spemann organizer, and Id2 and Msx1 in mammalian dominant-negative TGF-B type II receptor in bigenic MMTV- cells (4, 5). Further, mutations affecting the integrity of both the Wnt1/DNIIR mice increased mammary tumor latency and was Wnt and TGF-h pathways in human malignancies have been correlated with a decrease in expression of Gpc1, Inhba, and extensively reported. For instance, mutations in the human TGF-h Robo1, three of the TGF-B/Wnt cooperative targets. Our type II receptor, Smad2, and Smad4 occur in colon tumors; Smad4 results indicate that the TGF-B and Wnt/B-catenin pathways mutations are found in a high percentage of pancreatic cancers (6). are firmly intertwined and generate a unique gene expression Activation of the Wnt/h-catenin pathway through a variety of pattern that can contribute to tumor progression. [Cancer Res mechanisms is also widespread in human tumors. For instance, in 2007;67(1):75–84] sporadic colorectal cancers, biallelic loss of APC occurs in up to 85% of cases, with most remaining cases harboring h-catenin Introduction mutations (2). Although mutations in APC, Axin, and h-catenin are rare in human breast cancers, accumulation of nuclear h-catenin, a Growth factors belonging to the transforming growth factor-h hallmark of Wnt pathway activation, is seen in f60% of clinical (TGF-h) and Wnt family of ligands are involved in the numerous breast cancer samples (7). The most common mechanisms of Wnt aspects of embryonic development and are important regulators of pathway activation in breast cancers involve loss of expression of organ homeostasis in postnatal physiology (1, 2). TGF-h factors secreted frizzled-related protein 1 and Wnt inhibitory factor 1 through promoter methylation and overexpression of Wnt ligands (8–10). In addition, a number of signaling pathways with known h Note: Supplementary data for this article are available at Cancer Research Online alterations in breast cancers can affect -catenin function. These (http://cancerres.aacrjournals.org/). include the epidermal growth factor, insulin-like growth factor, L. Attisano is a Canada Research Chair. integrin-linked kinase, and Akt pathways, as well as the tumor Requests for reprints: Liliana Attisano, Department of Biochemistry, Terrence Donnelly Centre for Cellular and Biomolecular Research, Room 1008, 160 College suppressors p53 and PTEN (10). Street, University of Toronto, Toronto, ON, Canada M5S 3E1. Phone: 416-946-3129; Fax: Despite an established role in suppressing tumor formation, the 416-978-8548; E-mail: [email protected]. h I2007 American Association for Cancer Research. TGF- pathway can also act as a promoter of tumor invasion at doi:10.1158/0008-5472.CAN-06-2559 later stages of tumorigenesis (11). Whereas most tumors of www.aacrjournals.org 75 Cancer Res 2007; 67: (1). January 1, 2007

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research epithelial origin lose the ability to be growth inhibited by TGF-h and 4¶,6-diamidino-2-phenylindole staining. Nuclear versus cytoplasmic through various molecular or mutational mechanisms, retention of fluorescence was quantitated using the ArrayScan II reader and Cellomics a partially functional pathway favors a more aggressive tumor Nuclear Translocation software (Cellomics, Pittsburgh, PA). Phosphorylated behavior and consequently a less favorable prognosis (11). TGF-h Smad2/3 was detected by Western blotting using rabbit polyclonal anti– phospho-Smad2 antibody (1:2,000; Cell Signaling Technology, Danvers, MA). has been shown to synergize with oncogenic pathways in various Microarray processing and analysis. RNA was extracted from NMuMG systems. For example, transgenic mice overexpressing the HER2/ cells with TRI reagent (Sigma-Aldrich, St. Louis, MO) then purified using the h Neu oncogene and TGF- 1 under the control of the MMTV RNeasy kit (Qiagen, Valencia, CA). Indirect Cy3/Cy5 labeling of cDNAs, promoter have increased invasiveness and a higher frequency of hybridization, and array processing was done using standard protocols6 lung metastasis compared with single transgenics (12). Similarly, with in-house pair-spotted 70-mer oligonucleotide arrays (SLRI_MO7K) evidence suggests that Wnts can also cooperate with other representing 6,868 mouse genes from the UniGene database (Unigene Mm signaling pathways during tumorigenesis. For instance, in MMTV- build 83; Array-Ready Oligo set, OPERON). Scanning and analysis was done Wnt1 mice, targeted deletion of the estrogen receptor a delays with the GenePix 4000B scanner and software (Axon Instruments, tumor formation, and bigenic mice carrying fibroblast growth Molecular Devices, Sunnyvale, CA). For analysis, fluorescence data were factor-3 driven by MMTV and MMTV-Wnt1 develop tumors faster mean-centered and a ligand/control expression ratio was computed and log transformed. The data were sorted by decreasing or increasing median than mice with either transgene alone (13, 14). Interestingly, 2 h ratio to generate induced and repressed lists, respectively. Data were MMTV-Wnt1 mice crossed with MMTV-TGF- 1 mice show no obtained for six independent experiments with reciprocal Cy3/Cy5 labeling 5 h alterations in tumor formation, suggesting that TGF- cannot for a total of 12 hybridizations per condition. For the final analysis, all inhibit the growth of epithelial cells stimulated by Wnt1. We and experiments were used for individual Wnt3a and TGF-h treatments, whereas others have previously described a mechanism for cooperative seven hybridizations were used for the combined Wnt3a+TGF-h treatment. transcriptional regulation by mediators of the TGF-h and Wnt Mouse and human tumor samples. Min (many intestinal neoplasia; À pathways (15). Synergistic gene induction involves direct physical APC+/ ) mice bred on the C57BL/6J strain were obtained from The Jackson interaction between Smads and LEF/TCF proteins coincident with Laboratory (Bar Harbor, ME). Representative colon and small intestinal DNA binding by the complex to target promoters such as Xtwin, polyps and a matched normal sample were macroscopically harvested at Msx2, and Gastrin (16–18). Despite strong evidence that TGF-h and 5 months of age. The MMTV-Wnt1 transgenic mice, obtained from H.E. Varmus (Program Wnt factors cooperatively regulate developmental and homeostatic in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, processes, little is known about the specific transcriptional targets New York, NY), were bred to MMTV/DNIIRanimals (14) to generate MMTV- that are controlled by a combination of these ligands and whether Wnt1/DNIIRmice. The Wnt1 transgene was detected by Southern blot (14) a direct interplay of these two pathways regulates the various or PCR, whereas the DNIIR transgene was detected by PCR as previously stages of tumorigenesis. described (21). Virgin Wnt1 heterozygotes (24 females) and multiparous In this study, we have defined a novel transcriptional program Wnt1/DNIIRheterozygotes (19 females) were used in the tumor study. induced by TGF-h and Wnt that could not have been predicted by Mammary tumors were removed 3 to 4 weeks after detection. the analysis of the single ligand treatments alone. Further, we show Human familial adenomatous polyposis (FAP) tumor samples were that many of these cooperative targets display increased expression collected from eight clinically affected individuals with FAP from separate in tumors, suggesting that these two pathways may cooperate to kindreds. Informed consent for tissue use was obtained from each patient as per local Institutional Review Board approval. Matched normal (six promote tumor development. samples) and adenomatous tissues (eight samples) were dissected from fresh surgical colectomy specimens. Mouse and human tissues were fixed in 4% paraformaldehyde or 10% Materials and Methods buffered formalin, embedded in paraffin for sectioning, or frozen at À80jC Production of Wnt3a conditioned medium. Mouse L cells stably in RNAlater (Ambion, Austin, TX), and subsequently processed for RNA expressing mouse Wnt3a were generated using pPGK-neo-Wnt3a (19). extraction with TRI reagent (Sigma-Aldrich). Control or Wnt3a conditioned medium was collected from confluent cells Real-time quantitative reverse transcription-PCR analysis. Reverse cultured for 3 days in DMEM with 0.2% fetal bovine serum (FBS), transcription was done with random hexamers on DNaseI-treated total centrifuged to remove cell debris, and stored at 4jC for up to 3 months. RNA with Superscript II (Invitrogen). Real-time PCR was done using the ABI/ Human colorectal cell lines LS1034, HT29, and SW480 were grown in a- Prism 7000 Sequence Detection System (Applied Biosystems) and the Brilliant MEM with 10% FBS. SYBRGreen QPCRmaster mix (Stratagene, La Jolla, CA). Primer pairs Constructs, nuclear accumulation assays, and immunoblotting. The (Supplementary Table S3) designed using Primer Express (Applied Biosys- Smad-binding elements (SBE)-TOP plasmid was constructed by inserting tems) were used at 100 nmol/L and were validated against hypoxanthine two SBEs upstream of the LEF/TCF binding sites in the pGL3-OT version of phosphoribosyltransferase (HPRT) or glyceraldehyde-3-phosphate dehydro- TOPFLASH (20). For reporter assays, normal murine mammary gland genase (GAPDH) by comparative standard curve analysis. Relative quantita- DD (NMuMG) cells were transfected using LipofectAMINE (Invitrogen, tion was calculated by the Ct method normalized to HPRT or GAPDH. Carlsbad, CA) as previously described (16). LS1034 cells were transfected Immunohistochemistry, in situ hybridization, and cancer profiling using Fugene 6 according to the manufacturer’s instructions (Roche array hybridization. Paraffin-embedded tissue sections (7 Am) were Diagnostics, Indianapolis, IN). For nuclear accumulation assays, NMuMG prepared using standard protocols. Immunohistochemical staining was cells were incubated in control conditioned medium for 20 h, and TGF-h or done using the horseradish peroxidase-AEC kit (R&D Systems, Minneapolis, Wnt3a was added for the indicated times. The cells were fixed, MN) with mouse monoclonal anti–h-catenin (1:1,000; BD Transduction permeabilized, and proteins were detected using a mouse monoclonal Laboratories) or rabbit anti–phospho-Smad2 (1:500; Cell Signaling Tech- anti–h-catenin antibody (1:1,000; BD Transduction Laboratories, BD nology) and counterstained with Hematoxylin QS (Vector Laboratories, Biosciences, San Jose, CA) or rabbit polyclonal anti-Smad4 antibody Burlingame, CA). Digoxigenin-labeled RNA probes (Supplementary Table (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) followed by a FITC- S3) for in situ hybridizations were synthesized from sequence-verified conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) mouse cDNAs cloned from NMuMG into pBluescript(KS). Hybridizations

5 X. Chytil, Y. Chen, and H.L. Moses, unpublished data. 6 http://www.mshri.on.ca/microarray/.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. TGF-b and Wnt Transcriptional Cooperation were done using standard protocols. A pBluescript(KS)-InhibinhA human Results 32 cDNA construct was used to synthesize a [ P]dATP-labeled DNA probe B with the Rediprime II DNA labeling kit (Amersham Biosciences). The probe Identification and characterization of a Wnt3a- and TGF- – h was hybridized to the Cancer Profiling Array II (Clontech, Mountain View, responsive cell line. To understand how the TGF- and Wnt CA) according to the manufacturer’s instructions, scanned with the STORM signaling pathways cooperate to alter gene expression in mamma- phosphorimager reader, and analyzed with ImageQuant (Molecular lian cells, we first surveyed a variety of cell lines for their ability to Dynamics, Sunnyvale, CA). respondtobothpathwaysandtherebyidentifiedNMuMG

Figure 1. Identification of TGF-h and Wnt cooperatively induced genes in NMuMG cells. A, nuclear accumulation of endogenous h-catenin in response to Wnt3a (left) or Smad4in response to TGF-h (right) was examined in NMuMG cells using a quantitative immunofluorescence assay. Points and columns, mean nuclear-to-cytoplasmic ratio of arbitrary fluorescence units in 100 cells from three wells; bars, SD. B, NMuMG epithelial cells were transiently transfected with a luciferase reporter plasmid responsive to TGF-h (3TP-lux), Wnt3a (TOPFLASH), or both ligands (SBE-TOP). Transfected cells were incubated overnight in the presence of 100 pmol/L TGF-h and/or Wnt3a conditioned medium. Luciferase activity was normalized using a cotransfected h-galactosidase reporter. Columns, mean of quadruplicates; bars, SD. C, top 124 induced and repressed genes from each ligand treatment were sorted by decreasing fold change and organized in Venn groups according to responsiveness to Wnt3a, TGF-h, and a combination of Wnt3a and TGF-h. D, candidate Wnt3a+TGF-h cooperative target genes obtained from the microarray analysis were validated by real-time quantitative reverse transcription-PCR using RNAs obtained from ligand-treated NMuMG cells for the indicated times.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research epithelial cells. Stimulation of NMuMGs with Wnt3a and TGF-h Treatment of cells with TGF-h alone resulted in the regulation of resulted in nuclear accumulation of h-catenin and Smad4 (Fig. 1A), novel as well as known targets, such as Bmp1, Ctgf, Serpine1/ and activated the TOPFLASH and 3TP-lux promoters, respectively plasminogen activator inhibitor-1 (PAI-1), and Edn1 (22–25). (Fig. 1B, left). We previously showed that Smads interact with LEF1 Similarly, genes regulated by Wnt3a included both novel (Hs3st1, to synergistically activate promoter constructs containing Smad SerpinB9, Ccna2, Sgk) and previously characterized targets such as and LEF/TCF binding sites (16). Consistent with this, costimulation Ahr, Axin2, Spp1, Ccnd1, and Thbs1 (26–30). These results confirm with TGF-h and Wnt3a synergistically activated the SBE-TOP- that NMuMG cells respond appropriately to TGF-h and Wnt3a and FLASH reporter in which Smad-binding elements were introduced show the validity of our approach. Interestingly, cells costimulated upstream of the LEF/TCF sites of TOPFLASH (Fig. 1B). Together, with TGF-h and Wnt3a displayed an expression profile distinct these results confirm that TGF-h and Wnt3a induce nuclear from that observed with single ligand treatments (Fig. 1C). Thus, accumulation of their respective transducers and cooperatively simultaneous receipt of TGF-h and Wnt signals yields a gene activate specific transcriptional responses in NMuMG cells. expression profile that is not simply an additive effect of individual Wnt3a and TGF-B cooperate to induce expression of a ligand treatments. subset of target genes. We next examined the effects of Validation of TGF-B and Wnt3a-induced target genes by TGF-h and Wnt3a on global gene expression patterns following real-time quantitative reverse transcription-PCR. Given the 16-h treatment of NMuMG cells with either ligand alone or in limited sensitivity range typical of microarrays and the need for combination. RNA was isolated and processed with control-treated secondary validation, we next examined the expression of samples for pairwise hybridizations to oligonucleotide microarrays candidate targets using real-time quantitative PCR, focusing on composed of f7,000 known genes. Lists of regulated genes for induced genes. We selected the first 25 to 30 highest ranked genes each condition were determined (Supplementary Table S1) as from each treatment condition (Wnt3a, TGF-h, and Wnt3a+TGF-h) described in Materials and Methods. representing a total of 60 distinct genes. RNA was isolated from

Figure 2. A, a subset of Wnt3a/TGF-h target genes is induced by TGF-h in LS1034, a TGF-h–responsive APC-mutated colorectal cell line. LS1034 cells transfected with Wnt3a [TOPLASH (TOP) and mutant control FOPFLASH (FOP)], Wnt3a+TGF-h (SBE-TOP), or TGF-h–responsive [3TP-lux (3TP)] reporters in the presence or absence of APC were treated with TGF-h (left). Bars, SD of quadruplicate assays from a representative experiment. RNA from LS1034cells treated overnight with 100 pmol/L TGF-h was assayed for Wnt3a/TGF-h target gene expression using real-time quantitative reverse transcription-PCR (right). Gene expression in each sample was normalized to HPRT as the internal control and expressed as fold induction by TGF-h over control. Bars, SD for triplicate amplifications from a representative experiment. B to E, Wnt/ h-catenin and TGF-h/Smad pathways are active in hyperplastic cells in Min intestinal adenomas and MMTV-Wnt1 tumors. B, cell lysates from HT29 and SW480 colon cancer cell lines treated overnight with 100 pmol/L TGF-h were immunoblotted with anti–phospho-Smad2/3 (1:2,000) to show antibody specificity. C to E, paraffin-embedded sections of wild-type intestinal tissues (C), Min adenomas (D), wild-type mammary glands, and MMTV-Wnt1 mammary tumors (E) were stained for h-catenin and phospho-Smad2/3, revealing extensive costaining in normal and tumor tissues.

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NMuMG cells treated with ligands for 2, 4, 8, or 16 h and was used intestinal polyps of Min mice, strong cytoplasmic h-catenin to determine gene expression levels by quantitative PCR(Fig. 1 D; staining was observed throughout areas displaying abnormal Supplementary Table S2). Of 59 genes successfully amplified, 53 histology, whereas adjacent healthy epithelium showed only displayed z2-fold Wnt3a- and/or TGF-h–dependent regulation, membrane staining (Fig. 2D, top). Extensive staining for nuclear representing a 90% validation rate. Consistent with the use of a phospho-Smad2/3 was also detected in adenomatous lesions 16-h ligand treatment for our microarray screen, many of the (Fig. 2D, bottom). These results confirm that Wnt/h-catenin regulated genes displayed maximal activation at the 8 or 16 h time signaling is strongly activated in Min intestinal adenomas, whereas points. However, a few genes were induced early such as known normal epithelium only shows spatially limited activation within direct targets of TGF-h (Ctgf )andWnt(Axin2;Fig.1D; the crypt compartment. Surprisingly, however, the TGF-h/Smad Supplementary Table S2). Our quantitative PCRanalysis of gene pathway seems to be widely activated in both normal and expression in cells treated simultaneously with TGF-h and Wnt3a adenomatous epithelium of the small bowel and colon throughout revealed patterns consistent with the microarray data. Of particular the entire crypt-villus axis. interest, a number of genes were additively or cooperatively We next examined Wnt and TGF-h pathway activation status in induced when both TGF-h and Wnt3a were used together (Fig. 1D; the mammary epithelium of MMTV-Wnt1 mice. These transgenic Supplementary Table S2). Of note, several of these genes have been animals overexpress the Wnt1 oncogene in mammary epithelial previously implicated as modulators (Gpc1, Cyr61, Mmp14)or cells leading to ductal hyperplasia during late embryogenesis components (Axin2, Inhba) of TGF-h or Wnt pathways. and mammary adenocarcinomas in 50% of females by the age of Cooperative activation of TGF-B and Wnt target genes in 6 months (32). Staining of tissue sections from MMTV-Wnt1 colorectal cancer cells. Components of the Wnt and TGF-h mammary tumors revealed strong cytoplasmic h-catenin staining pathways are frequently mutated in human colon cancers. Thus, in most ductal and alveolar epithelial cells, suggestive of active Wnt we next investigated whether cooperative TGF-h/Wnt targets signaling (Fig. 2E). Extensive nuclear phospho-Smad2/3 staining identified in NMuMG epithelial cells might also be cooperatively enhanced in colorectal cancer cells. As most, if not all, established colorectal cell lines harbor activating mutations affecting the Wnt pathway, we focused on examining the effect of TGF-h in the responsive human colorectal line, LS1034, which harbors a mutation in APC. Transfection of LS1034 cells with the TOPFLASH reporter revealed a high level of basal luciferase activity that was abrogated in the presence of overexpressed APC or by a mutant version of the reporter lacking LEF/TCF binding sites (FOPFLASH), thereby confirming constitutive Wnt signaling in this line (Fig. 2A, left). Furthermore, TGF-h–dependent activation of both the 3TP- lux and SBE-TOP reporters showed that LS1034 cells can respond to TGF-h alone or in cooperation with the activated Wnt pathway to regulate target gene transcription (Fig. 2A, left). Consistent with this, a TGF-h–induced increase in endogenous Smad7 and PAI-1 levels, two known TGF-h target genes, was observed (Fig. 2A, right). Of 17 genes successfully amplified in LS1034 cells, incubation with TGF-h increased transcript expression of 12 cooperative targets by z2-fold (Fig. 2A, right). Thus, TGF-h can further stimulate the expression of 70% of cooperative target genes above the expression levels reached in the context of an activated Wnt/h-catenin pathway. These results are consistent with the notion that TGF-h and Wnt can cooperate to activate gene transcription in two epithelial cell systems, a mouse mammary epithelial line and a human colorectal cancer line. TGF-B and Wnt pathways are active in neoplastic intestinal and mammary gland epithelium. To investigate whether TGF-h and Wnt can cooperate in vivo, we first assessed pathway activation in normal and neoplastic epithelium of two mouse models of Wnt-induced tumors. For gastrointestinal cancers, we À used Min (APC+/ ) mice, which harbor a mutation in APC and develop multiple small-bowel adenomas and colon microadenomas (31). Consistent with previous reports, h-catenin staining was Figure 3. Wnt3a/TGF-h target genes are overexpressed in adenomas from detected mainly at cell-to-cell junctions of wild-type intestinal Min mice and MMTV-Wnt1 mammary tumors. The expression of Wnt3a/TGF- epithelial cells (Fig. 2C) and occasionally in the nucleus of crypt h–regulated genes in total RNA extracted from small bowel and colon adenomas of Min mice (left) and MMTV-Wnt1 mammary tumors (right) was analyzed by cells, which is indicative of active Wnt signaling (data not shown). real-time quantitative reverse transcription-PCR. Data are represented as Staining for phospho-Smad2/3 in these sections, using a specific log2-transformed mean ratio of expression in adenomas/tumors normalized to a antibody (Fig. 2B), revealed extensive nuclear localization, demon- matched healthy sample (for Min), or to the average of four normal mammary h gland samples (for MMTV-Wnt1). Points, mean of triplicate amplification strating active TGF- signaling in small bowel and in colonic reactions. Vertical bars, average expression of each gene across all samples. epithelial cells of both crypts and villi (Fig. 2C). In contrast, in *, genes significantly overexpressed in both tumor types. www.aacrjournals.org 79 Cancer Res 2007; 67: (1). January 1, 2007

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Cancer Research was also evident in a large proportion of hyperplastic alveolar cells both Min and MMTV-Wnt1 tumor models. In summary, these (Fig. 2E), indicative of an active TGF-h/Smad pathway. Thus, these results show that half of the genes identified as cooperatively results suggest that both the Wnt/h-catenin and TGF-h/Smad induced genes in NMuMG cells show marked in vivo overexpression pathways are fully activated in epithelial cells of Min intestinal in Wnt/h-catenin–induced intestinal and mammary tumors. adenomas and MMTV-Wnt1 mammary tumors, and may therefore Epithelial and stromal overexpression of cooperative targets cooperate to induce target genes in vivo. in Min adenomas. To confirm our quantitative PCRdata, we did Expression of TGF-B and Wnt cooperatively induced genes RNA in situ hybridization on a subset of genes in tumors from Min in mouse models of Wnt/B-catenin–induced tumors. As the mice. As previously reported, Axin2 was highly expressed in Min TGF-h and Wnt pathways are both active in Min and MMTV-Wnt1 adenomas (Fig. 4A; ref. 27). Interestingly, Gpc1 and Robo1 showed tumors, we next examined the expression of our cooperative target similar expression patterns in adenomas, suggesting that these genes in these tissues. For intestinal adenomas, we used macro- genes may be coregulated with Axin2 in adenomatous intestinal scopically dissected samples from Min mice. Of 18 genes examined, epithelial cells. Although we also identified Inhba as a TGF-h/ 13 displayed a z2-fold (P < 0.05) increase in expression in Wnt3a cooperative target in NMuMG epithelial cells, prominent adenomas versus normal samples. The average increase in Inhba expression was detected in stromal cells within and expression ranged from 4.7- to 43-fold, with the top seven most surrounding adenomas, but not in epithelial cells (Fig. 4B). highly overexpressed genes being IL11, Robo1, Inhba, Ankrd1, Immunohistochemistry with an antibody specific to Activin A Hs6st2, Mmp14, Gpc1, and Axin2 (Fig. 3, left). Overexpression of (Inhba homodimers) revealed that whereas Activin A marked the Axin2 and Ccnd1 in Min adenomas is consistent with previous stromal compartment of normal small bowel and colon villi, this reports (27, 33). secreted factor was evident within both stromal and epithelial The expression of cooperative targets was also examined in compartments of adenomas (Fig. 4B). mammary tumors derived from MMTV-Wnt1 mice (Fig. 3, right). Of Disruption of TGF-B signaling in Wnt1-induced mammary the 18 genes tested, 10 displayed a significant (P < 0.05) increase of tumors increases latency and alters expression of some Wnt/ z2-fold in tumors versus wild-type mammary glands. Ankrd1 and TGF-B cooperative target genes. Overexpression of a dominant- Gpc1 were the most highly overexpressed genes (14- and 7.5-fold, negative TGF-h type II receptor (DNIIR) driven by the MMTV respectively). Nine genes, namely Ankrd1, Ccnd1, Ctgf, Gpc1, Hs6st2, enhancer attenuates TGF-h signaling and results in the develop- IL11, Inhba, Mmp14, and Robo1, were significantly overexpressed in ment of mammary carcinomas with a median latency of >2 years

Figure 4. Wnt3a/TGF-h target genes are overexpressed in the dysplastic cells of Min adenomas. A, paraffin-embedded sections of Min small bowel (top) and colon (bottom) adenomas were analyzed by anti–h-catenin immunohistochemistry (left) and by RNA in situ hybridization (right) with the appropriate antisense digoxigenin-labeled RNA probes. Axin2, Gpc1, and Robo1 were strongly expressed in dysplastic epithelium with dysregulated h-catenin (circled areas). B, in situ RNA hybridization for Inhba in small bowel and colon adenomas revealed strong stromal staining restricted to adenomas (left), whereas immunohistochemical detection of the active Inhba dimer (Activin A) showed stromal localization in normal tissues (center), but strong stromal and epithelial staining in adenomas (right).

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(21). To examine the contribution of the TGF-h pathway to tumor latency in the context of activated Wnt signaling, we abrogated TGF-h signaling in MMTV-Wnt1–induced tumors using the MMTV/DNIIRtransgene. For this, we generated bigenic MMTV/ Wnt1/DNIIRmice and analyzed the development of mammary tumors. As previously reported (13), 50% of MMTV-Wnt1 females develop tumors within 24 weeks. In mice expressing both transgenes, we observed an increase in tumor-free survival, with tumors appearing in 50% of mice only after 45 weeks (P < 0.0001; Fig. 5A). These results indicate that endogenous TGF-h signaling in the mammary epithelium promotes Wnt1-driven tumorigenesis. We next examined the effect of blocking TGF-h signaling on the expression of cooperatively regulated TGF-h/Wnt target genes by quantitative PCR. MMTV-Wnt1 and MMTV-Wnt1/DNIIR tumor samples with similar levels of Wnt1 expression were identified and further analyzed (P = 0.2; Fig. 5B, top left). Our previous analysis revealed that nine of the Wnt/TGF-h cooperative target genes displayed increased expression in MMTV-Wnt1 tumors and Min adenomas (Fig. 3). Thus, we examined the expression of these genes to assess the effect of reduced TGF-h signaling. Although Ankrd1, Ccnd1, Ctgf, IL11, Hs6st2, and Mmp14 were expressed at similar levels in both tumor types (data not shown), Gpc1, Inhba, and Robo1 displayed significantly reduced expression (P < 0.05) in Wnt1/DNIIRtumors (Fig. 5 B). Of note, these three genes were among those showing the most dramatic TGF-h/Wnt3a cooperativity in NMuMG cells. Cooperative Wnt/TGF-B targets in FAP adenomas and human cancers. Our previous data showed that Wnt/TGF-h cooperative targets are overexpressed in intestinal adenomas of Min mice, a model for human FAP. We next investigated whether the same cooperative genes exhibited increased expression in eight human FAP samples using quantitative PCR. Consistent with previous studies (27), Axin2 was overexpressed in adenomas Â À10 by an average 10.1-fold (P =1.5 10 ). Of nine Wnt/TGF-h Figure 5. Tumor latency is increased in bigenic Wnt1/DNIIR compared with targets whose expression was increased in Min and MMTV-Wnt1 MMTV-Wnt1 mammary tumors and is associated with down-regulation of a subset of Wnt3a/TGF-h target genes. A, bigenic MMTV-WNT1/DNIIR mice tumors, Ccnd1, Hs6st2, and Inhba were also significantly overex- remain tumor-free for f20 wk longer than MMTV-Wnt1 transgenic mice pressed in FAP tumors by z2-fold and had mean expression (P < 0.0001). B, expression of Wnt1, Gpc1, Inhba, and Robo1 in wild-type ratios of 2.2-fold (P = 0.0001), 2.7-fold (P = 0.002), and 3.2-fold mammary glands. MMTV-Wnt1 and MMTV-Wnt1/DNIIR mammary tumors were analyzed by real-time quantitative reverse transcription-PCR. Although Wnt1 (P = 0.003), respectively. We also analyzed the Inhba status by levels are not significantly different in Wnt1 versus Wnt1/DNIIR tumors (P = 0.2), hybridization of a radiolabeled probe to a human cancer profiling decreased expression of Gpc1 (P = 0.003), Inhba (P = 0.02), and Robo1 array. Extensive overexpression of Inhba in colon cancers (average (P = 0.002) in bigenic Wnt1/DNIIR tumors was observed. 3.3-fold), as well as cancers of the small intestine (3.0-fold), rectum (4.1-fold), and, to a weaker extent, in breast malignancies (2.4-fold; Fig. 6B), was observed. Although our sample size is Interestingly, many of the identified cooperative genes encode for limited, these findings indicate that increased expression of TGF- proteins involved in signaling processes and it is particularly h/Wnt cooperative targets may also be involved in human cancer intriguing that several of these genes are modulators or progression. components of the TGF-h and Wnt pathways themselves. For instance, Axin2, an identified cooperative target, functions redundantly with Axin within the h-catenin destruction complex Discussion in the Wnt pathway (2) and can interact with Smad3 to facilitate its Involvement of Wnt and TGF-h signaling in embryonic dev- activation by the TGF-h receptor complex (34). Another cooper- elopment, tissue homeostasis, and pathologic conditions suggests ative target, the heparan-sulfate proteoglycan, Gpc1, is known to that these pathways may function within a complex interdepen- modulate the activity of various growth factors, including TGF-h dent and mutually regulating network (1, 2). To gain mechanistic and Wnts (35). Remodeling of the 6-O sulfation state of Gpc1 by insights into cooperative signaling by Wnt and TGF-h, we used a QSulf1 can modulate Wnt signaling (36) and, interestingly, we microarray-based approach to evaluate crosstalk effects on gene identified 6-O-sulfotransferase 2 (Hs6st2), a potential modifier of expression. We observed that costimulation of epithelial-derived Gpc1, as a cooperative target of Wnt and TGF-h. Knockdown of tissue culture cells with both factors was not simply a sum of the Hs6st activity in zebrafish leads to myogenic defects and up- individual responses, rather that the ligands both positively and regulation of the Wnt target genes myoD and engrailed2 (37). negatively modified each other’s gene responses. This highlights the Because both Wnt and TGF-h–related pathways are strongly complex, intertwined nature of the two signaling pathways. implicated in myogenesis, these results suggest that Hs6st activity www.aacrjournals.org 81 Cancer Res 2007; 67: (1). January 1, 2007

Figure 6. Overexpression of Wnt3a/ TGF-h target genes in FAP polyps and other human cancers. A, RNA extracted from FAP polyps was analyzed by real-time quantitative RT-PCR for the indicated genes. Results are expressed as the log2-transformed ratio of transcript expression in polyps normalized to the matched normal samples, or, if unavailable, to the mean of all normals analyzed in parallel. Vertical bars, average expression of each gene across all samples. *, genes overexpressed in mouse tumor models (Fig. 3). Ankrd1, Dusp1, and IL11 are not shown due to poor or no amplification. B, the expression of Inhba in human cancers was examined by hybridization of antisense 32P-labeled cDNA probe to a cancer profiling array. Densitometric analysis of the autoradiogram (bottom). Tumor/normal (T/N) expression ratios are graphed using the indicated ranges.

may be a novel modulator of these pathways. Differential sulfation types, and our expression analysis showed that nine cooperative of Gpc1 also regulates its affinity for Slit ligands (38), which elicit targets identified in tissue culture cells were also significantly their biological effects via binding to the Roundabout (Robo) family overexpressed in both Min adenomas and MMTV-Wnt1 tumors. of transmembrane receptors (39). Of note, we also identified Robo1 Furthermore, preliminary analysis in a small sampling of FAP as a cooperative target of Wnt and TGF-h. Finally, Inhba, another patients revealed that four of these genes (Axin2, Ccnd1, Hs6st2, cooperative target, is itself a member of the TGF-h family of and Inhba) also show increased expression. Examination of a ligands. Our results, therefore, show that cooperative signaling by larger number of samples will be required to determine the extent Wnt and TGF-h up-regulates the expression of several known and of overexpression of our set of cooperative targets in FAP potentially novel feedback regulators. adenomas and whether these genes might contribute to tumor Molecular alterations affecting TGF-h and Wnt pathway progression in other human cancers. components in human cancers and mouse tumor models have To directly assess the effect of TGF-h signaling on Wnt1- been widely reported (6). Activation of Wnt signaling is a feature induced tumors, we generated double-transgenic mice by crossing of several human malignancies with most colorectal tumors MMTV-Wnt1 to MMTV/DNIIRanimals. The MMTV/DNIIR harboring mutations in APC or h-catenin (40). In contrast, for the transgene alone does not delay ductal development, although it TGF-h pathway, mutations or epigenetic modifications that target causes lobulo-alveolar development in virgin animals (41). Thus, the receptors or Smads lead to loss or attenuation of TGF-h– the observed delay in tumor formation in the bigenic versus mediated growth inhibition. Although the selective loss of TGF- MMTV-Wnt1 mice is consistent with the model that endogenous h–mediated growth inhibition favors progression of epithelial TGF-h signaling in mammary epithelial cells contributes to Wnt1- tumors, maintenance of other aspects of TGF-h signaling induced tumorigenesis. Concomitant with the increase in tumor enhances tumor invasion and metastasis (11). This suggests that latency in bigenic animals, we observed that at least three an intact TGF-h pathway integrates two opposing actions: a cooperative target genes, namely Gpc1, Inhba, and Robo1, show dominant activity that prevents tumor progression and another reduced expression levels in Wnt1/DNIIRtumors compared with activity that favors it. This also implies that crosstalk events those from Wnt1 animals. Although a few gene targets may be between a fully functional TGF-h pathway and other tumor- primary contributors to the TGF-h effect in this model, we initiating oncogenic pathways, such as Wnt, may set up a gene believe that a combinatorial effect on overall gene expression expression program that facilitates early tumor progression. patterns is more likely responsible for the increase in tumor Consistent with this possibility, we observed that in two mouse latency. In contrast to our study, in two other mammary tumor models of Wnt-induced tumors, Min and MMTV-Wnt1 mice, the models, MMTV-TGF-a and MMTV-polyoma middle T antigen– Wnt/h-catenin and TGF-h/Smad pathways are active in tumor induced tumors, decreased TGF-h signaling has been shown to epithelial cells. The molecular pathology of distinct cancer types reduce rather than augment tumor latency (21, 42). Interestingly, is quite diverse; however, we were particularly interested in genes although targeted deletion of one Smad4 allele was shown to cooperatively regulated by TGF-h and Wnt in multiple tumor increase tumor size in Min mice (43), specific deletion of Smad4

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. TGF-b and Wnt Transcriptional Cooperation in T cells, but not in epithelial cells, leads to spontaneous protein was abundant throughout the tumor. We postulate that epithelial cancers of the gastrointestinal tract (44). Together with persistent stimulation of tumor cells by Activin A produced by the our data, these observations suggest that the molecular basis for stroma may establish a positive autoregulatory loop that induces the dichotomous role of TGF-h in tumorigenesis may not stem Inhba transcription in tumor epithelial cells in late tumorigenesis. from TGF-h–only initiated signals per se, but rather from the Consistent with this notion, overexpression of Inhba leading to combinatorial effects of TGF-h on gene expression patterns that overproduction of Activin A protein by epithelial tumor cells has occur in the context of distinct oncogenic pathways. been reported in stage IV colorectal cancer (45). A more widespread In our study, we examined cooperativity between TGF-h and role for Inhba in neoplasia is also suggested by the observation that Wnt. However, Activin A, which is composed of an Inhba dimer, Inhba is overexpressed in a range of human tumors (46–50). is a TGF-h family member that also signals through Smad2/3 to activate TGF-h–like signals. Thus, our identification of Inhba as a Acknowledgments cooperative target is of particular interest. We showed that Inhba Received 7/11/2006; revised 10/7/2006; accepted 11/3/2006. expression is increased in MMTV-Wnt1 and Min tumors, decreased Grant support: National Cancer Institute of Canada with funds from the Cancer Research Society and a Canada Research Chair (L. Attisano), USPHS grants CA085492 in MMTV-Wnt1/DNIIRtumors, and overexpressed in human and CA102162 (H.L. Moses), and a Canadian Institutes of Health Research Doctoral intestinal and breast malignancies. Thus, Activin is a candidate Research Award (E. Labbe´).R. Gryfe is a Charles H. Hollenberg Clinician Scientist with for a TGF-h superfamily factor that cooperates with activated Wnt/ funds from Eli-Lilly Canada, Cancer Care Ontario, and the Canadian Institutes of Health Research. h-catenin pathway in tumor epithelial cells. Although Inhba is a The costs of publication of this article were defrayed in part by the payment of page Wnt/TGF-h cooperative target in epithelial cells, in the Min mouse- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. derived adenomas, mRNA expression was detected primarily in the We thank C. Silvestri for insightful comments and C. Ash and K. Boras-Granic for stromal compartment. On the other hand, secreted Activin A expert technical advice.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2007 American Association for Cancer Research. Transcriptional Cooperation between the Transforming Growth Factor- β and Wnt Pathways in Mammary and Intestinal Tumorigenesis

Etienne Labbé, Lisa Lock, Ainhoa Letamendia, et al.

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