Tbx3 Is a Downstream Target of the Wnt/B-Catenin Pathway and a Critical Mediator of B-Catenin Survival Functions in Liver Cancer

Research Article

Claire-Ange´lique Renard,1,2 Charlotte Labalette,1,2 Carolina Armengol,1,2 Delphine Cougot,1,2 Yu Wei,1,2 Stefano Cairo,1,2 Pascal Pineau,1,3 Christine Neuveut,1,2 Aure´lien de Reynie`s,4 Anne Dejean,1,3 Christine Perret,5,6 and Marie-Annick Buendia1,2

1Institut National de la Sante et de la Recherche Medicale, U579; 2Oncogenesis and Molecular Virology Unit and 3Nuclear Organization and Oncogenesis Unit, Institut Pasteur; 4Ligue Nationale contre le Cancer; 5De´partementGe´ne´tique,De´veloppementet Pathologie Mole´culaire, Institut Cochin; and 6Institut National de la Sante et de la Recherche Medicale, U567, Paris, France

Abstract interacts with DNA-binding factors of the T-cell factor/lymphoid enhancer factor (Tcf/Lef) family to promote transcription of Wnt Tbx3 encodes a transcriptional repressor that is important for diverse patterning events during development, and Tbx3 target genes. Depending on cell context, the selective expression of mutation in humans causes the ulnar-mammary syndrome. distinct sets of Wnt-responsive genes is strictly controlled by the interplay of signaling pathways. Increasing numbers of candidate Here, we describe the identification of Tbx3 in array-based h search for genes downstream Wnt/B-catenin that are impli- -catenin targets include cell cycle and growth regulators and proteins involved in cell-matrix interactions, migration and cated in liver tumorigenesis. Overexpression of Tbx3 is closely 7 B invasion, and in the regulation of the Wnt pathway. associated with the mutational status of -catenin in murine h liver tumors induced by Myc as well as in human hepatocel- Recent studies have outlined the importance of Wnt/ -catenin lular carcinomas and hepatoblastomas. Moreover, Tbx3 signaling in regulating liver cell proliferation during development transcription is activated by ectopic expression of B-catenin (2–4) and in governing essential functions in the adult liver (5–7). in mouse liver and in human tumor cell lines. Evidence that Moreover, aberrant reactivation of Wnt signaling is a predominant B mechanism implicated in liver tumorigenesis. Mutations of the Tbx3 transcription is directly regulated by -catenin is h provided by chromatin immunoprecipitation and reporter -catenin and Axin genes leading to constitutive activation of h-catenin have been identified in hepatocellular carcinoma (HCC) assays. Although HepG2 cells stably transfected with Tbx3 display moderately enhanced growth rate, the dominant and hepatoblastoma, and frequent overexpression of the Wnt negative mutant Tbx3-Y149S drastically inhibits hepatoma receptor Frizzled-7 is a major early event in hepatocarcinogenesis in vitro in vivo (refs. 8–11; for reviews, see refs. 12, 13). Animal models have been cell growth and . Moreover, small interfering h RNAs (siRNA) directed against Tbx3 inhibit anchorage- instrumental in allowing the identification of -catenin target independent growth of liver and colon carcinoma cells. We genes in nondiseased or tumorous liver, including liver-specific further show that inhibition of Tbx3 expression by specific enzymes involved in glutamine and nitrogen metabolism, such as siRNAs blocks B-catenin–mediated cell survival and renders glutamine synthetase (GS), ornithine aminotransferase, the gluta- cells sensitive to doxorubicin-induced apoptosis. Conversely, mate transporter (GLT-1), and the cytochrome P450 enzymes ectopic expression of Tbx3 inhibits apoptosis induced by CYP1A2 and CYP2E1 (5, 6, 14–17). However, c-myc has not been found thus far to be regulated by h-catenin in the liver context, and B-catenin depletion. Marked overexpression of Tbx3 in a subset of hepatoblastomas is associated with chemotherapy- the oncogenic program triggered by Wnt signaling in hepatocarcinogenesis remains largely unknown. resistant phenotype and unfavorable patient outcome. These Myc results reveal an unsuspected role of Tbx3 as a mediator of We have shown previously that transgenic mice carrying a B-catenin activities on cell proliferation and survival and as oncogene controlled by woodchuck hepatitis virus (WHV) regulatory sequences were highly predisposed to liver cancer (18, 19). an important player in liver tumorigenesis. [Cancer Res h 2007;67(3):901–10] Activating mutations of -catenin were found at significant rates in these HCCs, as for other Myc transgenic strains, suggesting that the survival functions of h-catenin might rescue tumor cells from Introduction Myc-induced apoptosis (8, 19, 20). Consistent with this notion, The Wnt/Wingless pathway plays a pivotal role in embryonic inactivation of p53 was found to represent an alternative oncogenic development and adult homeostasis by regulating cell fate, mechanism (9, 19). To explore downstream genetic programs proliferation, and differentiation, and it is aberrantly reactivated activated by Wnt/h-catenin signaling in liver cancer, we employed in various human cancers (1). Mutations in adenomatous polyposis microarray profiling and found that the T-box protein 3 (Tbx3) was coli, Axin, and h-catenin genes that mimic Wnt activation result in specifically activated in murine tumors carrying mutant h-catenin. the stabilization of h-catenin, which then moves to the nucleus and The closely related genes TBX2 and TBX3 are members of the T-box gene family that play an important role in patterning events during development. Tbx3 is notably implicated in heart, limb, Note: Supplementary data for this article are available at Cancer Research Online and posterior digit specification and in mammary gland develop- (http://cancerres.aacrjournals.org/). Requests for reprints: Marie-Annick Buendia, Oncogenesis and Molecular ment (21, 22). In humans, Tbx3 mutations are responsible for the Virology Unit, Institut National de la Sante et de la Recherche Medicale U579, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, France. Phone: 33-145-688-866; Fax: 33-145-688-943; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-2344 7 http://www.stanford.edu/~rnusse/pathways/targets.html. www.aacrjournals.org 901 Cancer Res 2007; 67: (3). February 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research ulnar-mammary syndrome (UMS), an autosomal dominant disor- Total RNA was extracted from each sample using the RNeasy Kit der characterized by upper limb deficiencies and apocrine/ (Qiagen) and treated with RNase-free DNase (Ambion). mammary gland hypoplasia (23). Tbx2/Tbx3 contain a conserved Oligonucleotide microarray and statistical analysis. Fifty-five human transcription-repression domain and can repress basal and HCCs, 24 hepatoblastomas, and 9 pools of nontumorous livers (5 from adults and 4 from children) were selected on the basis of high RNA quality as activated transcription (24). Identification of p14ARF as a direct determined with the Agilent 2100 bioanalyzer (Agilent Technologies, Palo target of Tbx2/Tbx3 has linked these transcriptional repressors Alto, CA). Raw feature data from Affymetrix HG-U133A GeneChip micro- to the control of cell cycle and senescence and to the antipro- arrays were obtained for these samples. All samples were processed using the liferative response delivered by the ARF-Mdm2-p53 pathway in one-cycle target labeling protocol.8 We analyzed the data using the tumorigenesis (25, 26). R system software (v2.2.1), GeneSpring GX 7.3, and BRB ArrayTools (v3.3.1). In this study, we showed that the expression of Tbx3, but not Tbx2, Raw feature data were normalized using the Robust Multi-array method was induced by mutant h-catenin in different systems, including (R package affy, v1.4.32; ref. 27), which yielded log2 intensity expression normal murine liver, murine and human liver tumors, and human summary values for each of the 22,283 probe sets. Each set (HCC and carcinoma cell lines. We cloned the TBX3 promoter and used a hepatoblastoma) was normalized independently. Additional statistical anal- combination of chromatin immunoprecipitation (ChIP) and report- yses were done with SPSS 11.0 package (SPSS, Inc., Chicago, IL). Comparison between groups was done using the Fisher’s exact test. Probability of er assays that led to identify Tbx3 as a direct transcriptional target of h overall survival was determined using the Kaplan-Meier analysis and the log- the -catenin/Tcf complex. We also showed that Tbx3 plays a crucial rank and Breslow tests. Follow-up was closed at the time of death or last visit. role in cell proliferation and survival. Inhibition of Tbx3 expression Plasmids. The reporter plasmid L-Tbx3-Luc carrying the TBX3 promoter by RNA interference dramatically reduced anchorage-independent was generated by PCR amplification of human genomic DNA sequences growth and abolished h-catenin protection over doxorubicin- (University of California–Santa CruzGenome Bioinformatics Site) extend- induced apoptosis in tumor cells. Finally, we propose that strong ing 2,226 bp upstream and 619 bp downstream of the transcriptional start overexpression of Tbx3 contributes to the chemotherapy-resistant site of the human TBX3 gene (GenBank accession number NM_016569). We phenotype of a subset of hepatoblastomas. used the following primers: L-Tbx3F, 5¶-CTAGCTAGCGAAACCCTGCAGT- GACTTCCG-3¶; and L-Tbx3R, 5¶-GGAAGATCTGCTCGAAATAGACACTC- CAGC-3¶. The PCR product was cloned into pGL3-Basic (Promega, Materials and Methods Madison, WI). A shorter promoter construct (S-Tbx3-Luc) extending Transgenic mice and tumor samples. WHV/Myc transgenic mouse 500 bp upstream of the transcription start site was generated by using strains carry a woodchuck c-Myc or N-Myc2 transgene targeted to the liver the primers S-Tbx3F, 5¶-CTAGCTAGCGCGAGCGGAGTGCAAGAGAGG-3¶ by hepadnaviral regulatory elements and develop liver tumors almost and S-Tbx3R, 5¶-GGAAGATCTCGGCGGCTCTAGAAGGTCG-3¶.Athird invariably, as described previously (18, 19). WHV/Myc transgenic mice were reporter construct carrying sequences upstream of the proximal TBX3 genotyped by PCR on tail DNA and hybridization with WHV DNA as promoter (2226 to 206) was derived from L-Tbx3 construct with the described (19). Animals were housed at the pathogen-free facility of Institut primers L-Tbx3F and D-Tbx3R: 5¶-CCAGATCTCCTGTGAATATGTCA-3¶. Pasteur and maintained according to the recommendations established by Mutation known to abolish Tcf binding (GTCAAAG ! GCCAAAG) was the French government (Services Ve´te´rinaires de la Sante´etdela introduced in the putative Tcf/Lef binding site using the Quick Change XL Production Animale, Ministe`re de l’Agriculture). Mice carrying liver tumors site-directed mutagenesis kit (Stratagene, La Jolla, CA). All plasmid inserts were euthanized, and tumor samples were dissected free of liver were verified by sequencing. The reporter plasmid containing the cyclin D1 parenchyma. Tumors and nontumoral livers were snap-frozen in liquid promoter (-163CD1-Luc) was kindly provided by R. Pestell (Thomas nitrogen and kept at 80jC. Jefferson University, Philadelphia, PA). The expression vector for dominant RNA preparation and Northern blot analysis. Total RNA was stable h-catenin T41A was described previously (28). Full-length human extracted using the RNeasy system (Qiagen, Valencia, CA) and DNase Tbx3 cDNA was a generous gift of M. Van Lohuizen (The Netherlands treated (DNA-free Kit, Ambion, Austin, TX). Screening of h-catenin Cancer Institute, Amsterdam, The Netherlands), and expression vectors mutations was carried out as described previously (8). For Northern blot encoding wild-type (wt) HA-Tbx3 and HA-Tbx3 Y149S mutant were provided analysis, RNA samples (30 Ag) were resolved by agarose gel electrophoresis by T.R. Brummelkamp (The Netherlands Cancer Institute, Amsterdam, The and blotted onto Hybond N+ membranes. Blots were hybridized with Tbx3, Netherlands; ref. 26). Tcf4 and dominant negative DNTcf4 expression vectors Tbx2, and GS probes and with an 18S rDNA probe for loading control. were kindly provided by H. Clevers (Netherlands Institute for Developmental Signals were quantified using the Storm 840 PhosphorImager (Molecular Biology, Utrecht, The Netherlands). Dynamics, Sunnyvale, CA). Cell lines, transfections, and reporter assays. The human cell lines Murine cDNA arrays. Total RNA from 12 murine HCCs and matched HepG2, Huh6 and Huh7 (hepatomas), 293 (kidney), SW480 and HCT116 nontumor livers was labeled using the Atlas Pure Total RNA Labeling System (colon carcinomas), and U2OS (osteosarcoma) were maintained in DMEM (Clontech, Mountain View, CA) according to manufacturer’s recommenda- with 10% fetal bovine serum. For reporter assays, semiconfluent cells in six- tions. cDNA arrays spotted with the products of 1,185 genes (Clontech, well plates were transfected with expression vectors for h-catenin, Tcf4, and/ ATLAS Mouse 1.2 Array) were used for differential hybridization analysis or DNTcf4 and 0.5 Ag of luciferase reporter construct, using either calcium between tumors and matched livers. cDNA probe hybridization was done phosphate precipitation (293, SW480), Exgen (Euromedex, Souffelweyer- according to the manufacturer’s recommendations. The array results were sheim, France; HepG2) or LipofectAMINE (Invitrogen, Carlsbad, CA; Huh6, scanned using the Storm 840 PhosphorImager and analyzed using Atlas HCT116). Luciferase activity was determined 48 h later. All experiments Image 1.01 software (Clontech Laboratories). Distance measurements and were repeated at least thrice. A thymidine kinase-h-galactosidase plasmid hierarchical clustering computations were done using Cluster and TreeView was cotransfected to normalize luciferase activity for transfection efficiency, software. Differential gene expression between HCC and corresponding and total DNA amount was kept constant by adding pcDNA3. nontumoral liver was considered significant when signal ratio was >2. siRNA transfection. Cells were transfected 24 h after plating with small Human tumor samples. Human HCC and hepatoblastoma specimens interfering RNAs (siRNA) against Tbx3 or h-catenin, or nonsilencing control were obtained from patients undergoing surgical resection. Snap-frozen (luciferase). siRNAs designed by selecting specific sequences of these genes tumor tissues were collected from different French, Italian, and Chinese were synthesized by Eurogentec (Seraing, Belgium). The target sequences medical centers between 1998 and 2005. Most hepatoblastoma patients were enrolled in clinical trials of the International Society of Pediatric Oncology Liver Tumor Study Group. Informed consents were obtained at each hospital, and biological studies were approved by the review board of Institut Pasteur. 8 http://www.affymetrix.com.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Activation of Tbx3 by b-Catenin were as follows: for h-catenin, 5¶-AGC UGA UAU UGA UGG ACA G-3¶;for catenin–induced genes that are involved in liver tumorigenesis, Tbx3 (E), 5¶-AUG GAG AUG UUC UGG GCU G-3¶ and Tbx3 (F), 5¶-GAG GAU we generated gene expression profiles in 12 HCCs, including 6 GUA CAU UCA CCC G-3¶; for Luc, 5¶-CGU ACG CGG AAU ACU UCG A-3¶. cases harboring point mutation or interstitial deletion in the NH2 Cells were transfected with 100 nmol of siRNA per well using Oligofect- terminus of h-catenin and 6 cases with wt h- catenin. RNA from AMINE (Invitrogen). tumors and matched nontumoral livers was reverse transcribed, Western blotting. Frozen mouse tissues or cells were lysed in chilled lysis buffer [50 mmol/L Tris-HCl (pH 7.5); 250 mmol/L NaCl; 0.5% Nonidet P-40; 250 labeled, and hybridized to 1,185 gene cDNA arrays. The expression mmol/L EDTA; 1 mmol/L DTT] supplemented with protease inhibitors profile was compared between each tumor sample and corres- (Roche, Basel, Switzerland). Whole extracts were resolved in 8% or 10% ponding liver, and data were expressed as T/NT ratio. We found 10 polyacrylamide gels and transferred to Hybond-C extra (Amersham genes specifically deregulated (>2-fold) in the h-catenin mutant Pharmacia Biotech, Buckinghamshire, United Kingdom). The following tumor category (Supplementary Table S1). Besides known h- antibodies were used for immunodetection: mouse anti-h-catenin and mouse catenin targets such as the glutamate transporter GLT-1 and the anti-glutamine synthase (BD-Transduction Laboratories, San Jose, CA), mouse dynein light-chain genes, we found that Tbx3 was specifically up- anti-poly(ADP-ribose) polymerase (PharMingen, San Diego, CA), mouse anti- regulated in HCCs bearing mutant h-catenin. Enhanced expression actin (Abcam, Cambridge, United Kingdom), and goat anti-Tbx3 (Santa of Tbx3 was confirmed by Northern and Western blotting (Fig. 1). CruzBiotechnology, Santa Cruz,CA). Mouse and goat anti-immunoglobulin The mRNA levels of Tbx3 and the longer isoform Tbx3 + 2a (30) G conjugated to alkaline phosphatase were from Tropix (Applied Biosystems, h Foster City, CA) and Sigma (St. Louis, MO). Immunoreactive proteins were increased 2- to 10-fold in tumors with mutant -catenin h were visualized using the Western CDP-star kit (Tropix). compared with tumors with wt -catenin and normal liver Chromatin immunoprecipitation assay. ChIP assay was conducted as (Fig. 1A). The protein levels of Tbx3 showed parallel, significant described previously (29), with sheared DNA fragment size between 200 and variations between tumor samples (Fig. 1B). To validate these data, 1,000 bp. Briefly, Huh6 cells were fixed with 1.1% formaldehyde, and nuclear we analyzed Tbx3 expression in an additional set of 13 randomly extracts were isolated. The sonicated nuclear lysates were immunopreci- selected HCCs from WHV/Myc mice. Compared with normal liver, pitated with a Tcf4 antibody (Upstate Biotech, Charlottesville, VA) or Tbx3 mRNA levels were enhanced only in tumors carrying mutant h a -catenin antibody (Santa CruzBiotechnology). After purification of h-catenin (Fig. 1C). immunoprecipitated DNA, a 200-bp region of the Tbx3 promoter We then analyzed the expression of GS as a marker of activated encompassing the putative Tcf site was amplified by PCR using the h ¶ ¶ ¶ Wnt/ -catenin signaling in the hepatic context (5). The Tbx3 and following primers: f55, 5 -AGCTCTATCCCCCAGCACTCG-3 , and r59, 5 - WHV/ GAGAAAGCGAGAGCTCCTCGC-3¶. GS genes displayed strikingly similar expression patterns in Myc For control, two irrelevant regions of the Tbx3 locus located 10 kbp HCCs, both at transcriptional and protein levels (Fig. 1). upstream or 10 kbp downstream were amplified using the primers: Thus, the up-regulation of Tbx3 was associated to Wnt/h-catenin Upstream-F, 5¶-AGCAGACTTCTGTAAAACAGG-3¶ and Upstream-R, 5¶-ACT- signaling. By contrast, expression of the related gene TBX2 CAGTAAGCTCTCTAAACG-3¶; Downstream-F, 5¶-GGGGGCCAGATCAGG- remained constant in tumors and normal livers (Fig. 1C). CATGC-3¶ and Downstream-R, 5¶-GCTGCTGCTGGCAGCCTTTCC-3¶. Tbx3 expression is tightly linked to activated Wnt/B-catenin For siRNA-coupled ChIP assays, Huh6 cells were transfected with siRNA signal in human liver tumors. These findings prompted us to h for either -catenin or luciferase. Cells were harvested 48 h later and investigate Tbx3 expression in human primary liver tumors. processed as described above. Oligonucleotide array (Affymetrix HG U133A) analysis was done Cell proliferation and anchorage-independent growth assays. HepG2 in 55 HCCs and 24 hepatoblastomas specimens. Screening for h- cells stably expressing wt Tbx3 or the mutant Tbx3 Y149S were established by transfection using ExGen 500, followed by Geneticin selection. For cell catenin mutations revealed point mutations or interstitial deletions h proliferation studies, cells were harvested daily for 5 days and stained with in the NH2-terminal region of -catenin in 10:55 HCCs and 19:24 trypan blue, and viable cells were counted on a hemocytometer. For in vivo hepatoblastomas (Supplementary Tables S2 and S3). RNA from tumorigenesis assays, 107 cells were implanted s.c. into the flanks of these tumors and control pools of nontumoral livers from adults 6-week-old female athymic nu/nu mice (Charles River Laboratories, and children were used for microarray analysis. Detailed data of Wilmington, MA). Tumor size was determined 5 weeks later in groups of global gene expression profiles will be published elsewhere for five to six mice by measuring tumor diameter. human HCC9 and hepatoblastoma.10 Microarray data were h For soft-agar assays, Huh6 cells were transfected with siRNA for - validated by quantitative reverse transcription-PCR. catenin, Tbx3, or luciferase. After 24 h, cells were suspended in 0.3% agarose In human HCCs, expression of Tbx3 in the arrays was 2- to 10- in DMEM supplemented with 10% fetal bovine serum. They were plated in fold higher in most tumors carrying mutant h-catenin compared triplicates in six-well dishes onto solidified 0.6% agarose-containing bottom D layer medium at a density of 5 104 cells per well. Cultures were fed twice a with normal livers (Fig. 1 ). Tbx3 was expressed at normal levels in h week, and colonies were counted and photographed 10 days postplating. a majority of tumors with wt -catenin, but six HCCs in this group TUNEL assay. U2OS cells were plated on glass coverslips, and cells at 60% had highly elevated levels of Tbx3 mRNA. In these six cases like in confluence were transfected with h-catenin or empty vector together with the mutant h-catenin group, other known targets of h-catenin were siRNAs for Tbx3, h-catenin, or irrelevant control using LipofectAMINE. At overexpressed, including GS (Supplementary Fig. S1), pancreatitis- 24 h after transfection, cells were treated with 500 ng/mL doxorubicin (Sigma- associated protein, regenerating islet–derived-1a, neuronal cell Aldrich, Lyon, France) and incubated for an additional 24 h. Cells were fixed with adhesion molecule, bone morphogenic protein (BMP) 4, and 4% formaldehyde in PBS for 15 min at room temperature and permeabilized CYP2E1 (15, 17). It suggests that the Wnt/h-catenin pathway with 0.1% Triton X-100 in PBS for 2 min on ice. Terminal nucleotidyl was activated through other means, such as overexpression of transferase–mediated nick end labeling (TUNEL) staining was done using the Frizzled-7 or loss-of-function mutations of Axin (9). Overall, in the In Situ Apoptosis kit (Roche) according to manufacturer’s instructions. 55 human HCCs analyzed, Tbx3 expression was significantly associated with deregulated h-catenin signaling (P = 0.002). Results Tbx3 expression in murine HCC. We reported previously that WHV/Myc transgenic mice develop HCCs that carry frequent 9 P. Pineau, A. Dejean, unpublished data. activating mutation of the h-catenin gene (8, 19). To identify h- 10 C. Armengol, M.A. Buendia, unpublished data. www.aacrjournals.org 903 Cancer Res 2007; 67: (3). February 1, 2007

Figure 1. Tbx3 expression is activated by deregulated Wnt/h-catenin signaling in murine and human liver tumors. A, Northern blot analysis was used to verify the relative Tbx3 expression levels determined by microarray in murine HCCs from WHV/Myc transgenic animals. Total RNA (30 Ag) from seven tumors (T) and two normal livers (NL) was used. Hybridization with full-length mouse Tbx3 cDNA revealed two bands corresponding to recently identified isoforms (30). The blot was stripped and sequentially hybridized with a GS probe and a 18S rRNA probe. Tumors carrying a mutant h-catenin allele are marked with an asterisk. B, Western blot analysis of the tumors shown in (A) indicates that variations of Tbx3 and GS protein levels parallel those of RNA levels. Total protein extracts from tumors and livers were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted with h-catenin–, Tbx3-, or GS-specific antibodies. Besides the 92-kDa h-catenin protein present at similar levels in all lanes, h-catenin deletion mutants in tumors T2*, T6*, and T7* are seen as additional, slower-migrating bands. Actin served as loading control. C, expression of Tbx3 is correlated with h-catenin status in a random set of HCCs from WHV/Myc transgenic mice. Northern blot analysis showed that Tbx3 and GS were overexpressed specifically in tumors with mutant h-catenin. No significant variation of Tbx2 expression was detected in tumors and normal liver. D, distribution of Tbx3 expression levels in 55 human HCCs (left) and in 24 hepatoblastomas (right) according to h-catenin status. Unlog values derived from Affymetrix microarray data are expressed in arbitrary units (AU). In HCCs, mean expression values were significantly higher in tumors expressing mutant h-catenin (MUT) and in a group of six cases with wt h-catenin but elevated levels of GS (WT, Wnt on) than in other tumors carrying wt h-catenin (WT, Wnt off) and normal liver (P < 0.002). Increase in Tbx3 expression in hepatoblastomas carrying mutant h-catenin compared with other tumors and normal liver levels is also significant (P < 0.007). These data were validated by real-time PCR.

Similar analysis of 24 hepatoblastomas revealed the up- Tbx3 expression is induced by mutant B-catenin in mouse regulation of Tbx3 in 21:24 (87%) cases, including all tumors liver and human cell lines. Given the strong correlation between with mutant h-catenin and two tumors with wt h-catenin. We Tbx3 expression and h-catenin signal activation in liver tumors, we observed a 2- to 8-fold increase in the mutant h-catenin group extended our studies to different nontumoral conditions. It has compared with tumors carrying wt h-catenin (P = 0.007; Fig. 1D). been shown that injection of recombinant adenovirus expressing Hepatoblastomas carrying mutant h-catenin displayed increased the mutant S37A h-catenin strongly activated several Wnt target expression of known h-catenin targets, including GS (Supplemen- genes including GS (5). Northern blot analysis of livers from mice tary Fig. S1), pancreatitis-associated protein, the Dickkopf homo- infected with Adh-catS37A, or AdLacZ or AdGFP as controls, logue DKK1, BMP, and activin membrane-bound inhibitor indicated parallel up-regulation of Tbx3 and GS in the livers of mice homologue (BAMBI), BMP4, and the tyrosine kinase receptor 48 h after injection of Adh-catS37A (Fig. 2A). We next analyzed EPHB2.11 liver expression of Tbx3 in EAB/9K/h-catenin transgenic mice, Collectively, these results show that Tbx3 expression is tightly which overexpress the deletion mutant DN131 h-catenin in the linked to the status of Wnt/h-catenin signaling in human and liver, kidney, and intestine and develop hepatomegaly soon after murine liver tumors. By contrast, no such association was birth (14). On Northern analysis, expression of Tbx3 and GS was evidenced for other T-box genes, including Tbx1, Tbx2, Tbx4, high in the livers of two transgenic mice aged 3 to 4 weeks, but it Tbx5, Tbx10, Tbx19, and Tbx21 (data not shown). was barely detectable in matched nontransgenic livers (Fig. 2A). Both in Adh-catS37A infection and in EAB/9K/h-catenin transgenic conditions, Tbx3 mRNA levels were increased by 2- to 3-fold. To further address the regulation of Tbx3 by h-catenin, we inves- 11 Unpublished data. tigated the effect of an active h-catenin mutant on endogenous

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Tbx3 expression in human cell lines that display no h-catenin 74 (see Fig. 3B), which is conserved in the murine sequence. activation. U2OS cells were transfected with the pcDNA-h-catT41A Chromatin obtained from Huh6 hepatoblastoma cells, which carry vector or empty vector, and significant up-regulation of Tbx3 was mutant h-catenin G34V, was immunoprecipitated with either a seen at 48 h after transfection, both at mRNA and protein levels Tcf4 antibody or a h-catenin antibody. In both cases, we could (Fig. 2B). Similar data were obtained in Huh7 hepatoma cells amplify the 200-bp region containing the Tcf-binding element in (Supplementary Fig. S2). Thus, h-catenin–driven induction of Tbx3 the proximal Tbx3 promoter (Fig. 3A). As control of the specificity might be independent of the cellular context. of ChIP conditions, neither Tcf4 nor h-catenin was detected at B-catenin physically occupies and activates the Tbx3 distal upstream and downstream regions of the Tbx3 gene, and the promoter through Tcf/Lef. To determine whether Tbx3 is a Tbx3 promoter could not be amplified in the absence of the added direct transcriptional target of h-catenin, we adressed the antibody. Furthermore, h-catenin was no longer detected at the association of h-catenin with the Tbx3 promoter by ChIP assays. Tbx3 promoter after siRNA-mediated silencing of h-catenin in A presumptive Tbx3 transcription start site located 965 bp Huh6 cells, whereas irrelevant siRNA (siLuc) did not abolish h- upstream of the Tbx3 coding region was deduced from the 5¶ ends catenin detection (Fig. 3A). of human and murine cDNA clones (accession numbers Then, the ability of h-catenin to activate Tbx3 transcription was NM_016569 and XM_132317), and it was confirmed using primer investigated by reporter assay. We used PCR and human genomic extension (data not shown). Analysis of upstream sequences DNA to clone 2,845 bp of promoter sequences upstream of the revealed a potential Tcf-binding site (GTCAAAG) at position 69 to luciferase gene in pGL3 (L-Tbx3-LUC, Fig. 3B). We derived a shorter promoter construct covering 500 bp of the proximal promoter (S-Tbx3-LUC). Nucleotide substitution known to abolish Tcf binding (GCCAAAG) was introduced into the Tcf site (Lmut- and Smut-Tbx3-Luc). We also included in the analysis the DTbx3-Luc construct that is deleted of Tbx3 proximal promoter sequences (position 2,226 to 206, Fig. 3B). h-Catenin activity on Tbx3 transcription was examined in HCT116 and Huh6 cells, which carry activating h-catenin mutations. Cotransfection of the dominant negative mutant DNTcf4 with L- or S-Tbx3-Luc plasmids in HCT116 cells strongly decreased promoter activity in a dose- dependent manner, similar to the effects on the cyclin D1 promoter (Fig. 3C). By contrast, activity of the DTbx3-LUC reporter was low and insensitive to DNTcf4. Mutation of the putative Tcf-binding motif reduced L- and S-Tbx3 promoter activity by 2-fold and abolished sensitivity to DNTcf4. Similar data using the S-Tbx3 promoter were obtained in Huh6 cells (Fig. 3D). Moreover, in 293 cells, in which the Wnt pathway remains intact, the Tbx3 promoter was activated 3-fold by cotransfection of Tcf4 and h-catenin, but not by DNTcf4 and h-catenin (Supplementary Fig. S3). Taken together, these data provide evidence that Tbx3 is a direct transcriptional target of the h-catenin/Tcf complex. Tbx3 can regulate proliferation and anchorage-independent growth of hepatoma cells. h-Catenin has been shown to increase cell proliferation and protect epithelial cells from suspension-induced apoptosis (anoikis; refs. 31, 32). We therefore tested whether Tbx3 might be implicated in the regulation of cell proliferation by h-catenin. To this end, we established HepG2 cell clones stably expressing either wt Tbx3 or the dominant negative Tbx3 Y149S mutant isolated from a UMS patient (26). Analysis of Figure 2. Induction of Tbx3 expression by h-catenin in mouse livers and cell proliferation in representative HepG2 clones with similar human tumor cell lines. A, Northern blot analysis of Tbx3 in adenovirus-infected transgene expression levels showed that expression of wt Tbx3 mouse liver (Ad h-cat S37A) and in DN131h-catenin transgenic liver (Tg DN131h-cat). Left, mice were injected with the indicated adenovirus was associated with slightly enhanced growth rate, whereas (AdLacZ, AdGFP, and Adh-catS37A) and sacrificed 48 h after injection. mutant Tbx3 inhibited drastically cell proliferation (Fig. 4A). The Right, Tbx3 expression was compared between livers from transgenic ability of HepG2 cells expressing the Tbx3 mutant to form tumors D h N131 -cat and matched nontransgenic mice (NT). GS expression served as in vivo 6 control for Wnt pathway activation. Signal intensity of the upper Tbx3 band was investigated by injecting nude mice with 10 10 was quantified in each lane using a Storm PhosphorImager and normalized to cells. Groups of five or six mice were injected with either HepG2- 18S rRNA control. Fold activation of Tbx3 in Adh-catS37A–infected livers was Tbx3 Y149S or control HepG2 cells and examined 5 weeks later. determined by comparison with the mean intensity in AdGFP- and AdLacZ- infected livers, which was arbitrarily set to 1. Fold activation of Tbx3 in D131h-cat In two independent assays, we found that a total of 10:12 mice transgenic mice was determined by comparison with nontransgenic mouse injected with control HepG2 cells formed tumors, but only 1 of livers. B, U2OS cells were transfected with 1Ag of either T41Ah-catenin or empty pcDNA3 vector. Top, total RNA was extracted from cells harvested 10 mice injected with HepG2-Tbx3 Y149S developed a tumor 48 h posttransfection, and Tbx3 expression was analyzed by Northern blotting. (Fig. 4B). Ethidium bromide staining of 28S rRNA served as control for equal loading. Then, we examined the effects of Tbx3 on anchorage-inde- Bottom, protein extracts were prepared from U2OS cells harvested 48 h after transfection as above, and expression of Tbx3 was assessed by Western pendent growth of Huh6 cells in soft-agar assays. Cells were blot analysis using 20 Ag of protein extract and actin as loading control. transfected with siRNA for either h-catenin, Tbx3, or luciferase as www.aacrjournals.org 905 Cancer Res 2007; 67: (3). February 1, 2007

Figure 3. h-catenin is recruited to the TBX3 promoter and activates Tcf-dependent transcription. A, ChIP using anti-Tcf4 and anti-h-catenin antibodies was done in Huh6 cells. Eluted DNA fragments were purified and used for PCRusing primers specific for the TBX3 proximal promoter (f55/r59) or for sequences located at 10 kb distance, either upstream (Up) or downstream (Dn) of the promoter. When indicated, Huh6 cells were transfected with 100 nmol of h-cat siRNA or Luc siRNA as control and harvested 48 h later for ChIP analysis. NAC, no antibody control; input represents amplification of a 1:50 dilution of total input chromatin. Data are representative of three independent experiments. B, TBX3 promoter constructs in the pGL3 luciferase reporter plasmid. L-Tbx3-Luc contains a 2.2-kb sequence, and S-Tbx3-Luc contains a 500-bp sequence upstream of the Tbx3 transcription start site. In D-Tbx3-Luc, 206 bp of proximal promoter sequences were deleted from L-Tbx3-Luc. Point mutation that abolishes Tcf binding was introduced into the Tcf/Lef recognition site (position 74 to 69) in L-Tbx3 and S-Tbx3 reporter constructs. C, inhibition of Tbx3 promoter activity by DNTcf4 in HCT116 colorectal tumor cells. HCT116 cells were cotransfected with different TBX3 promoter constructs (0.5 Ag) and either DNTcf4 expression vector or empty vector (0.5 or 1 Ag). Cells were harvested 48 h after transfection for measurement of luciferase activity. Total amounts of plasmid DNA were kept constant by adding the empty pcDNA3 vector. The h-catenin–responsive cyclin D1 promoter reporter (CD1-Luc) was used as control. All experiments were done in duplicate and repeated at least thrice. Columns, means; bars, SD. D, the TBX3 promoter is regulated by Wnt signaling in Huh6 hepatoblastoma cells. Huh6 cells were cotransfected with the S-Tbx3-Luc or Smut-Tbx3-Luc reporter plasmid (0.5 Ag) and either DNTcf4 expression vector or empty vector (0.25 Ag) as detailed in (C). control and plated in soft agar 24 h later. Both h-catenin and Tbx3 Tbx3 is involved in B-catenin–induced protection against siRNAs dramatically reduced the growth of cell clones, whereas apoptosis. It has been reported that h-catenin protects cells robust clonal cell growth was seen in untransfected cells and in against p53-dependent apoptosis, and that Tbx3 has antiapoptotic cells transfected with Luc siRNA (Fig. 4C). Similar data were activity, probably through down-regulation of p19ARF and subse- obtained in four independent experiments, and reduction of quently p53 (33). To determine whether Tbx3 could mediate h-catenin and Tbx3 expression in cells transfected with the resistance to apoptosis conferred by h-catenin, we first examined corresponding siRNAs was verified by Western blotting (Fig. 4D). the influence of Tbx3 on apoptosis induced by doxorubicin, a DNA Similar results were obtained in HCT116, SW480, and 293 cells. damage–inducing agent known to induce p53-dependent apopto- Thus, impaired Tbx3 expression has potent inhibitory effects on sis. In U2OS cells, transfection of h-cateninT41A and irrelevant the growth of cancer cells. siRNA (siLuc) reduced doxorubicin-induced apoptosis from

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Figure 4. Tbx3 is required for proliferation of liver tumor cells. A, the proliferation of HepG2 cells stably expressing either wt Tbx3 or mutant Tbx3 Y149S and control HepG2 cells carrying the empty vector was compared during 5 d after plating. Growth curves were established by counting viable cells at daily intervals by trypan blue exclusion assay in triplicate wells. Points, means; bars, SD. Similar data were obtained for three individual clones of HepG2 cells expressing wt or mutant Tbx3. Expression level of the stably transfected HA-tagged Tbx3 proteins was verified by Western blotting (right). B, in vivo tumorigenesis assays. Groups of five to six nude mice were injected s.c. with 107 HepG2 cells stably expressing either the Tbx3 Y149S mutant or the empty vector (HepG2 Mock). Animals were examined at 5 weeks postinjection. In six HepG2 Mock animals, tumor diameters ranged from 4 to 11 mm. In HepG2 Tbx3Y149S animals, only one tumor was seen with a diameter of 5 mm. Reduced capacity of HepG2 cells expressing the Tbx3 mutant to form tumors in vivo was verified in a second assay, in which 4:6 mice injected with HepG2 Mock developed tumors, but no tumor was seen in five mice injected with HepG2-Tbx3Y149S cells. C, soft-agar assay. Huh6 cells were transfected with siRNAs that specifically target h-catenin, Tbx3, or luciferase as indicated. Cells were plated in soft agar at 24 h posttransfection, and colonies were photographed under microscopy 10 days postplating (left part). The number of colonies >125 Am in diameter was scored. Colony numbers from Luc siRNA-transfected cells were identical to those of cells transfected without siRNA duplexes (mock) and were set to 100%. Results in cells transfected with h-cat and Tbx3 siRNAs are expressed as percentage of mock-transfected cells. Columns, means from two independent experiments in triplicate wells; bars, SD (right). D, Western blotting shows down-regulation of h-catenin and Tbx3 in Huh6 cells at 48 h after transfection with 100 nmol/L of h-catenin siRNA, and 50 or 100 nmol/L of Tbx3 siRNA.

29 F 10% to 19.25 F 6.7% (mean F SD), as determined by the induced cell death, but they were equally sensitive to apoptosis frequency of TUNEL-positive cells (Fig. 5A). As expected, induced by siRNA-mediated depletion of h-catenin. At 48 h after suppression of h-catenin by siRNA in cells transfected with mutant transfection of siRNA against h-catenin, the ratio of cleaved/ h-catenin restored apoptosis close to levels in control siLuc-treated uncleaved poly(ADP-ribose) polymerase was increased 2.5-fold, cells. Strikingly, inhibition of Tbx3 by two different specific siRNAs and 3-fold with additional doxorubicin treatment. Interestingly, in cells transfected with h-catenin T41A strongly increased cotransfection of the Tbx3 expression vector with h-catenin doxorubicin-induced apoptosis to 47.5 F 0.7% and 49 F 1.4% siRNA decreased the level of apoptotic cells by 1.5-fold, both with (mean F SD). Similar data were obtained in two independent and without doxorubicin treatment. Efficient down-regulation of experiments done in triplicates. Illustrative examples of TUNEL- h-catenin and Tbx3 by h-catenin siRNA and overexpression of positive cells are shown in Fig. 5B. These data were confirmed by Tbx3 in cells transfected with the Tbx3 vector were verified by using the caspase-mediated cleavage of poly(ADP-ribose) polymer- Western blotting (Fig. 5C). Thus, expression of Tbx3 is required ase and Western blotting to evaluate apoptosis (Supplementary for suppression of apoptotic death by h-catenin in human tumor Fig. S4). It indicates that Tbx3 expression might be required for cells. h-catenin–mediated protection against apoptosis. Clinical relevance of Tbx3 overexpression in hepatoblas- We further analyzed the antiapoptotic activity of Tbx3 in HCT116 toma. Given the antiapoptotic activities of Tbx3, we sought to cells that express wt p53 and mutant h-catenin. Figure 5C shows investigate the relevance of Tbx3 in human liver tumors. We that these cells were more resistant than U2OS cells to doxorubicin- selected hepatoblastoma as a model because this tumor, unlike www.aacrjournals.org 907 Cancer Res 2007; 67: (3). February 1, 2007

HCC, is currently treated by chemotherapy. The relationship signaling in murine as well as in human liver tumors, confirming between Tbx3 expression and various clinical features was the value of mouse models to study molecular events that occur in investigated in the above-described panel of 24 hepatoblastomas HCC (34). Moreover, we provide evidence that Tbx3 is a direct (Supplementary Table S3). High-level Tbx3 expression (greater than transcriptional target of the h-catenin/Tcf complex by showing or equal to the median Tbx3 expression value in hepatoblastomas, that h-catenin physically occupies and activates the Tbx3 corresponding to a 4-fold increase compared with the median value promoter, and that a single Tcf/Lef binding motif in the proximal in normal livers) was significantly associated with advanced tumor Tbx3 promoter is critical for h-catenin responsiveness. These stage, poor response to chemotherapy, and shorter overall survival results add Tbx3 to the list of Wnt-responsive T-box genes, of patients (P < 0.05, Fisher’s exact test; Table 1). The Kaplan-Meier including the founding member T (Brachyury) and the Tbx3 survival estimate for patients in the high-level Tbx3 group was orthologs optomotor-blind (Omb) in Drosophila and LvTbx2/3 in about 2-fold lower than for other hepatoblastoma patients sea urchin (35–37). In this respect, it is of interest that deregulated (log-rank test, P = 0.021, Breslow test, P = 0.036; Fig. 5D). Thus, Wnt/h-catenin signaling did not activate the close relative Tbx2 or overexpression of Tbx3 might participate in tumor resistance to any other T-box gene in the liver tumors analyzed in this study. conventional chemotherapy, thereby influencing the outcome of During development, Tbx3 has been implicated in inductive hepatoblastoma disease. interactions at many stages of embryogenesis by its highly specific expression pattern. In particular, Tbx3 is expressed in the mesenchyme at the limb bud margins and in the mammary buds, Discussion correlating with defects in anteroposterior patterning and mam- In this study, we show that the T-box gene Tbx3 is a novel target mary gland induction in Tbx3-deficient mice and in UMS patients of h-catenin implicated in the proliferation and survival of cancer (22, 23, 38). Strikingly, published evidence placing Tbx3 down- cells. We show that Tbx3 expression is activated by Wnt/h-catenin stream of h-catenin in these processes is limited to the cooperative

Figure 5. Tbx3 protects cells against drug-induced apoptosis. A, apoptosis was measured by TUNEL assay in doxorubicin- treated U2OS cells after transfection with 2 Ag of either h-catenin or empty vector together with 100 nmol of siRNAs for h-catenin, Tbx3, or luciferase as control. The percentage of TUNEL-positive U2OS cells was determined by counting more than 100 cells from 10 randomly chosen fields. The experiment was done in triplicate wells and repeated twice with comparable results. Two different Tbx3 siRNAs yielded similar percentages of apoptotic cells. Columns, means of a representative assay; bars, SD. B, illustrative examples of TUNEL assays in U2OS cells in the different conditions. C, Tbx3 protects cells against apoptosis induced by h-catenin depletion and by doxorubicin. HCT116 cells were cotransfected with 100 nmol/L of h-catenin siRNA and Tbx3 vector or empty vector (2.5 Ag) and treated or not with 500 ng/mL doxorubicin. Cells were harvested 24 h later, and total protein extracts were analyzed by Western blot with anti-poly (ADP-ribose) polymerase antibody. Apoptosis was measured by quantification of cleaved/uncleaved (C/NC) poly(ADP-ribose) polymerase ratios using Syngene Photo Image System. Expression of h-catenin and Tbx3 in each sample was verified by Western blotting. Note that h-catenin siRNA inhibited also Tbx3 expression. D, correlation of Tbx3 expression with overall survival of patients with hepatoblastoma. Kaplan-Meier curves showed that cases with high Tbx3 expression (greater than or equal to 4-fold the median level in normal liver) have reduced survival probability compared with cases with lower Tbx3 expression. Log-rank test, P = 0.021; Breslow test, P = 0.036.

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Table 1. High-level expression of Tbx3 in HB (more than apoptosis, thereby facilitating transformation (44). Strikingly, other 4-fold over normal liver) is correlated with advanced tumor studies have pointed to similar activity of Tbx3 in counteracting stage, poor response to chemotherapy, and poor survival Myc-induced apoptosis (33). Our finding that a Tbx3 dominant of HB patients negative mutant could potently inhibit hepatoma cell growth in vitro and in vivo provides evidence that Tbx3 mediates, at least Variables Tbx3 < 4 Tbx3 z 4 P* in part, the growth-promoting effects of h-catenin in cancer cells. Moreover, siRNA-mediated depletion of Tbx3 dramatically inhibits n =16 n =8 the growth of hepatoblastoma cells in soft agar. We also show that h c Tbx3 expression is mandatory for the protective effect of -catenin PRETEXT stage over p53-dependent apoptosis induced by doxorubicin, and that I or II 9 0 0.014 the overexpression of Tbx3 rescues cells from apoptosis induced by III or IV 7 7 h-catenin depletion. Thus, Tbx3 seems to be a critical determinant Histology of cellular responses to proliferative and antiapoptotic signals Mixed 2 3 0.158 h Epithelial 14 5 delivered by -catenin. These effects can be explained by Vascular invasion transcriptional repression of the ARF tumor suppressor gene by No 8 2 0.184 Tbx3, leading in turn to the down-regulation of p53 and the Yes 8 6 activation of Myc-induced proliferation and transformation, but Multifocality not Myc-induced apoptosis (26, 45). No, solitary tumor 12 3 0.078 To address the question of how these activities manifest in vivo, Yes 4 5 we used hepatoblastoma, a tumor that responds generally well to Metastasis at diagnosis current chemotherapy regimens such as cisplatin and doxorubicin, No 14 4 0.062 although refractory cases (20–30% of patients) with advanced Yes 2 4 tumor stage or metastasis have poor survival rates (46). The Response to chemotherapy h Good 9 1 0.046 presence of -catenin mutations in a majority of hepatoblastomas Poor 4 5 is not associated with disease progression or any tumor charac- Survival teristic (47). We show that high overexpression of Tbx3 in a subset Alive 14 3 0.019 of hepatoblastomas carrying mutant h-catenin is correlated with Dead 2 5 advanced tumor stage, poor response to chemotherapy, and unfavorable prognosis. Overexpression of Tbx3 is not restricted to liver cancer because it has been described in breast and ovarian *Fisher’s exact test. cancer (30, 48) and in ovarian endometrioid adenocarcinomas cPRETEXT stage, as defined by SIOPEL, is a staging system based on characterized with respect to mutations affecting the Wnt/h- imaging at diagnosis that defines four categories depending on the number of liver sectors that are free of tumor (50). For one patient, no catenin pathway (49). Based on the key role of Tbx3 in data were available. carcinogenesis, inhibition of Tbx3 activity could be an effective therapeutic strategy for human cancer.

Acknowledgments effect of Wnt and fibroblast growth factor signals in mammary Received 6/27/2006; revised 10/28/2006; accepted 11/29/2006. gland initiation (39). Previous studies rather pointed to the role of Grant support: Ligue Nationale contre le Cancer (Carte d’Identite´des Tumeurs Sonic hedgehog and BMP-mediated signals in regulating Tbx3 Program), Association pour la Recherche sur le Cancer, Ecole Normale Supe´rieurede Lyon (C. Labalette), European Association for the Study of the Liver Sheila Sherlock expression (21, 40–42). Notably, it has been shown that Tbx3 is a Fellowship (C. Armengol), and GIS-Institut des Maladies Rares (S. Cairo). direct target of BMP Smads (43). Therefore, Tbx3 expression seems The costs of publication of this article were defrayed in part by the payment of page to be governed by a complex network of signals. The question of charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. whether regulatory cross-talks between Sonic hedgehog, Bmp, and We thank Laurence Le´vyand Oliver Bishof for stimulating discussion and Daniela Wnt pathways also operate in oncogenic processes to regulate Geromin and Jean-Yves Coppe´efor help with RNA quality control and quantitative PCR. We thank the French, Italian, and Chinese medical centers that provided us with Tbx3 expression warrants further investigation. tumor specimens, in particular Drs. M. Fabre (Hoˆpital de Biceˆtre,Le Kremlin-Biceˆtre, In this study, activation of Tbx3 by the Wnt/h-catenin pathway France), B. Terris (Hoˆpital Cochin, Paris, France), B. Turlin (Centre Hospitalier was initially recognized in Myc-induced murine liver tumors that Universitaire Pontchaillou, Rennes, France), V. Mazzaferro (Tumor National Institute, h h Milan, Italy), and L.X. Qin (Fudan University, Shangai, China). We are grateful to Bert carry second-hit mutations in the -catenin gene (8, 19). Wnt/ - Vogelstein, Thijn Brummelkamp, Maarten van Lohuizen, Hans Clevers, and Richard catenin signaling is known to protect cells from Myc-induced Pestell for providing reagents.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Tbx3 Is a Downstream Target of the Wnt/β-Catenin Pathway and a Critical Mediator of β-Catenin Survival Functions in Liver Cancer

Claire-Angélique Renard, Charlotte Labalette, Carolina Armengol, et al.

Cancer Res 2007;67:901-910.

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