Catenin/T-Cell Factor Signaling Identified by Gene Expression Profiling of Ovarian Endometrioid Adenocarcinomas1

[CANCER RESEARCH 63, 2913–2922, June 1, 2003] Novel Candidate Targets of ␤-Catenin/T-cell Factor Signaling Identified by Gene Expression Profiling of Ovarian Endometrioid Adenocarcinomas1

Donald R. Schwartz,2 Rong Wu,2 Sharon L. R. Kardia, Albert M. Levin, Chiang-Ching Huang, Kerby A. Shedden, Rork Kuick, David E. Misek, Samir M. Hanash, Jeremy M. G. Taylor, Heather Reed, Neali Hendrix, Yali Zhai, Eric R. Fearon, and Kathleen R. Cho3 Comprehensive Cancer Center and Departments of Pathology [D. R. S., R. W., H. R., N. H., Y. Z., E. R. F., K. R. C.], Internal Medicine [E. R. F., K. R. C.], Pediatrics and Communicable Diseases [R. K., D. E. M., S. M. H.], and Biostatistics [C-C. H., J. M. G. T.], School of Medicine, Department of Epidemiology [S. L. R. K., A. M. L.], School of Public Health and Statistics [K. A. S.], College of Literature Science and the Arts, University of Michigan, Ann Arbor, Michigan 48109-0638

ABSTRACT specific molecular and pathobiological features [reviewed by Feeley and Wells (2) and Aunoble et al. (3)]. ␤ ␤ The activity of -catenin ( -cat), a key component of the Wnt signaling OEAs4 are characterized by frequent (16–54%) mutations of pathway, is deregulated in about 40% of ovarian endometrioid adenocar- CTNNB1, the gene encoding ␤-cat, a critical component of the Wnt cinomas (OEAs), usually as a result of CTNNB1 gene mutations. The signaling pathway (4–9). Wnts are a highly conserved family of function of ␤-cat in neoplastic transformation is dependent on T-cell factor (TCF) transcription factors, but specific genes activated by the secreted growth factors that bind members of the frizzled family of interaction of ␤-cat with TCFs in OEAs and other cancers with Wnt transmembrane receptors and, through downstream signaling, modu- pathway defects are largely unclear. As a strategy to identify ␤-cat/TCF late many developmental and adult tissue processes including cell fate transcriptional targets likely to contribute to OEA pathogenesis, we used specification, proliferation, and differentiation (10). ␤-cat plays a oligonucleotide microarrays to compare gene expression in primary OEAs critical role in both cell adhesion and Wnt signaling. The cytoplasmic/ to OEAs with intact nuclear pool of ␤-cat involved in Wnt signaling is largely regulated by (11 ؍ with mutational defects in ␤-cat regulation (n ,Both hierarchical clustering and a multiprotein complex consisting of the APC tumor suppressor .(17 ؍ regulation of ␤-cat activity (n principal component analysis based on global gene expression distin- AXIN, and GSK3␤ proteins (11–18). In the absence of Wnt signals, ␤ ␤ guished -cat-defective tumors from those with intact -cat regulation. this protein complex promotes degradation of free cytosolic ␤-cat via We identified 81 potential ␤-cat/TCF targets by selecting genes with at GSK3␤-mediated phosphorylation of NH -terminal ␤-cat residues least 2-fold increased expression in ␤-cat-defective versus ␤-cat regula- 2 and subsequent ubiquitination and degradation of ␤-cat by the pro- tion-intact tumors and significance in a t test (P < 0.05). Seven of the 81 genes have been previously reported as Wnt/␤-cat pathway targets (i.e., teasome. Wnt ligands, upon binding to a Frizzled-LRP (lipoprotein- BMP4, CCND1, CD44, FGF9, EPHB3, MMP7, and MSX2). Differential receptor-related protein) transmembrane receptor complex, activate a expression of several known and candidate target genes in the OEAs was pathway that inhibits GSK3␤ activity, with resultant stabilization and confirmed. For the candidate target genes CST1 and EDN3, reporter and nuclear localization of ␤-cat. Nuclear ␤-cat cooperates with members chromatin immunoprecipitation assays directly implicated ␤-cat and TCF of the TCF/lymphoid enhancer factor transcription regulator proteins in their regulation. Analysis of presumptive regulatory elements in 67 of (hereafter referred to collectively as TCFs) to activate transcription of the 81 candidate genes for which complete genomic sequence data were specific target genes. The Wnt pathway is deregulated in many types available revealed an apparent difference in the location and abundance of human cancers (17), including melanomas (19), hepatoblastomas of consensus TCF-binding sites compared with the patterns seen in control (20), medulloblastomas (21) and carcinomas of the colon (22), pros- genes. Our findings imply that analysis of gene expression profiling data tate (23, 24), uterine endometrium (25–27), and ovary (4–9). Presum- from primary tumor samples annotated with detailed molecular informa- ably, many of the proteins encoded by ␤-cat/TCF transcriptional tion may be a powerful approach to identify key downstream targets of signaling pathways defective in cancer cells. targets play important roles in effecting neoplastic transformation. Many of the previous studies aimed at identifying novel Wnt pathway target genes have been carried out in developmental systems INTRODUCTION or using various in vitro or animal tumor models (28–33). The relatively few studies based on analysis of primary human cancers The vast majority of ovarian cancers are derived from epithelial have focused mainly on colorectal carcinomas, the majority of which cells. These malignant epithelial tumors (carcinomas) are typically manifest Wnt pathway defects (34–36). We recently found diverse gland-forming and can be divided into four major morphological mechanisms of ␤-cat deregulation in approximately one-third of pri- types, serous, endometrioid, clear cell, and mucinous adenocarcino- mary OEAs, including frequent mutations of CTNNB1 and, less mas. Recently, we used oligonucleotide microarrays to characterize commonly, mutations of APC, AXIN1, and AXIN2 (9). Expression of global gene expression in 113 ovarian adenocarcinomas (1). Our six previously reported ␤-cat/TCF-regulated genes (CCND1, c-MYC, results and those of other studies offer support for the concept that MMP-7, CX43, ITF2, and PPAR-␦) was subsequently evaluated in different histological types of ovarian carcinoma likely represent OEAs with and without documented Wnt pathway defects (37). All of distinct, albeit overlapping, disease entities, with each type exhibiting these genes except c-MYC were significantly increased in expression in OEAs with deregulated ␤-cat. These studies suggest that compar- Received 1/28/03; accepted 4/14/03. ison of comprehensive gene expression data from primary OEAs with The costs of publication of this article were defrayed in part by the payment of page and without deregulated ␤-cat may provide a robust strategy for charges. This article must therefore be hereby marked advertisement in accordance with ␤ 18 U.S.C. Section 1734 solely to indicate this fact. identifying as-yet-undetermined -cat/TCF target genes with roles in 1 Supported by funds from the Department of Defense (DAMD 17-1-1-0727), the the pathogenesis of OEAs and perhaps other types of human cancers National Cancer Institute (U19 CA84953, RO1 CA94172, and RO1 CA85463), and in part by the Tissue Core of the University of Michigan Comprehensive Cancer Center (NIH P30 CA46952). 4 The abbreviations used are: OEA, ovarian endometrioid adenocarcinoma; TCF, 2 Both authors contributed equally to this work. T-cell factor; APC, adenomatous polyposis coli; GSK3␤, glycogen synthase kinase 3␤; 3 To whom requests for reprints should be addressed, at Department of Pathology, RT-PCR, reverse transcription-PCR; q-RT-PCR, quantitative RT-PCR; HC, hierarchical University of Michigan Medical School, 4301 MSRB III, 1150 West Medical Center clustering; PCA, principal component analysis; PC, principal component; ␤-cat, ␤- Drive, Ann Arbor, MI 48109-0638. Phone: (734) 764-1549; Fax: (734) 647-7979; E-mail: catenin; CMV, cytomegalovirus; dn-, dominant negative; ChIP, chromatin immunopre- [email protected]. cipitation; PI3K, phosphatidylinositol 3Ј-kinase; UTR, untranslated region. 2913

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. ␤-CATENIN/TCF TARGET GENES IN OVARIAN CANCER with Wnt pathway defects. We applied this approach using gene of California at Santa Cruz working draft of the human genome sequence,6 expression data from oligonucleotide microarrays, and we report here with highly repetitive sequences masked. Pointers to the transcription start and on several novel candidate ␤-cat/TCF transcriptional targets. stop sites provided within the RefSeq portion of the University of California at Santa Cruz human genome browser database were used to extract specific gene sequences for analysis (42). We obtained sequence starting from 5000 bp Ј Ј MATERIALS AND METHODS upstream (5 ) of the start of transcription to 1000 bp downstream (3 ) from the stop of translation. For genes with multiple transcripts, only the longest Tumor Samples and RNA Isolation. Forty-one snap-frozen primary transcript was used for TCF site annotation. In-house software was used to OEAs were analyzed using oligonucleotide microarrays and/or RT-PCR (37 extract genomic DNA sequences and to identify consensus TCF sites within from the Cooperative Human Tissue Network/Gynecologic Oncology Group them. We compared the proportion of sites (the number of sites within a gene Tissue Bank, 2 from the University of Michigan Health System, and 2 from the region divided by the total number of possible sites within the same gene Johns Hopkins Hospital). Gene expression in 28 of the 41 tumors evaluated region) within candidate Wnt pathway genes and control genes using a one- with Affymetrix HuGeneFL oligonucleotide microarrays has been reported sided standard normal Z test. The ratio of the proportions of sites in candidate previously (1, 38). Of these 28 OEAs, 11 have a documented Wnt pathway target genes relative to control genes was estimated, and 95% confidence defect and nuclear localization of ␤-cat protein (9). The subset of 11 Wnt intervals were computed using the delta method. pathway-defective tumors includes 10 tumors (OE-2, -13, -17, -18, -37, -44, PCR-based Gene Expression Expression Analyses. We used real-time -47, -48, -55, and -71) with stabilizing mutation of CTNNB1, and one tumor q-RT-PCR analysis to validate differential expression of selected putative Wnt (OE-32) with biallelic inactivation of APC. The remaining 17 OEAs lacked pathway target genes in RNA samples from 31 primary OEAs, including 18 of Wnt pathway defects based on immunohistochemical studies showing lack of the 28 OEAs analyzed by oligonucleotide microarrays. Of these 31 tumors, 14 nuclear ␤-cat protein and mutational analyses of CTNNB1, APC, AXIN1, had documented ␤-cat deregulation, and 17 had intact ␤-cat/TCF signaling. and/or AXIN2 (9). Primary tumor tissues were manually microdissected before The detailed methods and sequences of forward primer (f), reverse primer (r), RNA extraction to ensure each tumor sample contained at least 70% neoplastic and probe (p) for the genes validated by q-RT-PCR [CNND1 (cyclin D1), cells. Total RNA was extracted from frozen tissue biopsies with Trizol (Life FGF9 (fibroblast growth factor 9), EDN3 (endothelin 3), SFN (stratifin), and 5 Technologies, Inc., Carlsbad, CA) and then further purified using RNeasy spin HPRT1, which served as an internal control] are available online. Differences columns (Qiagen, Valencia, CA) according to the manufacturers’ protocols. between tumors with intact versus deregulated TCF/␤-cat were tested using the Analysis of tissues from human subjects was approved by the University of Student’s t test. Pearson product-moment correlations were used to estimate Michigan’s Institutional Review Board (IRB-MED #2001-0568). the degree of association between the microarray and q-RT-PCR data. Semi- Data Processing. Acquisition and processing of oligonucleotide microar- quantitative multiplex RT-PCR for CST1 was performed as follows. Each PCR 32 ray data from the 28 OEAs described above has been reported previously (1). reaction contained 2 ␮Ci of [␣- P]dCTP (ICN, Costa Mesa, CA); 25 ␮M A detailed description of the methods, as well as the freely available code, can dCTP; 200 ␮M dATP, dGTP, and dDTP; 1.5 ␮M of each primer (both target be found online.5 The analyses for the present study included data from 6955 gene and internal control; see website5 for primer sequences); 1 unit of noncontrol probe sets (of 7069 available on the chip), after elimination of platinum Taq polymerase (Invitrogen, Carlsbad, CA); and 20 ng of first-strand probe sets that contained fewer than 8 probe pairs (n ϭ 57) and those that were cDNA. HPRT1 was used as the reference gene. Reactions were performed at invariant (SD Ͻ10Ϫ6; n ϭ 57). These probe sets represent approximately 5700 95°C for 3 min; denaturing at 95°C for 30 s, annealing at 58°C for 30 s, and unique genes. elongation at 72°C for 45 s for 28 cycles; followed by 7 min of extension. PCR Statistical Analysis. Gene expression values were log-transformed by products were resolved on 6% denaturing polyacrylamide gels. After vacuum ϩ ϩ logarithm10(max[X 100,0], 100). Two-sample t tests of the log-transformed drying, gels were exposed to phosphorimager screens (Amersham Biosciences data were used to compare Wnt pathway-defective with pathway-intact sam- Corp., Piscataway, NJ). To determine the ratio of target gene amplification in ples. Fold change in gene expression is the ratio (Wnt defect samples:Wnt tumors, the values from the target gene (CST1) were normalized with the intact samples) of the relative gene expression values, which were computed as values of internal control gene (HPRT1) from each sample. All reactions were the antilogarithm of the mean log-transformed data. We selected candidate performed at least twice. Wnt pathway target genes by demanding that their relative gene expression Plasmids. Expression constructs for the mutant form of ␤-cat (codon 33 levels yield P Ͻ 0.05 for the t test, as well as giving at least a 2-fold increase substitution of tyrosine for serine, S33Y) and dn-TCF-4 (TCF-4r N31) have in the gene expression ratio. been described previously (43). The reporter constructs pTOPFLASH, which To view relationships of the samples based on global gene expression, we contains three copies of an optimal TCF/lymphoid enhancer factor binding performed both HC (39) and PCA (40) on the log-transformed data from all motif (CCTTTGATC), and pFOPFLASH, which contains three copies of a 6955 probe sets. Both HC and PCA were performed using S-PLUS 2000 mutant motif (CCTTTGGCC), were generously provided by Bert Vogelstein software (MathSoft, Inc., Cambridge, MA). HC is a method that finds rela- (Johns Hopkins University) and have been described previously (44). pCH110 tionships between samples based on correlation of their gene expression and (Amersham, Arlington Heights, IL) contains a functional LacZ gene cloned then displays the sample relationships in a taxonomy-like dendrogram. PCA downstream of a CMV early region promoter-enhancer element. DNA frag- contracts multidimensional gene expression data into ranked statistically in- ments containing human CST1 and EDN3 promoter sequences were obtained dependent projections, called components. The first PC captures the greatest by PCR amplification of genomic DNA, using primers for CST1 (accession fraction of variance from the expression data compared to any other projection, number AL591074) and EDN3 (accession number AL035250) from sequences whereas the second PC captures the greatest variance remaining in the data, in GenBank. PCR fragments were cloned into pCR-Blunt II-TOPO (Invitro- independent of the first projection, and so on. Any two PCs can be used to plot gen) and then subcloned upstream of the luciferase reporter gene in the a two-dimensional view of the multidimensional gene expression data such pGL3Basic vector (Promega, Madison, WI). Mutations of presumptive TCF- that samples located close to each other have more similar gene expression binding sites in the CST1 and EDN3 promoters were obtained in vitro by than samples that are further apart. PCR-based mutagenesis using primers containing the desired mutations. All Search for Consensus TCF Sites in Candidate Wnt Pathway Target construct sequences were confirmed by automated sequencing of double- Genes. We searched for consensus TCF sites (5Ј-WWCAAWG-3Ј), as de- stranded DNA templates. fined by Roose and Clevers (41), in selected regions of putative Wnt pathway Cell Culture. The cells lines 293 (transformed human kidney epithelium), target genes in silico. Similar studies were carried out on control genes DLD-1 (colon cancer), SW480 (colon cancer), and TOV112D (OEA) were insensitive to Wnt pathway status, i.e., genes with a geometric mean gene obtained from the American Type Culture Collection (Manassas, VA). Cell expression ratio of 1 Ϯ 0.01 when Wnt pathway-deregulated samples were lines with stable expression of dn-TCF-4 (TCF-4⌬N31/pPGS-CMV-CITE- compared with Wnt pathway-intact samples. Genomic sequences of only fully neo), DLD-1/dn-TCF, SW480/dn-TCF, and TOV112D/dn-TCF or empty vec- mapped genes were downloaded from the June 2002 version of the University tor (pPGS-CMV-CITE-neo) were generated as described previously (45). All

5 http://dot.ped.med.umich.edu:2000/pub/Ovary/index.html. 6 http://genome.ucsc.edu. 2914

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. ␤-CATENIN/TCF TARGET GENES IN OVARIAN CANCER cell lines were grown in DMEM containing 10% fetal bovine serum and penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD). Luciferase Reporter Gene Assays. Reporter assays for ␤-cat/TCF- mediated transcriptional activity were performed in triplicate as described previously (9, 46). Briefly, cells were plated into 6-well dishes and then transfected with various constructs using FuGENE 6 reagent (Roche, Indian- apolis, IN). Cells were co-transfected with 0.5 ␮g of pCH110 (Amersham) to determine transfection efficiency based on ␤-galactosidase expression. After 48 h of incubation at 37°C, transfected cells were washed with PBS and lysed with reporter lysis buffer (Promega). Luciferase assay reagent (Promega) was added to the cell lysates, and then luciferase activity was measured with a luminometer (model TD-20E; Turner Corp., Mountain View, CA). To correct for variation in transfection efficiency, luciferase activity was normalized to ␤-galactosidase activity, which was measured using the ␤-galactosidase en- zyme assay system (Promega) and a microplate reader. To assess the response of human CST1 and EDN3 promoter sequences to ␤-cat, 293 cells were cotransfected with pCH110, 0.2 ␮g of pcDNA3/S33Y, and 0.5 ␮g of various CST1 or EDN3 reporter constructs (see Figs. 3A and 4A)or0.5␮gofthe pGL3Basic vector as a negative control. As a positive control for ␤-cat activation of a TCF responsive promoter, pTOPFlash or pFOPFlash was cotransfected into 293 cells. Reporter gene assays using dn-TCF-mediated transcriptional repression were performed in triplicate as described previously (45). Cell lines stably expressing dn-TCF (pPGS-CMV-CITE-TCF-4r N31) or empty vector (pPGS-CMV-CITE-neo) were cotransfected (0.5 ␮g) with reporter constructs CST1-A, EDN3-A, or pGL3Basic empty vector as a negative control. Transfection efficiency was assessed as described above. The total mass of transfected DNA in each well was kept constant by adding empty vector plasmid DNA. Fold activation of TCF transcriptional activity was calculated by normalizing the luciferase activity to the empty vector (pGL3Basic) control after adjusting for transfection efficiency. ChIP Assay. ChIP assays were performed according to the manufacturer’s protocol (kit 17-925; Upstate Biotechnology, Lake Placid, NY), with some modifications as reported previously (47). Briefly, 1% formaldehyde was added to the culture medium of 2 ϫ 106 adherent cells for 10 min at 37°C. Cells were washed twice with ice-cold PBS (Life Technologies, Inc.) containing proteinase inhibitors. Cells were collected and suspended in 200 ␮l of lysis Fig. 1. Tumor relationships based on global gene expression data from HuGeneFL oligonucleotide microarrays. A, dendrogram of a HC analysis using average linkage. B, buffer containing proteinase inhibitors, and lysed cells were sonicated to yield PCA showing the first two PCs. The Wnt/␤-catenin pathway status of each tumor is 200-1000-bp DNA fragments. After centrifugation to eliminate cell debris, the annotated as indicated at the bottom of the figure. samples were diluted 1:10 in ChIP dilution buffer containing proteinase inhibitors and 0.1 volume of the diluted sample was removed and saved to assess input DNA. Salmon sperm DNA/protein A-agarose (80 ␮l) was added alternative method for viewing relationships between tumors based on to 1.8 ml of the diluted sample and gently mixed for 30 min at 4°C to reduce multidimensional gene expression data. We have plotted the first two nonspecific binding. Then, 4 ␮l of polyclonal anti-␤-cat antibody (06-734; PCs, which represented 23% of the variation in gene expression, in Upstate Biotechnology) was added to the precleared ChIP solution and incu- Fig. 1B. Tumor samples with deregulated ␤-cat occupy a space that bated overnight at 4°C with gentle agitation, followed by the addition of the minimally overlaps the area defined by OEAs without ␤-cat deregu- salmon sperm DNA/protein A-agarose slurry for 1 h. Immunoprecipitates were lation. The observation that the first PC for this large gene collection washed, and the DNA-protein complexes were eluted as reported previously is strongly associated with Wnt pathway status was unexpected and (47). NaCl (final concentration, 200 mM) was added, and the samples were suggests that this feature is a critical, but not sole, determinant of gene incubated at 65°Cfor4htoreverse the formaldehyde cross-link. After expression in OEAs. treatment with proteinase K, DNA was extracted from the samples with Identification of Candidate Wnt Pathway Target Genes in phenol/chloroform and precipitated with ethanol. DNA pellets were suspended ␮ OEAs. We required that candidate Wnt/␤-cat pathway target genes in 40 lofH2O. The primer pairs used to amplify EDN3 promoter sequences and “irrelevant” DNA downstream and upstream of the EDN3 promoter from meet both of the following criteria: (a) expression value increased at the immunoprecipitated chromatin are specified on our website.5 least 2-fold in OEAs with deregulated ␤-cat compared with OEAs with apparently intact ␤-cat regulation; and (b) significant Student’s t Ͻ RESULTS test (P 0.05). Eighty-one genes satisfying these criteria were identified (Table 1). We performed a randomization procedure to Wnt Pathway Status Is a Major Determinant of Global Gene demonstrate that 81 genes is more than could be expected by chance Expression in OEAs. We generated gene expression profiles of 28 alone. Specifically, the Wnt pathway status labels were randomly primary OEAs using Affymetrix HuGeneFL oligonucleotide microar- permuted across the 28 OEA samples 2000 times, and in each case, rays. To visualize relationships of tumor samples based on global gene the number of genes in the randomized data that satisfied our gene expression, both HC and PCA of the microarray data were performed. selection criteria was determined. In the randomized data, the maxi- The analyses included data from nearly all of the probe sets on the mum number of genes satisfying our criteria was 65, the minimum chip (6955 of 7069 noncontrol probe sets as described in “Materials number was 0, and the average number was 6.9. Compared with the and Methods”) to obtain views of gene expression unbiased by pre- number of genes found by chance alone, the observed number selection of genes. Fig. 1A shows a HC dendrogram. Notably, all of (n ϭ 81) of candidate genes was highly significant (P Ͻ 0.0005). the OEAs with deregulated ␤-cat are found on one branch of the To assess whether the 81 genes are more highly associated with primary division between the two main clusters. PCA provides an each other in the tumors with deregulated ␤-cat than the other group, 2915

Table 1 Candidate Wnt pathway target genes in OEA Relative mean gene expression

Gene Intact Deregulated Affymetrix probe set symbol Description Wnt pathway Wnt pathway Ratio P LocusLink Refa X54667࿝s࿝at CST4 Cystatin S 927.1 9065.9 9.8 0.0000 1472 U23070࿝at NMA Putative transmembrane protein 346.8 2835.7 8.2 0.0000 25805 X57348࿝s࿝at SFN Stratifin 483.0 3918.3 8.1 0.0000 2810 M81883࿝at GAD1 Glutamate decarboxylase 1 (brain, 67 kDa) 320.0 2480.9 7.8 0.0000 2571 U65932࿝at ECM1 Extracellular matrix protein 1 463.5 2712.3 5.9 0.0000 1893 D89377࿝s࿝at MSX2 msh (Drosophila) homeo box homologue 2a 246.6 1425.0 5.8 0.0000 4488 53 M31682࿝at INHBB Inhibin, ␤ B (activin AB ␤ polypeptide) 137.8 770.4 5.6 0.0001 3625 X52001࿝at EDN3 Endothelin 3 209.1 1122.6 5.4 0.0000 1908 L22524࿝s࿝at MMP7 Matrix metalloproteinase 7 (matrilysin) 1267.0 6059.6 4.8 0.0066 4316 52,28 U14528࿝at SLC26A2 Solute carrier family 26 (sulfate transporter), member 2 476.7 2263.6 4.7 0.0000 1836 S79219࿝s࿝at PCCA Propionyl coenzyme A carboxylase, ␣ polypeptide 1225.0 5661.3 4.6 0.0000 5095 D14838࿝at FGF9 Fibroblast growth factor 9 423.1 1950.8 4.6 0.0000 2254 51 K01396࿝at SERPINA1 Serine (or cys) proteinase inhibitor, clade A, member 1 1661.8 7340.6 4.4 0.0007 5265 M68516࿝rna1࿝at SERPINA5 Serine (or cys) proteinase inhibitor, clade A, member 5 705.4 2973.1 4.2 0.0060 5104 X68742࿝at ITGA1 Integrin, ␣ 1 448.4 1847.2 4.1 0.0000 3672 M21389࿝at KRT5 Keratin 5 (epidermolysis bullosa simplex) 425.5 1688.1 4.0 0.0068 3852 HG880-HT880࿝s࿝at MUC6 Mucin 6, gastric 130.3 512.2 3.9 0.0038 4588 L38517࿝at IHH Indian hedgehog (Drosophila) homologue 176.0 675.2 3.8 0.0005 3549 M55593࿝at MMP2 Matrix metalloproteinase 2 (gelatinase A) 1057.0 3934.7 3.7 0.0000 4313 U33147࿝at MGB1 Mammaglobin 1 570.3 2117.6 3.7 0.0009 4250 J05257࿝at DPEP1 Dipeptidase 1 (renal) 202.0 731.4 3.6 0.0038 1800 X91117࿝rna1࿝at SLC6A2 Solute carrier family 6 (noradrenalin transporter), member 2 265.6 923.9 3.5 0.0000 6530 U53506࿝at DIO2 Deiodinase, iodothyronine, type II 109.4 376.0 3.4 0.0004 1734 X51441࿝s࿝at SAA1 Serum amyloid A1 302.1 1004.6 3.3 0.0311 6288 X67697࿝at SPAG11 Sperm associated antigen 11 406.9 1342.0 3.3 0.0016 10407 X61118࿝rna1࿝at LMO2 LIM domain only 2 (rhombotin-like 1) 469.9 1507.7 3.2 0.0000 4005 HG2981-HT3127࿝s࿝at CD44 CD44 antigen (Indian blood group system) 396.9 1239.1 3.1 0.0000 960 49 X59798࿝at CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1) 1390.7 4318.4 3.1 0.0081 893 29,30 X16354࿝at CEACAM1 Carcinoembryonic antigen-related cell adhesion molecule 1 589.7 1734.8 2.9 0.0010 634 L13286࿝at CYP24 Cytochrome P450, subfamily 24 (vitamin D 24-hydroxylase) 211.3 613.9 2.9 0.0001 1591 J02611࿝at APOD Apolipoprotein D 787.4 2285.9 2.9 0.0013 347 M21624࿝at TRD@ T cell receptor ⌬ locus 366.1 1057.5 2.9 0.0005 6964 D50840࿝at UGCG UDP-glucose ceramide glucosyltransferase 1613.0 4520.1 2.8 0.0001 7357 U59321࿝at DDX17 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 17 427.9 1177.4 2.8 0.0056 10521 X13255࿝at DBH Dopamine ␤-hydroxylase 203.2 526.3 2.6 0.0029 1621 X68733࿝rna1࿝at SERPINA3 Serine (or cysteine) proteinase inhibitor, clade A, 3 1172.8 3010.1 2.6 0.0153 12 X78706࿝at CRAT Carnitine acetyltransferase 872.0 2213.1 2.5 0.0035 1384 AB000220࿝at SEMA3C Sema domain, immunoglobulin domain, (semaphorin) 3C 1517.5 3840.1 2.5 0.0038 10512 M31994࿝at ALDH1 Aldehyde dehydrogenase 1, soluble 1522.8 3809.8 2.5 0.0128 216 X07730࿝at KLK3 Kallikrein 3, (prostate specific antigen) 545.9 1362.6 2.5 0.0105 354 M57710࿝at LGALS3 Lectin, galactoside-binding, soluble, 3 (galectin 3) 7561.3 18451.6 2.4 0.0006 3958 L27479࿝at X123 Friedreich ataxia region gene X123 205.3 499.9 2.4 0.0020 9413 D42073࿝at RCN1 Reticulocalbin 1, EF-hand calcium binding domain 1952.3 4737.2 2.4 0.0003 5954 M14539࿝at F13A1 Coagulation factor XIII, A1 polypeptide 549.7 1327.9 2.4 0.0098 2162 S39329࿝at KLK2 Kallikrein 2, prostatic 193.8 464.0 2.4 0.0204 3817 HG1067-HT1067࿝r࿝at NULL Homo sapiens clone 24747 mRNA sequence 125.2 298.4 2.4 0.0287 NULL U53347࿝at SLC1A5 Solute carrier family 1 (neutral A.A. transporter), 5 1495.1 3556.5 2.4 0.0009 6510 X68314࿝at GPX2 Glutathione peroxidase 2 (gastrointestinal) 245.7 582.3 2.4 0.0047 2877 U83115࿝at AIM1 Absent in melanoma 1 1147.9 2685.3 2.3 0.0153 202 M22490࿝at BMP4 Bone morphogenetic protein 4 840.0 1950.7 2.3 0.0002 652 48 S62539࿝at IRS1 Insulin receptor substrate 1 525.5 1217.1 2.3 0.0003 3667 L11708࿝at HSD17B2 Hydroxysteroid (17-␤) dehydrogenase 2 267.9 616.5 2.3 0.0003 3294 M27492࿝at IL1R1 Interleukin 1 receptor, type I 1579.7 3624.7 2.3 0.0019 3554 X76717࿝at MT1L Metallothionein 1L 2966.1 6801.2 2.3 0.0020 4500 M35252࿝at TM4SF3 Transmembrane 4 superfamily member 3 549.6 1254.8 2.3 0.0478 7103 D64109࿝at TOB2 Transducer of ERBB2, 2 1082.9 2458.5 2.3 0.0007 10766 M97925࿝rna1࿝at DEFA5 Defensin, ␣ 5, Paneth cell-specific 515.6 1166.1 2.3 0.0018 1670 U90911࿝at NULL H. sapiens cDNA: FLJ23260 fis, clone COL05804 1311.3 2946.8 2.2 0.0005 NULL HG2755-HT2862࿝at PLS3 Plastin 3 (T isoform) 838.7 1846.4 2.2 0.0276 5358 J03779࿝at MME Membrane metallo-endopeptidase (neutral, CD10) 520.3 1129.1 2.2 0.0032 4311 X75208࿝at EPHB3 EphB3 1070.5 2304.6 2.2 0.0001 2049 50 U46689࿝at ALDH10 Aldehyde dehydrogenase 10 (fatty aldehyde dehydrogenase) 630.0 1353.6 2.1 0.0000 224 U73799࿝at NULL H. sapiens mRNA, (from clone DKFZp434B1620) 115.0 245.0 2.1 0.0014 NULL X16832࿝at NULL H. sapiens cDNA: FLJ22499 fis, similar to Hu cathepsin H 2622.9 5520.0 2.1 0.0111 NULL X69111࿝at ID3 Inhibitor of DNA binding 3, dominant negative H-L-H protein 1670.5 3503.4 2.1 0.0014 3399 HG2167-HT2237࿝at LBC Lymphoid blast crisis oncogene 1455.2 3028.9 2.1 0.0000 3928 U07807࿝at NULL H. sapiens pseudogene for metallothionein and AG/CT repeat 196.8 405.5 2.1 0.0001 NULL M94151࿝at CTNNA2 Catenin (cadherin-associated protein), ␣ 2 308.1 634.7 2.1 0.0154 1496 U35048࿝at TSC22 Transforming growth factor ␤-stimulated protein TSC-22 3889.6 7986.1 2.1 0.0008 8848 J04164࿝at IFITM1 Interferon induced transmembrane protein 1 (9-27) 16201.5 33164.5 2.0 0.0471 8519 X81892࿝at GPR64 G protein-coupled receptor 64 353.8 723.9 2.0 0.0313 10149 M14745࿝at BCL2 B-cell CLL/lymphoma 2 662.6 1355.5 2.0 0.0020 596 U52828࿝s࿝at CTNND2 Catenin, ⌬ 2 (neural plakophilin-related arm-repeat protein) 335.2 684.4 2.0 0.0012 1501 X71125࿝at QPCT Glutaminyl-peptide cyclotransferase (glutaminyl cyclase) 303.5 619.4 2.0 0.0001 25797 D83735࿝at CNN2 Calponin 2 1416.7 2882.3 2.0 0.0047 1265 U43148࿝at PTCH Patched (Drosophila) homologue 404.5 822.9 2.0 0.0004 5727 U73960࿝at ARL4 ADP-ribosylation factor-like 4 725.2 1473.4 2.0 0.0003 10124 M80482࿝at PACE4 Paired basic amino acid cleaving system 4 891.8 1800.0 2.0 0.0018 5046 M34455࿝at INDO Indoleamine-pyrrole 2,3 dioxygenase 1402.3 2812.3 2.0 0.0249 3620 D86980࿝at KIAA0227 KIAA0227 protein 288.0 576.6 2.0 0.0000 23508 D87258࿝at PRSS11 Protease, serine, 11 (IGF binding) 2996.2 5994.7 2.0 0.0127 5654 a Bold text indicates genes that have been previously reported as potential ␤-cat/TCF transcriptional targets in other systems. 2916

Table 2 The distribution of consensus TCF (5Ј-WWCAAWG-3Ј) sites differs between candidate Wnt pathway target genes and control genes Up-regulateda (ur), n ϭ 67 Controlb (c), n ϭ 100 95% Confidence interval

c d e f Gene region N T Pur NT Pc Pur/Pc Lower Upper P Ϫ5000:transcript-start 303 335000 9.044 413 500000 8.30 1.09 .93 1.26 0.115 Ϫ500:transcript-start 32 33500 9.55 42 50000 8.40 1.14 .61 1.66 0.292 Ϫ1000:Ϫ501 41 33500 12.239 40 50000 8.00 1.53 .86 2.20 0.027g Ϫ1500:Ϫ1001 33 33500 9.851 44 50000 8.80 1.12 .61 1.62 0.312 Ϫ2000:Ϫ1501 24 33500 7.164 53 50000 10.60 0.68 .35 1.00 0.946 Ϫ3000:Ϫ2001 53 67000 7.91 90 100000 9.00 0.88 .58 1.18 0.772 Ϫ4000:Ϫ3001 53 67000 7.91 65 100000 6.50 1.22 .78 1.66 0.144 Ϫ5000:Ϫ4001 67 67000 10.00 79 100000 7.90 1.27 .85 1.68 0.077 5Ј-UTR 203 162313 12.51 281 289367 9.70 1.29 1.06 1.52 0.003 3Ј-UTR 112 95710 11.702 289 245374 11.80 0.99 .78 1.21 0.523 Ϫ1000 from translation-stop 72 67000 10.746 135 100000 13.50 0.80 .57 1.02 0.942 translation-stop:ϩ500 43 33500 12.836 82 50000 16.40 0.78 .49 1.07 0.904 ϩ501:1000 29 33500 8.657 53 50000 10.60 0.82 .45 1.19 0.810 a Genes from Table 1 for which complete genomic sequence was available. b Control genes that have a gene expression ratio of 1 Ϯ 0.01 (Wnt deregulated/Wnt intact). c N, number of consensus (5Ј-WWCAAWG-3Ј) TCF sites within a specified gene region. d T, total number of possible sites of a 7-mer sequence within the specified gene region. e ϫ 4 P, proportion of consensus TCF sites within the specified up-regulated (Pur) or control (Pc) gene region (N/T) 10 . f P is from one-sided Z test. g Bold indicates significance.

we performed a permutation procedure. We randomly selected 81 invariant expression in OEAs with respect to ␤-cat regulation status). genes from the set of 6954 genes and calculated the pairwise corre- Consequently, we obtained publicly available genomic sequence lation (Spearman’s rho) coefficient between every pair of 81 genes. spanning 5000 bp upstream of the inferred start of transcription to We counted the proportion of these correlation coefficients that had an 1000 bp downstream of the translation stop codon for genes in Table absolute value greater than 0.3. This procedure was repeated 400 1 as well as 100 randomly selected control genes. We were able to times. In the observed data, this proportion is 0.47 for the Wnt acquire full genomic sequences for 67 of the 81 candidate target pathway-defective group and 0.29 for the Wnt pathway-intact group. genes. We then compared the proportion of consensus TCF-binding In the permutations, the range of proportions was 0.35–0.45 in the sites between the candidate target genes and control genes in various Wnt pathway-defective group and 0.25–0.32 in the Wnt pathway- regions, based on annotation in the RefSeq database (Table 2). In intact group. This demonstrates that these 81 genes are more highly candidate ␤-cat/TCF target genes, we found a significantly higher associated with each other in tumors with deregulated ␤-cat than in proportion of consensus TCF-binding sites in the region from Ϫ501 to ␤ tumors with intact -cat regulation. Ϫ1000 bp with respect to the transcriptional start site and in the Importantly, 7 (Table 1, bold text) of the 81 candidate Wnt pathway 5Ј-UTR. Notably, a recent study has shown the presence of function- ␤ target genes have been reported previously as potential -cat/TCF ally relevant TCF-binding sites outside the promoter (i.e., in the first transcriptional targets in other systems, namely, BMP4 (48), CCND1 intron) of the ␤-cat/TCF target gene, AXIN2 (55). Our results show (29, 30), CD44 (49), EPHB3 (50), FGF9 (51), MMP7 (28, 52), and that certain presumptive regulatory regions of our candidate Wnt/ MSX2 (53). To the best of our knowledge, the oligonucleotide mi- ␤-cat target genes have more consensus TCF-binding sites compared croarray used for this study includes probe sets for 34 previously with control genes. As such, the data offer further evidence that our implicated Wnt pathway target genes (represented by 55 probe sets) list of candidate target genes contains many genes likely to be directly reported to date (for a list of these genes and references, see our regulated by ␤-cat/TCF. website5 specified in “Materials and Methods”). These previously Validation of Microarray Data. Five of the most highly up- reported target genes were identified through our review of the liter- regulated candidate genes shown in Table 1 [namely, cyclinD1 ature and from those listed on the Nusse laboratory website (Stanford (CCND1), cystatin 1/cystatinSN (CST1),8 endothelin 3 (EDN3), fi- University).7 Because 7 of the 34 putative Wnt pathway target genes broblast growth factor 9 (FGF9), and stratifin (SFN, “14-3-3 sigma”)] were found on our list of 81 candidate target genes, our list is highly were selected for analysis. Semiquantitative multiplex RT-PCR enriched with presumptive Wnt pathway targets (P Ͻ 0.00001, Fish- (CST1) and real-time q-RT-PCR (CCND1, EDN3, FGF9, and SFN) er’s exact test). Notably absent from the list, among others, is c-MYC, which has been reported to be a Wnt pathway target gene in another were used to validate the microarray data (Fig. 2). The microarray system (31, 54), but not in OEAs (37). data (Fig. 2, A, C, E, G, and I) reveal, not unexpectedly, some Localization of Consensus TCF-binding Sites in Candidate heterogeneity of candidate gene expression within the two groups of ␤-Cat/TCF Target Genes. At least some of the genes with increased tumors. Nonetheless, it can be seen that the majority of samples with ␤ expression in OEAs with deregulated ␤-cat might be expected to be deregulated -cat express these genes at much higher levels than the direct targets of ␤-cat/TCF-mediated transactivation. Presumably, this majority of the samples with an intact Wnt pathway and, importantly, occurs through sequence-specific binding of the ␤-cat/TCF complex that the same conclusions can be reached from the RT-PCR data to TCF recognition sites in key regulatory elements, perhaps including (Fig. 2, B, D, F, H, and J, respectively). Moreover, for all of the genes, those at or near the genes’ proximal promoters. Based on these the RT-PCR data were highly correlated (P Ͻ 0.01) with the micro- considerations, we hypothesized that if a significant proportion of the array data (r ϭ 0.9, 0.77, 0.87, 0.65, and 0.58, respectively) as genes on our list were direct ␤-cat/TCF transcriptional targets, then estimated by the 18 samples included in both the PCR and microarray these genes might show differences in TCF-binding site number and/or distribution when compared with control genes (i.e., genes with 8 The HuGeneFL microarray does not contain a probe set specific for CST1.We determined that the probe set for CST4 (“X54667_s_at”) was primarily measuring CST1, and not CST4 gene expression in our OEAs [see the website specified in “Materials and 7 http://www.stanford.edu/ϳrnusse/wntwindow.html. Methods” (footnote 5)]. 2917

Fig. 2. Comparison of CCND1, FGF9, CST1, EDN3, and SFN gene expression from microarray and RT-PCR analyses. For each gene, relative expression based on the microarray data (A, C, E, G, and I) is shown, and mean fold gene expression (normalized to HPRT) based on the q-RT-PCR (B, D, H, and J) or multiplex-RT-PCR (F) is shown. For q-RT-PCR, 17 and 14 OEAs with an intact or deregulated Wnt pathway, respectively, were used in the analysis, and for multiplex-RT-PCR, 9 and 10 OEAs with an intact or deregulated Wnt pathway, respectively, were analyzed. Expression differences for CCND1 (P Ͻ 0.001), FGF9 (P Ͻ 0.026), CST1 (P Ͻ 0.00003), EDN3 (P Ͻ 0.016), and SFN (P Ͻ 0.0004), validated by RT-PCR between tumors with intact versus deregulated ␤-cat/TCF signaling, were readily apparent.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. ␤-CATENIN/TCF TARGET GENES IN OVARIAN CANCER experiments. Validation experiments were conducted only on the five genes specified above. Hence, we found no examples of discordance between the microarray and q-RT-PCR data. The CST1 and EDN3 Proximal Promoters Are Responsive to ␤-Cat/TCF. We sought to demonstrate that some of the novel genes on our target gene list were responsive to ␤-cat/TCF. Two genes, CST1 and EDN3, were selected for further evaluation. Analysis of the sequence of the CST1 (NT_011387) and EDN3 (NT_011362) promoter regions obtained via NCBI Entrez Nucleotide view revealed several potential TCF-binding sites within 2000 bp upstream of the genes’ purported transcriptional start sites. We used PCR to clone promoter sequences from both CST1 and EDN3, and sequence analysis of the cloned fragments confirmed the presence of the potential TCF-binding sites, which are shown schematically in Figs. 3A and 4A. To determine whether these minimal promoter sequences were responsive to ␤-cat, we generated luciferase reporter gene constructs containing the CST1 and EDN3 promoter fragments. For both genes, constructs containing mutations or deletions of the putative TCF recognition sites were also generated (Figs. 3A and 4A). We found that a mutant constitutively active form of ␤-cat (S33Y mutant), when coexpressed with a CST1 or EDN3 reporter construct in 293 cells, strongly stimulated reporter activity (Figs. 3B and 4B). Mutation of all four potential TCF-binding sites in the CST1 promoter (CST1-Am1–4) completely abrogated the response to ␤-cat (Fig. 3B). The other mutation and deletion constructs all showed reduced luciferase reporter activity compared with the construct containing the intact CST1 promoter fragment. Similar studies using mutated and deleted ver- sions of the EDN3 promoter fragment suggest that the TCF-binding site at –1614 is most critical for imparting TCF responsiveness to the EDN3 promoter (Fig. 4B). These data indicate that the proximal promoters of CST1 and EDN3 are responsive to ␤-cat/TCF and support the notion that both genes are directly activated by deregulated ␤-cat upon its interaction with TCF in OEAs with Wnt pathway defects. The DLD-1 and SW480 colorectal cancer-derived and TOV112D OEA-derived cell lines have deregulated ␤-cat, as a result of biallelic inactivation of APC (DLD-1 and SW480) or activating mutation of CTNNB1 (TOV112D). To further demonstrate that the CST1 as well as EDN3 promoters are responsive to TCF/␤-cat, we established cell lines that stably express a dn-mutant form of TCF (dn-TCF), which should diminish transcriptional activity of TCF-responsive promoters. Transcriptional activity, as measured by luciferase activation of CST1-A (Fig. 3C) and EDN3-A (Fig. 4C) reporter constructs, was reduced in the presence of dn-TCF in all three cell lines. These Fig. 3. Upstream regulatory sequences of CST1 are TCF responsive. A, schematic experiments provide additional evidence that proximal promoter se- diagram of the human CST1 minimal promoter indicating the locations of the transcrip- ␤ tional start, canonical TCF-binding sites, and extent of sequences cloned into pGL3Basic quences of CST1 and EDN3 are indeed TCF/ -cat responsive, con- luciferase reporter vector. Designations of the nine CST1 reporter constructs and the status sistent with the view that CST1 and EDN3 are likely to be directly [wild-type or mutant (m)] of candidate TCF-binding sites in the constructs are indicated. regulated by ␤-cat and TCF in OEAs. The bold/underlined nucleotides within candidate TCF-binding sites indicate the position of the mutations in the mutant reporter constructs. B, effects of ␤-cat on human CST1 ChIP Assays Demonstrate Interaction of ␤-Cat with an EDN3 promoter reporter constructs in 293 cells. The relative activity of the CST1 constructs Promoter Region Containing Presumptive TCF-binding Sites. To shown in A was assessed after transient transfection of the cells with either pcDNA3 ␤ Ј (empty vector) or pcDNA3 construct containing constitutively active ␤-cat(S33Y). C, cell document direct interaction of -cat with sequences in the 5 -flanking lines expressing dn-TCF were transiently cotransfected with CST1-A promoter construct region of EDN3, we performed ChIP assays using an antibody directed (or empty vector) and pCH110. Luciferase activity was normalized to ␤-galactosidase against ␤-cat and chromatin obtained from SW480/neo or SW480/ activity and reported as the relative fold activation in transcriptional activity compared with empty vector (pGL3Basic) control. dn-TCF cells. The positions of the various primers used to amplify and detect specific regions of immunoprecipitated EDN3 promoter fragments are shown in Fig. 4A. TCF-binding site-containing DNA fragments from promoter fragment and further bolster the case that EDN3 is a direct the EDN3 promoter were readily recovered using primers flanking the transcriptional target of ␤-cat/TCF. two putative TCF-binding sites at –1693 and Ϫ1614 and, as expected, were reduced in the presence of dn-TCF (Fig. 4D). Moreover, irrelevant DISCUSSION upstream and downstream DNA fragments lacking TCF-binding sites were not recovered after ChIP with ␤-cat (Fig. 4C). These findings The Wnt signaling pathway is frequently deregulated in certain support results from the reporter assays suggesting a role for the TCF- types of human cancer. In tumors with Wnt pathway defects, stabi- binding site at –1614 in mediating TCF responsiveness of the EDN3 lized ␤-cat interacts with TCF transcription factors to mediate in- 2919

Fig. 4. TCF elements in the promoter of human EDN3 help regulate transcriptional activity and directly interact with ␤-cat. A, schematic diagram of the human EDN3 minimal promoter indicating locations of the transcriptional start, canonical TCF-binding sites, and extent of sequences cloned into pGL3Basic luciferase reporter vector. Designations of the six EDN3 reporter constructs and the status [wild-type or mutant (m)] of candidate TCF-binding sites in the constructs are indicated. The bold/underlined nucleotides within candidate TCF-binding sites indicate the position of the mutations in the mutant reporter constructs. B, effects of ␤-cat on human EDN3 promoter reporter constructs in 293 cells. The relative activity of the EDN3 constructs shown in A was assessed after transient transfection of the cells with either pcDNA3 (empty vector) or pcDNA3 construct containing constitutively active ␤-cat(S33Y). C, cell lines expressing dn-TCF were transiently cotransfected with EDN3-A pGL3Basic luciferase reporter construct (or empty vector) and pCH110. Luciferase activity was normalized to ␤-galactosidase activity and reported as the relative fold activation in transcriptional activity compared with empty vector (pGL3Basic) control. D, ChIP assay. Cross-linked DNA from SW480/neo and SW480/dn-TCF cell lysates were combined with antibody against ␤-cat (or no antibody), and the im- munoprecipitate was subjected to PCR to amplify various regions of the EDN3 promoter.

creased expression of specific genes, at least some which likely play be due to the consequences of the disrupted signaling pathway under critical roles in cancer pathogenesis. Not surprisingly, identification of scrutiny. downstream targets of activated ␤-cat has become a subject of intense Other signaling pathways, such as those mediated by PTEN and, to a interest. Recent studies have identified several putative targets of lesser degree, KRAS, are likely to be aberrant in some of our OEAs, based ␤-cat signaling, primarily through analysis of cultured cells or animal on the reported frequency of PTEN and K-RAS mutations in this tumor model systems. We have used a novel strategy to discover potential type (57, 58). Clearly, defects in signaling pathways other than Wnt will ␤-cat/TCF target genes, comparing gene expression in otherwise contribute to variation of gene expression in individual tumors. Nonethe- similar primary tumors with and without Wnt/␤-cat pathway defects. less, our global gene expression analysis provides strong evidence that Our results suggest that the approach is a robust one for identifying Wnt pathway status, in and of itself, is a major determinant of global gene novel candidate genes worthy of further investigation as direct ␤-cat/ expression in OEAs. In the future, it will be interesting to survey genes TCF transcriptional targets with functional roles in cancer develop- in other pathways that may modulate the one mediated by APC/␤-cat/ ment or progression. This type of approach may be more likely to TCF. For example, the PI3K/Akt pathway has been shown to modulate yield useful insights into cancer pathogenesis than simple compari- ␤-cat/TCF-mediated gene expression in prostate cancer cells through sons of gene expression in tumor cells with their normal cell coun- phosphorylation and inhibition of GSK3␤, a downstream substrate of terparts, given the substantial variation in many phenotypic features PI3K/Akt, resulting in stabilization of ␤-cat (59). PTEN, an inhibitor of between neoplastic and nonneoplastic cells. Moreover, as is the case the PI3K/Akt pathway, may inhibit nuclear accumulation of ␤-cat in for epithelial ovarian cancer (56), the cell of origin for many cancers certain settings (60). may be unclear or difficult to obtain in sufficient quantities for global We recognize that some of the candidate Wnt/␤-cat pathway target gene expression profiling. By comparing a sufficiently large number genes shown in Table 1 may not be direct targets. However, 2 of the of otherwise similar tumors with and without defects in a particular top 10 genes on our candidate list not previously suggested as Wnt/ signaling pathway, nonspecific variations in gene expression are min- ␤-cat pathway targets, namely, CST1 and EDN3, were shown to have imized, and a significant proportion of the variation under study may ␤-cat/TCF-responsive upstream regulatory sequences. The protein 2920

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. ␤-CATENIN/TCF TARGET GENES IN OVARIAN CANCER product encoded by CST1, cystatin 1 (Cst1), is one of several salivary 9. Wu, R., Zhai, Y., Fearon, E. R., and Cho, K. R. Diverse mechanisms of ␤-catenin cysteine protease inhibitors and a member of a cysteine protease deregulation in ovarian endometrioid adenocarcinomas. Cancer Res., 61: 8247–8255, 2001. inhibitor superfamily (61). Very little is known about any role Cst1 10. Cadigan, K. M., and Nusse, R. Wnt signaling: a common theme in animal develop- may have in cancer. Based on serial analysis of gene expression ment. Genes Dev., 11: 3286–3305, 1997. 11. Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S. H., Masiarz, F. R., (SAGE), CST1 was determined to be up-regulated in a metastatic Munemitsu, S., and Polakis, P. 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Donald R. Schwartz, Rong Wu, Sharon L. R. Kardia, et al.

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