[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