The cyclin D1 gene is a target of the b-cateninyLEF-1 pathway

b-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell–cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated b-catenin levels in colorectal cancer caused by mutations in b-catenin or by the adenomatous polyposis coli molecule, which regulates b-catenin degradation, result in the binding of b-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the b-cateninyLEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of b-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by b-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased b-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle. b-Catenin is a major component of adherens junctions linking the actin cytoskeleton to members of the cadherin family of transmembrane cell–cell adhesion receptors (1, 2). In addition, b-catenin can translocate into the nucleus (3–7), where it can complex with transcription factors of the LEF-1 family and regulate the expression of specific genes (8, 9). By playing such a dual role—a structural role in cell–cell junctions and a regulatory role in the nucleus—b-catenin can transduce changes in cell adhesion and junction formation to control transmembrane signaling and gene expression (1, 10–12). b-Catenin-mediated signaling depends on its accumulation and subsequent translocation into the nucleus. The level of b-catenin in the cell is regulated by its association with the tumor suppressor molecule adenomatous polyposis coli (APC; refs. 13 and 14), axin (15, 16), and glycogen synthase kinase 3b (GSK-3b; ref. 17). Phosphorylation of b-catenin by the APC– axin–GSK-3b complex (18, 19) leads to its degradation by the ubiquitin–proteasome system (6, 20). The failure of this degradation in cells expressing mutant APC or b-catenin leads to the accumulation of b-catenin and is common in human colon cancer and melanoma (21–23). Elevated b-catenin levels in such tumors are suggested to confer uncontrolled activation of gene transcription by the b-cateninyLEF-1 complex that may contribute to tumor progression (1, 24, 25). The nature of the target genes of the b-cateninyLEF complex is, however, largely unknown, except for the recently discovered c-MYC that was shown to contain LEF-1-binding sequences in its promoter (26). Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle (27). Although the cyclin D1 gene is not amplified in human colon cancer, the expression of cyclin D1 is elevated in about 30% of human adenocarcinomas and in adenomatous polyps of the colon (28, 29), and expression of anti-sense cyclin D1 cDNA abolished the growth of SW480 colon cancer cells in nude mice, indicating a critical role for cyclin D1 in tumorigenesis (30). In this study, we investigated the possibility that the cyclin D1 gene is a target for the b-cateninyLEF-1 complex and show that the cyclin D1 promoter contains a LEF-1 binding sequence that is activated in human colon cancer cells. We further show that transcriptional activation of the cyclin D1 gene in such cells can be inhibited by enhancing the degradation of b-catenin with wild-type APC and axin or by its binding to the cadherin cytoplasmic tail. Unscheduled activation of cyclin D1 transcription by the b-cateninyLEF-1 complex in colon cancer cells may result in uncontrolled cell proliferation and thus contribute to tumor progression in these cells. MATERIALS AND METHODS Plasmid Constructions. Plasmids containing the luciferase reporter under the cyclin D1 promoter and the deletion constructions derived from it have been described (31). The mutated LEF-1 binding site at nucleotides 275 and 274 from AT to GC (2163mtLefCD1LUC) and the deletion of the LEF-1 binding site from 281 to 273 (2163DLefCD1LUC) were constructed by PCR-based site directed mutagenesis by using the 2163CD1LUC plasmid. Reporter plasmids containing luciferase under a multimeric consensusor mutant inactive-LEF-1 binding site (TOPFLASH and FOPFLASH; ref. 9) and vectors expressing various forms of b-catenin (7), LEF-1 (5), axin, APC (15), and the N-cadherin tail (32) have been described. Cell Lines and Transfections. SW480, 293T, and Neuro 2A cell lines were maintained in DMEM with 10% (volyvol) calf serum. Transient transfections were performed by the calcium phosphate precipitation method with 293T and Neuro 2A cell lines and by Lipofectamine with SW480 cells. A b-galactosidase-expressing plasmid (0.5 mg) was included in each transfection to monitor the transfection efficiency. We found that b-galactosidase expression was not affected significantly by either LEF-1 or b-catenin cotransfection. After 48 h, the cells were lysed, and luciferase and b-galactosidase activities were determined by enzyme assay kits from Promega. Luciferase activity was normalized to b-galactosidase activity as an internal transfection control. N-cadherin-tail-expressing cell lines were generated by stably transfecting the pECE-Ncadherin tail and pSV-hygro expression vectors into SW480 cells (7, 32). Individual clones were selected for resistance to 100 mgyml hygromycin. The green fluorescent protein (GFP)– DN-b-catenin and the mutant b-catenin constructs (b-cat Y33) were HA-tagged (7). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas. org. Abbreviations: APC, adenomatous polyposis coli; GSK-3b, glycogen synthase kinase 3b; GFP, green fluorescent protein. ‡To whom reprint requests should be addressed. e-mail: lgbenzev@ weizmann.weizmann. ac.il.