Wnt/β-Catenin Signaling Enhances Cyclooxygenase-2 (COX2) Transcriptional Activity in Gastric Cancer Cells

De Ferrari GV (2011) Wnt/b-Catenin Signaling Enhances Cyclooxygenase-2 (COX2) Transcriptional Activity in Gastric Cancer Cells. PLoS ONE 6(4): e18562. doi:10.1371/journal.pone.0018562 Wnt/b-Catenin Signaling Enhances Cyclooxygenase-2 (COX2) Transcriptional Activity in Gastric Cancer Cells Felipe Nun ez 0 Soraya Bravo 0 Fernando Cruzat 0 Martn Montecino 0 Giancarlo V. De Ferrari 0 Moray Campbell, Roswell Park Cancer Institute, United States of America 0 1 Centro de Tecnolog a e Innovacio n para el Ca ncer (CTI-Ca ncer), Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences, Universidad de Concepcio n , Concepcio n, Chile , 2 Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andre s Bello , Santiago , Chile Background: Increased expression of the cyclooxygenase-2 enzyme (COX2) is one of the main characteristics of gastric cancer (GC), which is a leading cause of death in the world, particularly in Asia and South America. Although the Wnt/bcatenin signaling pathway has been involved in the transcriptional activation of the COX2 gene, the precise mechanism modulating this response is still unknown. Methodology/Principal Findings: Here we studied the transcriptional regulation of the COX2 gene in GC cell lines and assessed whether this phenomenon is modulated by Wnt/b-catenin signaling. We first examined the expression of COX2 mRNA in GC cells and found that there is a differential expression pattern consistent with high levels of nuclear-localized bcatenin. Pharmacological treatment with either lithium or valproic acid and molecular induction with purified canonical Wnt3a significantly enhanced COX2 mRNA expression in a dose- and time-dependent manner. Serial deletion of a 1.6 Kbp COX2 promoter fragment and gain- or loss-of-function experiments allowed us to identify a minimal Wnt/b-catenin responsive region consisting of 0.8 Kbp of the COX2 promoter (pCOX2-0.8), which showed maximal response in genereporter assays. The activity of this pCOX2-0.8 promoter region was further confirmed by site-directed mutagenesis and DNA-protein binding assays. Conclusions/Significance: We conclude that the pCOX2-0.8 minimal promoter contains a novel functional T-cell factor/ lymphoid enhancer factor (TCF/LEF)-response element (TBE Site II; -689/-684) that responds directly to enhanced Wnt/bcatenin signaling and which may be important for the onset/progression of GC. - Funding: This work was supported by CTI-Cancer, Programa Bicentenario en Ciencia y Tecnologa (PBCT) - Comisio n Nacional de Investigacio n Cientfica y Tecnolo gica (CONICYT) grant number PBCT-6 from the Chilean government (M.M. and G.V.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Gastric cancer (GC) is a multifactorial disease, characterized by highly malignant neoplasms in the gastric mucosa, and represents the second leading cause of cancer death worldwide with the highest prevalence in Asia and South America [1,2,3]. Environmental associated risk factors include diet, snuff consumption, obesity and Helicobacter pylori infection [4]. Several mutations in tumor-suppressor genes, including P53, adenomatous polyposis coli (APC), E-cadherin and RUNX3 [3,5], as well as in oncogenes like k-ras, HER2 and b-catenin [3,6,7], have been documented in GC. In addition, over expression of various genes has been documented, including WNT2B [8], TC1 (C8orf4) [9], and the cyclooxygenase 2 (COX2) enzyme, which catalyzes the crucial step in the production of prostaglandin E2, a key mediator of joint inflammation [10,11]. It has been observed that the expression of the COX2 gene is significantly increased in human gastric adenocarcinoma tissues, when compared with paired gastric mucosal specimens devoid of cancer cells [10]. Such increased expression has been proposed to affect the intensity of invasion, size, lymph node metastases, tumor development and bad prognosis [4,12,13]. In this regard, large amounts of data describe chemo-preventive and anticancer activity of non steroidal anti-inflammatory drugs (NSAID) including selective COX2 inhibitors as potential treatments for GC [10,11,14]. Transcriptional control of the COX2 gene depends on the molecular machinery interacting with the COX2 promoter, which seems to be controlled through the activity of various signaling pathways [15,16,17]. Indeed, it was initially established that CRE (259/253), NF-IL6 (2132/2124) and NF-kB (2233/2214) consensus sequences in the COX2 promoter were necessary for the expression of the gene [16]. Subsequent functional studies in the COX2 promoter identified a series of regulatory elements participating in the transcription of the gene, including AP-1, AP2, Sp-1, C/EBPb [18,19,20] and proteins belonging to the T-Cell factor/Lymphoid enhancer factor (TCF/LEF) family of transcription factors, which are crucial for Wnt/b-catenin signal transduction [21]. The Wnt/b-catenin signaling pathway is widely acknowledged as playing a major role in human disease, particularly in the onset and development of cancer [22,23,24]. Interestingly, recent experiments in GC derived cells have shown a relationship between COX2 expression and the inhibition of the Glycogen Synthase Kinase-3b (GSK3b) enzyme [25], which is a key Wnt component that phosphorylates b-catenin and promotes its subsequent degradation via proteasome [26]. The relationship between Wnt/b-catenin and COX2 expression in different cancer cell models is further supported from the following studies. First, it has been observed in the mammary epithelium that Wnt/bcatenin play an indirect effect on COX2 transcription, which could be mediated by up-regulation of an intermediary factor PEA3 [27]. Second, and in contrast to an indirect mode of action, Araki and cols. [21] reported that in colon cancer cells there is an induction in COX2 expression through a b-catenin/TCF dependent mechanism, and partially characterized a consensus TCF/LEF binding site (TBE: core CTTTG) positioned 1,079 bp upstream from the transcriptional start site in the COX2 promoter. Third, it was observed that in colon cancer patients and derived cell lines there is an association between overexpression of the Wnt pathway-associated proteins LEF-1 and Pontin52/ TIP49a and up-regulation of COX2 expression [28]. Finally, using chondrocytes, it has been demonstrated that LEF-1, together with b-catenin, regulated COX2 expression by direct binding of the LEF-1/b-catenin complex to the 39UTR region of the COX2 genomic locus [29]. Therefore, at present there is not a clear picture as to whether Wnt signaling is involved in COX2 gene expression, or as its role in GC onset/progression. Here we sought to understand whether there is a direct regulation of the COX2 gene expression via Wnt/b-catenin signaling a (...truncated)