Activating Mutations in b-Catenin in Colon Cancer Cells Alter Their Interaction with Macrophages; the Role of Snail Pawan Kaler, Leonard Augenlicht, Lidija Klampfer* Department of Oncology, Albert Einstein Cancer Center, Montefiore Medical Center, Bronx, New York, United States of America Abstract Background: Tumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment. Principal Findings: We showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1b. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for b-catenin (HCT116WT/MT), harboring one wild type (WT) and one mutant (MT) allele, but isogenic lines that carry only the WT (HCT116WT) or MT b-catenin allele (HCT116MT) have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT b-catenin allele, but not in HCT116WT cells. Consistent with this observation, macrophages and IL1b failed to stabilize Snail in HCT116WT cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4) or HCT116 cells with silenced Snail failed to stimulate IL1b production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor. Significance: Our data demonstrate that oncogenic b-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages. Citation: Kaler P, Augenlicht L, Klampfer L (2012) Activating Mutations in b-Catenin in Colon Cancer Cells Alter Their Interaction with Macrophages; the Role of Snail. PLoS ONE 7(9): e45462. doi:10.1371/journal.pone.0045462 Editor: Masaru Katoh, National Cancer Center, Japan Received June 18, 2012; Accepted August 22, 2012; Published September 21, 2012 Copyright: 2012 Kaler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by CA 111361 (to LK), U54 CA 100926 (to LA) and P30-13330 from NCI (National Cancer Institute (U.S.)). 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. * E-mail: lidija.klampfer@einstein.yu.edu Introduction Beta catenin (b-catenin) is an E-cadherin binding protein and thus plays an important role in cell-cell adhesion [1]. In addition, it acts as an effector of Wnt signaling [2]. In the absence of Wnt signaling b-catenin is phosphorylated by GSK3b, resulting in its ubiquitination and subsequent degradation in the proteasome [3]. The activity of b-catenin is controlled by the tumor suppressor Adenomatous Polyposis Coli (Apc) and inactivating mutations in the Apc gene, which prevent b-catenin degradation, are found in the large majority of sporadic colorectal cancers [4,5]. Further, approximately 10% of colorectal cancers carry mutations in the GSK3b phosphorylation site located in the N-terminus of b- catenin [6]. Apc mutations and b-catenin mutations are mutually exclusive in colon cancer, as they both lead to stabilization of b- catenin and in constitutive b-catenin/TCF transcriptional activity. HCT116 cells are heterozygous for b-catenin, harboring one wild type (WT) allele and one mutant (MT) allele with inactivation of SER45, one of the residues phosphorylated by GSK3b that is frequently mutated in tumors [6,7]. Isogenic HCT116 cell lines that carry only WT or MT allele have been generated to study the role of oncogenic b-catenin signaling in colon cancer [8,9]. As expected, deletion of the MT b-catenin allele in HCT116 cells significantly reduced b-catenin/TCF mediated transcriptional activity in these cells. While the total levels of b-catenin were similar in cells with disrupted WT or MT b-catenin allele, inactivation of the MT allele resulted in redistribution of b-catenin to cell membranes associated with cell junctions [8,9]. Surprising- ly, expression of typical Wnt target genes, such as c-myc, was not reduced upon deletion of the mutant b-catenin allele, and growth characteristics of these cells under standard conditions were not significantly affected in vitro [8]. These results suggested that mutations in b-catenin, in addition to initiating a plethora of cell intrinsic alterations, may also affect the interaction of tumor cells with the components of the tumor microenvironment and thus contribute to tumor development in vivo. We recently reported that tumor cell-derived factor(s) activated macrophages to produce IL1b, which in turn inactivated GSK3b, increased the levels of unphosphorylated b-catenin and stimulated PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45462