Wnt Signaling and Colon Cancer Stem Cells Figure 5. Snail is stabilized in cells that harbor the mutant b-catenin allele. A: Macrophages and IL1b inactivate GSK3b activity in HCT116 and Hke-3 cells. B: The levels of Snail were determined in HCT116 cells that were cultured with THP1 macrophages or were stimulated with IL1b in the absence or the presence of LY294002 as indicated. C: HCT116WT and HCT116MT cells were cultured with THP1 macrophages or were treated with IL1b or TNF as indicated for 24 hours. D: The ability of THP1 macrophages and recombinant IL1b to induce Wnt signaling was compared in cells transfected with nonspecific (NSP) or Snail specific siRNA. doi:10.1371/journal.pone.0045462.g005 propagate a self-amplifying loop that sustains tumor growth. However, much less is known about the nature of the tumor- derived factors that blunt the cytotoxic ability of macrophages and stimulate their tumor promoting properties. Tumor-derived CCL2 promotes the recruitment of inflammatory monocytes and [25], and tumor-derived versican has been shown to activate myeloid cells via TLR2-dependent signaling [26]. We showed that macrophage-induced activation of Wnt signaling in colon cancer cells promotes their growth and survival, consistent with a prominent role of Wnt signaling in colon cancer, and recurrent mutations of Apc or b-catenin in the majority of sporadic colon cancers [27]. In this study we demonstrated that activation of Wnt signaling in colon cancer cells not only initiates a plethora of cell-intrinsic alterations in epithelial cells, but also impacts how epithelial cells communicate with macrophages. Inactivation of the MT b-catenin allele in HCT116 cells significantly altered their interaction with macrophages. We showed that macrophages failed to promote Wnt signaling and to protect HCT116WT cells from TRAIL-induced apoptosis. This is consistent with our earlier report that expression of dnTCF4 in tumor cells prevented signaling between macrophages and tumor cells [11,13] and confirms that acquisition of oncogenic mutations alters the interaction of epithelial cells with the adjacent stroma. Like IL1b, FGF19 has been shown to modulate Wnt signaling in HCT116 cells [28]. However, in contrast to our finding, FGF19 increased b-catenin/TCF transcriptional activity only in parental HCT116 cells and in HCT116 cells that retained the WT b- catenin allele [28]. Together these data underscore the importance of b-catenin mutations for the response of tumor cells to autocrine and paracrine signals and thus for their interaction with cells in the tumor microenvironment. Our data confirmed that activation of oncogenic pathways, such as Wnt, not only triggers cell-intrinsic changes, but also induces stromal alterations that are required for tumor progression. In contrast to b-catenin mutations, oncogenic activation of kRas in intestinal epithelial cells did not impact the crosstalk between epithelial cells and stroma. We showed that macrophage-derived factors stabilize Snail in colon cancer cells [13], which regulates epithelial mesenchymal transition and thereby promotes stemness of cancer cells and their ability for metastatic seeding [29,30]. Snail is a Wnt target gene PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45462