Wnt Signaling and Colon Cancer Stem Cells Figure 3. Macrophage-derived factors do not protect HCT116WT cells from TRAIL induced apoptosis. A: Parental HCT116 cells or HCT116 with deleted WT or MT b-catenin allele were treated with TRAIL in the absence of THP1 macrophages or IL1 as indicated. B: The extent of apoptosis was determined in 4 independent experiments. C: HCT116WT and HCT116MT cells were treated with TRAIL in the absence or the presence of TNF and the extent of apoptosis was determined by PI staining. doi:10.1371/journal.pone.0045462.g003 TRAIL-induced apoptosis, which is consistent with the fact that Wnt signaling protects cells from TRAIL-induced apoptosis [20]. In contrast to parental HCT116 cells and HCT116 cells with deleted WT b-catenin allele (HCT116MT), HCT116WT cells were not protected from TRAIL-induced apoptosis by macrophage, IL1b (Fig. 3A, B) or by TNF (Fig. 3C). Despite harboring mutant b-catenin HCT116 cells display predominantly plasma membrane localization of b-catenin [8]. Treatment of cells with TRAIL triggered the loss of distinct membrane staining in both HCT116MT and HCT116WT cells (Fig. 4A), consistent with the cleavage of b-catenin in TRAIL- treated cells (Fig. 4B). However, while TNF and IL1b restored membrane localization of b-catenin in HCT116MT cells, they failed to counteract the effect of TRAIL in HCT116WT cells (Fig. 4A). Accordingly, macrophages and IL1b failed to inhibit TRAIL-induced cleavage of b-catenin, activation of caspase-8, and subsequent processing of PARP in HCT116WT cells (Fig. 4B). These data confirmed that cells with active Wnt signaling display an altered response to macrophage-derived factors, potentially affecting their susceptibility to therapeutic agents. Lack of Snail stabilization in HCT116 cells with inactivated MT b-catenin allele Wnt signaling has been shown to promote the transcription, the stability and nuclear localization of Snail [15,16]. Indeed, we showed that inactivation of GSK3b by LiCl or by a specific GSK3b inhibitor, AR-A014418, was sufficient to increase the levels of Snail in HCT116 cells [13]. We showed that overex- pression of Snail promotes TCF4-b-catenin-driven transcription, and that silencing of Snail interferes with Wnt signaling in HCT116 cells [13], consistent with the ability of Snail to associate with b-catenin and to co-regulate the expression of Wnt target genes [17,21]. Macrophages and IL1b inactivate GSK3b in both HCT116 and Hke-3 cells (Fig. 5A) and, consistently, stabilize Snail independently of the presence kRas mutations in tumor cells [13]. Activation of GSK3b by a specific PI3K inhibitor, YL294002, precluded macrophage- and IL1b- induced stabiliza- tion of Snail in HCT116 cells, confirming that macrophage- derived factors stabilize Snail through their ability to inactivate GSK3b (Fig. 5B). PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45462