5A). Consistently, normal peripheral blood monocytes and THP1 ZD1839 ic50 macrophages failed to induce Wnt signaling in tumor cells that were transfected with dnAKT (Fig. 5B), confirming that AKT mediates the crosstalk between tumor cells and macrophages. Consistent with the inability of IL-1 or THP1 macrophages to promote Wnt signaling in HCT116

cells transfected with dnAKT, these cells did not respond to IL-1 or THP1 macrophages with phosphorylation of GSK3β or activation of β-catenin (Fig. 5C). Finally, we showed that the expression of a constitutively active AKT (CA AKT) was sufficient to drive Wnt signaling (Fig. 5D). Fig. 5 AKT is required for IL-1 or macrophage-induced Wnt signaling. a and PR-171 order b HCT116 cells were transfected with the TOP-FLASH reporter gene and were co-transfected with an empty vector (neo) or dnAKT as indicated. Cells were left untreated (CTRL) or were treated with IL-1 or were co-cultured with normal human peripheral blood monocytes (Mo) or THP1 macrophages. c Cell lysates from HCT116 cells transfected with an empty vector (neo) or dnAKT

were tested for the expression of pGSK3β and active β-catenin. The expression of dnAKT was confirmed by immunoblotting for HA. d HCT116 JNK high throughput screening cells were transfected with the TOP-FLASH reporter gene together with increasing concentrations of an empty vector (neo) or constitutively active AKT (CA AKT). The expression of CA AKT was confirmed by immunoblotting for HA (see the inset). E: HCT116 cells were transfected with an empty plasmid (neo), dnIκB, dnAKT or CA AKT and were cultured with THP1 macrophages or were treated with IL-1 or TNF for 1 h. The levels of c-myc, c-myc Thr58/Ser62, c-jun and βactin were determined by immunoblotting We showed

previously that macrophages and IL-1 induce the expression of Wnt target genes in tumor cells, including c-myc (Kaler et al, in press). c-Myc activity is also regulated at the posttranslational level through GSK3β mediated inhibitory phosphorylation of c-myc at Thr58, and ERK activating phosphorylation at Ser62 [43]. We demonstrated that macrophages and IL-1 induced c-myc phosphorylation on Thr58/Ser62 in tumor cells (Fig. 5E), demonstrating that factors in the tumor microenvironment also regulate the stability of Myc protein in tumor cells. The ability of THP1 macrophages and IL-1 to induce the expression of c-myc and c-jun from and to increase c-myc phosphorylation was abrogated not only in tumor cells transfected with dnIκB (Fig. 5E), but also in cells transfected with dnAKT (Fig. 5F), confirming the requirement of AKT for Wnt signaling. The expression of CA AKT was not sufficient to significantly increase the basal expression of c-myc or c-jun, but it augmented the responsiveness of tumor cells to IL-1 and macrophages (Fig. 5F). TNF acted as a poor inducer of c-myc and c-jun, consistent with its weaker ability to induce Wnt signaling in HCT116 cells (not shown).