7B). Similar results were obtained in Hke-3 cells (not shown). Fig. 7 NF-κB (a, b) and AKT (c, d) mediate the growth promoting activity Selleckchem THZ1 of macrophages: HCT116 cells were transfected with an empty vector (neo), dnIκB, or dnAKT as indicated, and were either

cultured with macrophages or were stimulated with IL-1. The number of colonies and their volume were determined as described in Material and Methods. A and C show representative colonies. B: *, p < 0.0001, #, p < 0.0001: **,p = 0.013, ##, p = 0.022, D: *, p =< 0.0001, #, p = 0.0001: **, p = 0.0003, ##, p = 0.00003 Since we demonstrated that AKT is downstream of NF-κB, we next tested whether inhibition of AKT activity in tumor cells alters their interactions with macrophages. Macrophages and IL-1 increased both the size and the number of colonies in HCT116 cells transfected with an empty vector, but not

in cells transfected with dnAKT (Fig. 7C and D), demonstrating that AKT mediates the growth promoting activity of macrophages/IL-1. In two independent experiments, each performed in duplicate, both HCT116 and Hke-3 cells transfected with dnAKT MGCD0103 yielded colonies with significantly larger volume; the reason for this increase remains, for now, unknown. In summary, our data demonstrate that, as tumor cells recruit normal peripheral blood monocytes to the tumor microenvironment, they stimulate them to release IL-1β. We showed that tumor associated macrophages and recombinant IL-1 exert their protumorigenic activity through NF-κB/AKT dependent activation of Wnt signaling in tumor cells (Fig. 8), establishing a novel molecular link among inflammation, Wnt signaling and tumor progression. Fig. 8 Signaling pathway selleck inhibitor whereby tumor Branched chain aminotransferase associated macrophages promote Wnt signaling in tumor cells. Peripheral blood monocytes (Mo) were cultured with control

medium or with conditioned medium from HCT116 or Hke-3 cells for 48 h. As shown here, soluble factor(s) from HCT116 and Hke-3 cells induced maturation of normal peripheral blood monocytes (Mo), demonstrated by phalloidin/DAPI staining, coupled to the release of IL-1β. IL-1β, through activation of NF-κB, induced phosphorylation of PDK1 and AKT, which inactivates GSK3β, leading to enhanced β-catenin/TCF4 transcriptional activity, and increased expression of Wnt target genes in tumor cells, including c-myc and c-jun Discussion We recently reported that macrophages promote growth of colon cancer cells through IL-1 mediated, STAT1 dependent, activation of Wnt signaling (Kaler et al, in press). Here we show that peripheral blood monocytes, direct precursors of the tumor associated macrophages, and IL-1 activate Wnt signaling in tumor cells in a NF-κB/AKT dependent manner.