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OP13. TLR4 signaling activates the Wnt pathway in a mouse model of colitis-associated neoplasia

R. Santaolalla1, J. Ruiz1, M. Fukata1, C. España1, J.M. Davies1, C. Pastorini1, D.A. Sussman1, J. Ko2, R. Dirisina2, T.A. Barrett2, M.T. Abreu1

1University of Miami, Miller School of Medicine, Division of Gastroenterology, Miami, United States; 2Northwestern University, Feinberg School of Medicine, Division of Gastroenterology, Chicago, United States

Background: In patients with colitis cancers, we have shown that there is an increased expression of toll-like receptor‑4 (TLR4) in intestinal epithelial cells (IECs). The Wnt pathway regulates intestinal cell proliferation and the development of neoplasia. We have previously shown constitutive expression of TLR4 in a mouse model increases inflammation in the mucosa and drives colitis-associated tumorigenesis. We hypothesized that cross-talk between TLR4 and the Wnt pathway could have a synergistic tumorigenic effect in an inflammatory environment.

Methods: Villin-TLR4 mice, which have increased TLR4 signaling in the intestinal epithelium, were compared to wild-type (WT) littermates. To induce tumorigenesis, the mice were treated with an initial injection of the genotoxic agent azoxymethane (AOM) followed by two cycles of 3% dextran sodium sulfate (DSS). Proliferation was assessed by BrDU labeling. Western blots were performed on intestinal IEC lines for total and phosphorylated β-catenin, GSK3-β and AKT. Immunohistochemistry was done for total β-catenin, active β-catenin phosphorylated at Ser552 and the Wnt target cyclin-D1. Villin-TLR4 mice were also crossed to Lgr5-EGFP mice. Lgr5 is Wnt target gene and stem cell marker.

Results: Compared to WT mice, villin-TLR4 mice have a higher cell proliferation rate in the colon crypts and show a higher expression of nuclear β-catenin and cyclin-D1, indicating Wnt pathway activation. Given this, we asked whether TLR4 activity regulated expression of Lgr5. Our data demonstrate that whereas Lgr5 expression is normally located at the base of the crypts in WT mice, there is a dramatic expansion of Lgr5+ cells in the crypts of villin-TLR4 mice at baseline. To understand the mechanism underlying Wnt pathway activation, we found that TLR4 activates the phosphatidylinositide 3‑kinase (PI3K)/AKT pathway to phosphorylate β-catenin at activation sites (Ser552 and Ser675). In vivo, we also see that villin-TLR4 AOM-DSS mice show increased nuclear β-catenin and phosphorylation of β-catenin at Ser552, an event associated with stem cell activation and colitis-associated cancer.

Conclusions: In the intestinal mucosa, tonic stimulation of TLR4 by its ligand LPS can lead to activation of the Wnt signaling pathway and thereby promote neoplasia. In addition, TLR4 is linked to Lgr5 expression in the colon and is associated with an increase in proliferation of the colon crypts. These results highlight the role of bacterial signaling in stimulation of oncogenic pathways.