TLR4 Activates the B-Catenin Pathway to Cause Intestinal Neoplasia

Abstract

Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide, is over-expressed in humans with ulcerative colitis-associated cancer. We aimed to determine whether TLR4 plays a role as a tumor promoter in the absence of inflammation. In vivo studies were performed using villin-TLR4 mice, which we generated to over-express TLR4 in the intestinal epithelium, and C57BL/6J wild type mice as controls. Colonic tumorigenesis was induced using the genotoxic agent azoxymethane (AOM). Cell proliferation was assessed by BrdU labeling, and β-catenin was stained by immunohistochemistry. Colon specimens at baseline and after AOM were scored for histological inflammation by three pathologists blinded to the mouse strain and treatment. In vitro experiments were performed using SW480 and IEC-6 intestinal epithelial cell lines. We found that at baseline villin-TLR4 mice had increased epithelial proliferation as well as longer crypts, compared to wild type mice. In addition, villin-TLR4 mice developed spontaneous duodenal adenomas. In wild-type mice, AOM treatment rarely resulted in tumors. By contrast, AOM induced robust colonic tumorigenesis in villin-TLR4 mice. Furthermore, villin-TLR4 mice were more likely to develop larger quantities of tumors than wild-type mice. Histologically, these tumors ranged from low-grade to high-grade dysplasia and carcinoma in situ similar to adenomatous polyps in humans. Moreover, histological scoring of villin-TLR4 and wild-type mice before and after AOM did not reveal any acute inflammation. Tumors arising in villin-TLR4 mice are characterized by intense nuclear β-catenin staining. In addition, villin-TLR4 mice showed an increase in β-catenin nuclear translocation as well as a greater cell proliferation in non-dysplastic colon compared to wild-type mice. Biochemical studies in colonic epithelial cells lines revealed that TLR4 activates β-catenin in a PI3K-dependent manner. Over-expression of TLR4 increases the risk of colon neoplasia even in the absence of inflammation. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat colorectal cancer.