The Toll-like receptor (TLR) 4 variant D299G promotes inflammation-associated colon cancer progression in the intestinal epithelium

Abstract

In active IBD colitis, TLR4 expression is significantly upregulated in intestinal epithelial cells (IEC). The variant TLR4-D299G has recently been associated with increased IBD risk. However, the phenotypic consequences of this mutation in IEC have not yet been resolved. The AIM OF THIS STUDY was to determine the effects of TLR4-D299G on IEC function. IEC lines (Caco-2) stably overexpressing HA-tagged wildtype TLR4, mutant TLR4-D299G or TLR4-T399I were generated. Gene expression profiling using DNA microarray analysis (Human Gene 1.0 ST; Affymetrix) was performed. Findings were confirmed by qRT-PCR and validated by immunoblot, ELISA, confocal immunofluorescence and functional analyses. In-vivo tumorigenicity was tested using the CD-1 nu/nu mice xenograft model. Human colon cancer specimens (n=214) were genotyped and assessed for disease stage. In contrast to control clones, TLR4-D299G IEC exhibited a mesenchymal phenotype with multipolar mitotic spindles. TLR4-D299G induced Wnt/ß-catenin activation, leading to IEC de-differentiation: a) phosphorylated ß-catenin translocated from the membrane to the cytoplasm and accumulated in the nucleus, b) E-cadherin redistributed to the cytoplasm which correlated with loss of cell polarity, and c) the transcriptional factor Snail2 anchored to the plasma membrane. The major TLR4-D299G targets were dominated by genes interconnected with “tumorigenesis” and “inflammation” (A2M, ALCAM, ANXA1, C5, CD47, CHI3L1, HPX, IL2RG, TF, TFPI, SERPINC1). TLR4-D299G IEC constitutively secreted large protein amounts of coagulation and complement factors (A2M, TFPI, C3a, C5a). TLR4-D299G induced Wnt-dependent STAT3 activation, which triggered IEC invasion. All of these effects were not evident in the control clones (TLR4-WT, TLR4-T399I, mock). Xenografts in mice revealed TLR4-D299G-mediated acceleration of intestinal tumor growth - which was blocked by a specific STAT3 inhibitor. At time of diagnosis human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (UICC≥III: 70% vs. 46%; P=0.0142) with metastasis (UICC=IV: 42% vs. 19%; P=0.0065) than those with wild-type TLR4. Expression of STAT3 mRNA was significantly increased among human colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. However, expression of TLR4 mRNA was elevated in all primary human colon cancers, regardless of genotype, when compared to normal tissues. Our findings show that TLR4-D299G drives inflammation-associated epithelial-mesenchymal transition and neoplastic progression in the IEC line Caco-2 in-vitro and in-vivo and associates with advanced colon cancer in humans. Future studies must determine whether TLR4-D299G puts IBD patients at increased risk to develop a more aggressive phenotype of colitis - associated neoplasia. [funded in part by the CCFA (SRA-1790)]