Abstract
Background: Trefoil factor 1 protein (TFF1) is a small secreted protein. It is constitutively and strongly expressed in the stomach, where it has a key role in maintaining mucosal integrity. Our previous studies using TFF1-knockout mice indicated a mucosal pro-inflammatory phenotype with a sequence of morphological and signaling changes leading to the development of gastric cancer. Methods and Results: In the current study, we hypothesized that the tumorigenic phenotype obtained in the TFF1-deficient gastric tissues is induced through regulation of b-catenin/TCF pathway. Using immunohistochemistry for b-catenin expression in the pyloric antrum region of the stomach in TFF1 wild type and TFF1 knockout mice, our results showed strong nuclear accumulation of b-catenin in the epithelial cells of TFF1 knockout mice; this accumulation was absent in TFF1 wild type. Using In Vitro cell models, we investigated the transcriptional regulation of b-catenin/TCF activity by TFF1 using the pTOP flash and its mutant pFOP flash luciferase reporter assays. MKN28 and SNU1 TFF1 stable cell lines showed 80% and 50% decrease in b-catenin activity, respectively, as compared to their corresponding pcDNA control cells (P<0.001). These results were confirmed using TFF1 adenovirus system inMKN28 cell line where transient TFF1 expression decreased b-catenin/TCF luciferase activity by 60% relative to control (P<0.01). Using quantitative real time PCR, we found that stable TFF1 expression led to a significant downregulation of b-catenin/TCF target genes, c-Myc and Cyclin D1 in MKN28 and SNU1 cell lines, as compared to their pcDNA control cells (P<.01). The immunofluorescence assay data indicated that reconstitution of TFF1 expression in MKN28 cell line by TFF1 adenovirus (5MOI) abrogated translocation of b-catenin to the nucleus and induced strong staining in the cell membrane as compared to the control cells where the b-catenin showed a strong staining in the nucleus. Conclusion: Our data suggest that b-catenin activation could play a role in the development of gastric neoplasms in TFF1 knockout mice. In addition, our In Vitro and In Vivo studies unveil a novel mechanism by which TFF1 regulates b-catenin/TCF activity, thereby implicating its tumor suppressor function in gastric cancer. Ongoing studies using In Vivo, ex-vivo and In Vitromodels are underway to dissect the regulatory mechanisms that mediate TFF1 suppression of b-catenin/TCF.
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