Abstract B49: Smad4 Suppresses Wnt Signaling Through Downregulation of Β-Catenin

Abstract Introduction: Transforming Growth Factorβ (TGFβ) and Wnt signaling pathways modulate differential responses during embryonic development, and later in intestinal epithelial cell differentiation, proliferation and colorectal tumor promotion. β-catenin, the central mediator of canonical Wnt signaling, has been shown to pathologically promote intestinal epithelial cell de-differentiation and induce sustained proliferation in the absence of functional APC. Smad proteins are key developmental proteins involved in TGFβ/Bone Morphogenetic Protein (BMP) signaling involved in epithelial cell growth inhibition. Smad4, in particular, functions as a tumor suppressor and mutation of the Smad4 gene is a frequent and potentially late event in colon cancer. We recently found that expression of wild type Smad4 in Smad4-mutant colon cancer cells results in profound inhibition of β-catenin/TCF-driven transcriptional activation. Purpose: In the current study, we set out to determine the mechanism of Smad4 inhibition of canonical Wnt signaling. Experimental Procedures: We examined steady-state levels of β-catenin mRNA and protein in cells and human tissues, followed by analysis of components of β-catenin transcriptional and degradation machinery in the presence and absence of Smad4, and with and without inhibition of GSK3-β, a critical regulator of cytoplasmic β-catenin. Results: We observed that wild type Smad4 expression resulted in significant repression of Wnt-specific transcriptional activity that could be restored with GSK-3β inhibition and overexpression of wild-type and mutant β-catenin in S W480 cells that have inactivating mutations in both APC and Smad4 genes. Transient overexpression of Smad4 also inhibited β-catenin-dependent transcriptional activity in HEK 293 cells that have wild-type APC and Smad4 genes. β-catenin phosphorylation is intact and unchanged in comparison of controls and Smad4-expressing S W480 cells, but the total level of β-catenin mRNA and protein is decreased in the Smad4-expressing cells. Assessment of human colorectal cancers using tissue microarray analysis revealed an inverse relationship between nuclear Smad4 and tumor cell β-catenin immunostaining. Furthermore, we observed that high Smad4 expression is associated with better disease-free survival and that high β-catenin expression is associated with worse disease-specific survival in analysis of outcomes from 250 patients. Conclusions: Our data suggest that the tumor suppressor role of Smad4 involves reduction of β-catenin levels with resultant reduction of β-catenin/TCF-mediated transcriptional activity, and this regulatory mechanism has an important impact on clinical outcomes in colorectal cancer. Citation Information: Cancer Res 2009;69(23 Suppl):B49.