Smad4 Dependency Defines Two Classes of Transforming Growth Factor β (TGF-β) Target Genes and Distinguishes TGF-β-Induced Epithelial-Mesenchymal Transition from Its Antiproliferative and Migratory Responses

TLDR

Smad4 complexes with activated Smad2/3 to regulate TGF‑β target genes in the nucleus. The study aimed to determine whether Smad4 is essential for all TGF‑β responses and to assess the impact of its loss in tumor development. Cell lines derived from HaCaT and Colo‑357 were engineered to express a tetracycline‑inducible siRNA that effectively silences Smad4. Loss of Smad4, observed in about 50 % of pancreatic and 15 % of colorectal cancers, silences a subset of TGF‑β target genes, abolishes TGF‑β–induced cell‑cycle arrest and migration but not EMT, indicating that Smad4 loss may promote tumorigenesis by removing tumor‑suppressive but preserving tumor‑promoting TGF‑β responses.

Abstract

In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. Mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.