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

Abstract

In response to transforming growth factor β (TGF-β), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-β 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-β/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-β 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-β 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-β-induced functions; TGF-β-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-β-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-β while maintaining some tumor-promoting TGF-β responses.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank Hans Clevers for the pTER siRNA Tet-inducible vector, Ed Leof for the antibody against phosphorylated Smad3, and Mike Howell for the c-junSBR₆-Luc construct. We are grateful to the Cancer Research UK Affymetrix Facility at the Paterson Institute, in particular Yvonne Hey, for performing the microarray analysis; to Simon Tomlinson for help in analyzing the microarray data; to the London Research Institute FACS lab for help with cell cycle analysis, in particular Ayad Eddaoudi; and to Debbie Aubyn for help with the video microscopy and the tracking analysis. We thank Julian Downward, Richard Treisman, and members of the Hill lab for useful discussions and comments on the manuscript.

This work was funded by Cancer Research UK.