Convergence of p53 and transforming growth factor β (TGFβ) signaling on activating expression of the tumor suppressor gene maspin in mammary epithelial cells

Using two-dimensional difference gel electrophoresis, we identified the tumor suppressor gene maspin as a transforming growth factor β (TGFβ) target gene in human mammary epithelial cells. TGFβ up-regulatesMaspin expression both at the RNA and protein levels. This up-regulation required Smad2/3 function and intact p53-binding elements in the Maspin promoter. DNAaffinity immunoblot and chromatin immunoprecipitation revealed the presence of both Smads and p53 at the Maspin promoter in TGFβ-treated cells, suggesting that both transcription factors cooperate to induce Maspin transcription. TGFβ did not activate Maspin-luciferase reporter in p53-mutant MDA-MB-231 breast cancer cells, which exhibit methylation of the endogenous Maspin promoter. Expression of ectopic p53, however, restored ligand-induced association of Smad2/3 with a transfected Maspin promoter. Stable transfection of Maspin inhibited basal and TGFβ-stimulated MDA-MB-231 cell motility. Finally, knockdown of endogenous Maspin in p53 wild-type MCF10A/HER2 cells enhanced basal and TGFβ-stimulated motility. Taken together, these data support cooperation between the p53 and TGFβ tumor suppressor pathways in the induction of Maspin expression, thus leading to inhibition of cell migration.