Definition of Smad3 Phosphorylation Events That Affect Malignant and Metastatic Behaviors in Breast Cancer Cells

Smad3, a major intracellular mediator of transforming growth factor-β (TGF-β) signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGF-β, the TGF-β receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase (CDK) activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGF-β-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial-mesenchymal transition (EMT), and invasive activity. Moreover, all TGF-β responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer.