TIMP-1 induces an epithelial mesenchymal transition via upregulation of the transcription factor Twist in human breast epithelial cells

5374 Turnover of the extracellular matrix (ECM) is regulated by the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs1-4). Previous in vitro and mouse studies demonstrated that TIMP-1 reduces tumor cell invasion through MMP inhibition. Therefore, it was unexpected to discover in clinical studies that TIMP-1 overexpression correlates with a poor prognosis in certain malignancies, including metastatic breast cancer. Importantly, we and others found that TIMP-1 is a potent inhibitor of apoptosis in a variety of cell types through either a MMP-dependent or -independent mechanism. Recently, we identified CD63, a member of the tetraspanin family of proteins, as a cell surface binding partner for TIMP-1 which modulates the integrin-mediated survival pathway in the human breast epithelial cell line MCF10A. In addition, we have made a novel finding that TIMP-1 expression in MCF10A cells induces phenotypic changes in cell morphology, cell-cell adhesion, and cytoskeletal remodeling taking on the hallmarks of an epithelial-mesenchymal transition (EMT). This is evidenced by loss of the epithelial cell adhesion molecules E-cadherin and beta-catenin and an increase in the mesenchymal markers vimentin, N-cadherin, and fibronectin. Treatment with rTIMP-1 also led to a loss of E-cadherin in MCF10A cells. Interestingly, TIMP-1 signaling induced Twist expression, a transcription factor known to suppress E-cadherin gene transcription and upregulate mesenchymal markers. SiRNA-mediated Twist knockdown restored E-cadherin expression in TIMP-1 overexpressing MCF10A cells, demonstrating a functional significance of Twist in TIMP-1 mediated EMT. We further demonstrated that TIMP-1 induces Twist expression and EMT in a CD63-dependent manner. Furthermore, analysis of TIMP-1 structural mutants unveiled that TIMP-1 interaction with CD63, activation of cell survival signaling, and induction of EMT in MCF10A cells does not require the MMP-inhibitory domain of TIMP-1. Taken together, we hypothesize that TIMP-1 binding to CD63 activates intracellular signal transduction pathways and leads to inhibition of apoptosis and induction of EMT in breast epithelial cells, facilitating breast cancer progression. Our study of the pleiotropic activities of TIMP-1 at the molecular level may enhance our understanding of TIMP-1’s multi-functions during tumor progression and metastasis. This information may also be useful in designing more rational, mechanism-based therapeutic interventions aimed at modulating activities of MMPs and TIMPs.