Abstract A25: Insights into TIMP-1/CD63 interaction and their functional relationship with MT1-MMP in cancer progression

Purpose: To unveil multi-faceted roles of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in cancer progression, particularly, those apart from Matrix Metalloproteinase (MMP) inhibition. Introduction: Clinical studies show increased TIMP-1 to associate with poor prognoses in many types of cancer, contrary to its well-known anti-invasive activity via MMP inhibition. Consistent with clinical data, we have made novel findings that TIMP-1 induces epithelial cell survival signaling and epithelial-mesenchymal transition (EMT) phenotype independent of MMP-inhibitory activity. Importantly, TIMP-1 signaling is dependent on interaction with the tetraspanin CD63, on the cell surface. Interestingly, CD63 was previously shown to bind and induce endocytosis of Membrane-type 1 MMP (MT1-MMP) to the lysosome. We have recently shown that over-expression of TIMP-1 can lead to increased MT1-MMP expression/activity which contribute to the resultant EMT phenotype. However, it has not yet been clarified if TIMP-1 and MT1-MMP have cooperative or competitive interactions with CD63. Methods/Results: To map the interacting domains of TIMP-1, or MT1-MMP with CD63, we have utilized yeast-2-hybrid (Y2H) and protein complementation assay (PCA). For Y2H, the proteins were fused to complementary fragments of the GAL-4 transcription factor. For PCA, partner proteins were fused to complementary fragments of a luciferase enzyme. Our results indicate that the Large Extracellular Loop (LEL) of CD63 may be responsible for TIMP-1 interaction; while the N-terminus or else transmembrane domains interact with MT1-MMP. For TIMP-1, the C-terminal end of the protein shows importance for CD63 interaction; which our preliminary results show can be specifically interrupted by use of a blocking antibody. Interestingly, despite differences in direct binding sites, TIMP-1 and MT1-MMP display competitive and not mutual interaction with CD63 according to our preliminary results. Conclusions: Taken together, we present a novel finding that TIMP-1/CD63 interaction and MT1-MMP/CD63 interaction are not compatible in their occurrence with each other. And that TIMP-1 over-expression in cancer therefore may disrupt the MT1-MMP/CD63 complex which offers MT1-MMP protection from lysosomal degradation. The C-terminus of TIMP-1 is critical to CD63 interaction and may provide a non-MMP-inhibitory site for therapeutically targeting TIMP-1 in cancer. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule via CD63 interaction and as an indirect regulator of MT1-MMP, in addition to its well-known function as an inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression. [Support from: Ruth L. Kirschtein National Research Service Award T-32 CA009531, Thomas C. Rumble University Graduate Fellowship, and NIH/NCI RO1 089113] Citation Format: Richard B. Warner, Young-Suk Jung, Hyeong-Reh C. Kim. Insights into TIMP-1/CD63 interaction and their functional relationship with MT1-MMP in cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A25.