Abstract 1210: TIMP-1 regulation of breast cancer cell survival and its co-expression with CD63 in vivo

Abstract

Abstract Purpose: The purpose of our study is to unveil the multi-faceted roles of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in cancer progression with a particular focus on anti-apoptotic activity of TIMP-1. Introduction: TIMP-1 has been thought to inhibit cancer invasion/metastasis via its inhibition of Matrix Metalloproteinases (MMPs). However, clinical data showed that TIMP-1 is a poor prognostic marker in many human cancers. Recently, our lab has made a novel finding that TIMP-1 promotes MCF10A cell survival independent of its MMP-inhibitory activity through its interaction with the trans-membrane protein CD63. To investigate TIMP-1's anti-apoptotic effects in the cancerous setting, we have analyzed apoptosis in MDA-MB-231 breast cancer cells. Also, we have examined TIMP-1 and CD63 expression in MCF10DCIS.com tumor xenografts, a human breast cancer progression model. Experimental Procedures: TIMP-1 is highly expressed in the malignant human breast cancer cell line, MDA-MB-231. To evaluate TIMP-1 regulation of cell survival, we generated shRNA-mediated TIMP-1 knockdown MDA-MB-231 cell line. Upon apoptosis induction by staurosporine treatment or growth factor (GF) withdrawal, cell survival and apoptosis were examined by WST-1 assay and caspase activity assay, respectively. Expression and localization of TIMP-1 and CD63 in MCF10DCIS.com tumors was assessed in invasive and pre-invasive ductal lesions by immunohistochemical (IHC) analysis. Co-localization between TIMP-1 and CD63 was detected by confocal microscopic analysis of immunofluorescent staining and quantitated using a statistics-based software, Volocity. Results: Knockdown of TIMP-1 expression greatly increased the rate of apoptotic cell death in MDA-MB-231 cells after staurosporine treatment or GF withdrawal. IHC analysis of MCF10DCIS.com tumors revealed TIMP-1 cell surface staining in ducts without apoptotic/necrotic cores and fewer cells with surface TIMP-1 staining in comedo-necrotic ducts. CD63 staining pattern was punctate within cells as well as on the cell surface throughout the lesions. Confocal microscopic analysis of TIMP-1 and CD63, revealed positive correlation (Pearson's) and increased co-localization at the cell surface (Kx, Ky, and Mx, My -Volocity parameters). Conclusions: We previously demonstrated a novel function of TIMP-1 in cell survival via activation of the CD63 signaling complex in “normal” human breast epithelial cell line MCF10A, independent of its well-known MMP inhibitory activity. Here we report a similar protective role of TIMP-1 in human breast cancer cells. In addition, we show that TIMP-1 is co-expressed with CD63 in MCF10DCIS.com ductal tumors and co-localizes at the cell surface. Taken together, our findings further depict a dual nature of TIMP-1 in cancer progression: tumor-promoting activity via increased cell survival, and tumor-suppressing activity via its MMP-inhibitory activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1210.