TIMP-1 Induces an EMT-Like Phenotypic Conversion in MDCK Cells Independent of Its MMP-Inhibitory Domain

Abstract

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) regulate epithelial-mesenchymal transition (EMT) critical for the development of epithelial organs as well as cancer cell invasion. TIMP-1 is frequently overexpressed in several types of human cancers and serves as a prognostic marker. The present study investigates the roles of TIMP-1 on the EMT process and formation of the lumen-like structure in a 3D Matrigel culture of MDCK cells. We show that TIMP-1 overexpression effectively prevents cell polarization and acinar-like structure formation. TIMP-1 induces expression of the developmental EMT transcription factors such as SLUG, TWIST, ZEB1 and ZEB2, leading to downregulation of epithelial marker and upregulation of mesenchymal markers. Importantly, TIMP-1′s ability to induce the EMT-like process is independent of its MMP-inhibitory domain. To our surprise, TIMP-1 induces migratory and invasive properties in MDCK cells. Here, we present a novel finding that TIMP-1 signaling upregulates MT1-MMP and MMP-2 expression, and potentiates MT1-MMP activation of pro-MMP-2, contributing to tumor cell invasion. In spite of the fact that TIMP-1, as opposed to TIMP-2, does not interact with and inhibit MT1-MMP, TIMP-1 may act as a key regulator of MT1-MMP/MMP-2 axis. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule and also as an endogenous 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.

TIMP-1 via TWIST1 Induces EMT Phenotypes in Human Breast Epithelial Cells

“…Importantly, we have previously showed that TIMP-1 activates CD63/integrin survival signaling pathways involving the PI3K/Akt pathways for the regulation of apoptosis. In fact, our recent report also showed TIMP-1 induces an EMT-like process independent of the MMP-inhibitory domain in MDCK cells (15), although we could not investigate the involvement of CD63 in the TIMP-1 mediated EMT process in this model because of the limitation of the unidentified canine CD63. However, the MDCK study demonstrated similar results to the TIMP-1 interaction with CD63 and subsequent activation of signal transduction pathways we have found previously in breast epithelial cells.…”

“…Transwell units with polycarbonate filters (Corning Costar, Cambridge, MA) were used for all cell migration assays according to the published protocol (15). Cells on the filters were fixed and stained using the Diff-Quik Stain Set (American Scientific Products, McGaw Park, IL), following the direction of the manufacturer.…”

“…Importantly, we and others have demonstrated a role for TIMP-1 in the regulation of cell growth and apoptosis inhibition in many different cell types. TIMP-1 regulation of cell survival appears to occur through two co-existing pathways: an MMP-dependent pathway (6–8) or MMP-independent mechanism (9–15). Our discovery of CD63, a member of the tetraspanin family of proteins, as a TIMP-1 interacting protein provided molecular insight as to the action of TIMP-1 as a signaling molecule distinct from their MMP inhibitory activity (16).…”

TIMP-1 Induces α-Smooth Muscle Actin in Fibroblasts to Promote Urethral Scar Formation

“…Although TIMP-1 has been recently recognized to play a role in the urethral scar formation, it is mainly thought to inhibit collagenase activity [5][6][7][8]23]. The effect of TIMP-1 on fibroblast transformation, however, has not been studied.…”

Basal/HER2 breast carcinomas

“…3). 92 96,97 analyzed a data set of 52 breast cancer cell lines, including three cell lines that were not included in Neve's study, 94 using a 50-gene supervised risk predictor of breast cancer based on intrinsic subtypes (i.e., luminal A, luminal B, upregulation of the expression of α(5)-integrin (ITGA5), a fibroblast marker and the natural receptor for fibronectin, whose upregulation generally occurs in parallel with an upregulation of α-smooth muscle actin, 106 and caldesmon (CALD1), an actin-linked regulatory protein found in smooth muscle and non-muscle cells that has numerous functions in cell motility exerted via the reorganization of the actin cytoskeleton, 107 compared with trastuzumab-responsive SKRB3 cells, trastuzumab-refractory JIMT1 cells were also found to exhibit increased levels of TIMP1, an endogenous inhibitor of matrix metalloproteinases (MMPs) that also functions as a signaling molecule that induces the expression of developmental EMT transcription factors, such as SNAIL2/SLUG, TWIST1 and ZEB, 108 and of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein involved in extracellular matrix remodeling and invasion that is directly involved in the acquisition of mesenchymal traits that favor metastatic dissemination.…”