Membrane-type matrix metalloproteinases (MT-MMP) constitute a subfamily of six distinct membrane-associated MMPs. Although the contribution of MT1-MMP during different steps of cancer progression has been well documented, the significance of other MT-MMPs is rather unknown. We have investigated the involvement of MT4-MMP, a glycosylphosphatidylinositol-anchored protease, in breast cancer progression. Interestingly, immunohistochemical analysis shows that MT4-MMP production at protein level is strongly increased in epithelial cancer cells of human breast carcinomas compared with normal epithelial cells. Positive staining for MT4-MMP is also detected in lymph node metastases. In contrast, quantitative reverse transcription-PCR analysis reveals similar MT4-MMP mRNA levels in human breast adenocarcinomas and normal breast tissues. Stable transfection of MT4-MMP cDNA in human breast adenocarcinoma MDA-MB-231 cells does not affect in vitro cell proliferation or invasion but strongly promotes primary tumor growth and associated metastases in RAG-1 immunodeficient mice. We provide for the first time evidence that MT4-MMP overproduction accelerates in vivo tumor growth, induces enlargement of i.t. blood vessels, and is associated with increased lung metastases. These results identify MT4-MMP as a new putative target to design anticancer strategies.

Angiogenesis, the formation of new blood vessels from preexisting ones, is a key event in tumor progression controlled by a balance between positive and negative regulators (1, 2). From this observation has emerged the concept of the "angiogenic switch" in which endothelial activation status is determined by the induction of angiogenic factors and/or the loss of inhibitors (3). Positive regulators include at least vascular endothelial growth factor family (VEGF-A, -B, -C, -D), 1 fibroblast growth factors, placental-like growth factor (PlGF), angiopoietins, their tyrosine kinase receptors (VEGF-R1, -R2; Tie1 and Tie2) and neuropilin-1 (NRP-1), a co-receptor for VEGF (1, 4). An increasing number of negative regulators have been identified such as inhibitors of proteinases, thrombospondins, interferons, chemokines (IP-10 and PF-4), and bioactive fragments of the extracellular matrix (2, 4). Once a tumor has acquired an angiogenic phenotype, tumor cell migration, invasion, and vessel sprouting require the dynamic and coordinated action of many cell surface molecules, including proteinases and integrins mediating cell-matrix interactions.Among the cell-associated proteinases, matrix metalloproteinases (MMPs) anchored to the plasma membrane, called membrane-type MMPs (MT-MMPs) play a pivotal role in pericellular proteolysis. Of the six MT-MMPs that have been identified to date, four (MT1-, MT2-, MT3-, and MT5-MMP) contain a transmembrane domain followed by a cytoplasmic tail and two (MT4-and MT6-MMP) are tethered to the cell surface via a glycosylphosphatidylinositol link (for review, see . It now appears that all MT-MMPs may have the ability to activate pro-MMP2 (9 -13). Although, each of the four physiological tissue inhibitors of MMPs (TIMPs) can non selectively bind to all active MMPs, TIMP1 is unable to inhibit effectively MT1, 2, 3, and 5-MMPs (14). TIMP-2 has dual functions, inhibiting the activity of each MT-MMP and participating in the activation of pro-MMP2 through the formation of a ternary complex formed of MT1-MMP, TIMP-2, and pro-MMP2 (for review, see Refs. 7,15,and 16). MMP2 activation by MT1-MMP could be promoted by interaction with integrin (17) and oligomerization of 19).

The restricted view of matrix metalloproteinases (MMPs) as simple destroyers of extracellular matrix components has largely ignored their substantial contribution in many aspects of cancer development and metastatic dissemination. Over the last few years, the relevance of MMPs in the processing of a large array of extracellular and cell surface-associated proteins has grown considerably. Our knowledge about the complex functions of MMPs and how their contribution may differ throughout cancer progression is rapidly expanding. These new findings provide several explanations for the lack of success of MMP inhibition in clinical trials. A complete understanding of MMP biology is needed before considering them, their substrates or their products as therapeutic targets. In this review, we explore the different faces of MMP implication in breast cancer progression by considering both clinical and fundamental aspects.

Cell surface proteolysis is an important mechanism for generating biologically active proteins that mediate a range of cellular functions and contribute to biological processes such as angiogenesis. Although most studies have focused on the plasminogen system and matrix metalloproteinases (MMPs), recently there has been an increase in the identification of membrane associated proteases, including serine proteases, ADAMs, and membrane-type MMPs (MT-MMPs). Normally, protease activity is tightly controlled by tissue inhibitors of MMPs (TIMPs) and plasminogen activator inhibitors (PAIs). The balance between active proteases and inhibitors is thought to determine the occurrence of proteolysis in vivo. High concentrations of proteolytic system components correlate with poor prognosis in many cancers. Paradoxically, high (not low) PAI-1 or TIMP concentrations predict poor survival in patients with various cancers. Recent observations indicate a much more complex role for protease inhibitors in tumour progression and angiogenesis than initially expected. As knowledge in the field of protease biology has improved, the unforeseen complexities of cell associated enzymes and their interaction with physiological inhibitors have emerged, often revealing unexpected mechanisms of action.

The present study aims at investigating the mechanism by which membrane-type 4 matrix metalloproteinase (MT4-MMP), a membrane-anchored MMP expressed by human breast tumour cells promotes the metastatic dissemination into lung. We applied experimental (intravenous) and spontaneous (subcutaneous) models of lung metastasis using human breast adenocarcinoma MDA-MB-231 cells overexpressing or not MT4-MMP. We found that MT4-MMP does not affect lymph node colonization nor extravasation of cells from the bloodstream, but increases the intravasation step leading to metastasis. Ultrastructural and fluorescent microscopic observations coupled with automatic computer-assisted quantifications revealed that MT4-MMP expression induces blood vessel enlargement and promotes the detachment of mural cells from the vascular tree, thus causing an increased tumour vascular leak. On this basis, we propose that MT4-MMP promotes lung metastasis by disturbing the tumour vessel integrity and thereby facilitating tumour cell intravasation.