James P. Quigley

Localization of Matrix Metalloproteinase MMP-2 to the Surface of Invasive Cells by Interaction with Integrin αvβ3

Abstract Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin αvβ3. MMP-2 and αvβ3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of αvβ3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind αvβ3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.

Matrix metalloproteinases and tumor metastasis.

Functions of individual matrix metalloproteinases (MMPs) differentially expressed by tumor cells and stromal cells, are finely regulated by their spatial as well as temporal interactions with distinct cellular and extracellular components of the tumor microenvironment and also distant pre-metastatic sites. Certain aspects of MMP involvement in tumor metastasis such as tumor-induced angiogenesis, tumor invasion, and establishment of metastatic foci at the secondary site, have received extensive attention that resulted in an overwhelming amount of experimental and observational data in favor of critical roles of MMPs in these processes. In particular, dependency of tumor angiogenesis on the activity of MMPs, especially that of MMP-9, renders this step possibly the most effective target of synthetic MMP inhibitors. MMP functioning in other stages of metastasis, including the escape of individual tumor cells from the primary tumor, their intravasation, survival in circulation, and extravasation at the secondary site, have not yet received enough consideration, resulting in insufficient or controversial data. The major pieces of evidence that are most compelling and clearly determine the role and involvement of MMPs in the metastatic cascade are provided by molecular genetic studies employing knock-out or transgenic animals and tumor cell lines, modified to overexpress or downregulate a specific MMP. Findings from all of these studies implicate different functional mechanisms for both tumor and stromal MMPs during distinct steps of the metastatic cascade and indicate that MMPs can exhibit pro-metastatic as well as anti-metastatic roles depending on their nature and the experimental setting. This dual function of individual MMPs in metastasis has become a major focus of this review.

Activation of Matrix Metalloproteinase-9 (MMP-9) via a Converging Plasmin/Stromelysin-1 Cascade Enhances Tumor Cell Invasion

Matrix metalloproteinase-9 (MMP-9) may play a critical catalytic role in tissue remodeling in vivo, but it is secreted by cells as a stable, inactive zymogen, pro-MMP-9, and requires activation for catalytic function. A number of proteolytic enzymes activate pro-MMP-9 in vitro, but the natural activator(s) of MMP-9 is unknown. To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells (MDA-MB-231 breast carcinoma cells) that were induced to produce MMP-9 over a 200-fold concentration range (0.03–8.1 nm). The levels of tissue inhibitors of metalloproteinase (TIMPs) in the induced cultures remain relatively constant at 1–4 nm. Quantitation of the zymogen/active enzyme status of MMP-9 in the MDA-MB-231 cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether pro-MMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous urokinase-type plasminogen activator, is not an efficient activator of pro-MMP-9, neither the secreted pro-MMP-9 nor the very low levels of pro-MMP-9 associated with intact cells. Although plasmin can proteolytically process pro-MMP-9, this limited action does not yield an enzymatically active MMP-9, nor does it cause the MMP-9 to be more susceptible to activation. Plasmin, however, is very efficient at generating active MMP-3 (stromelysin-1) from exogenously added pro-MMP-3. The activated MMP-3 becomes a potent activator of the 92-kDa pro-MMP-9, yielding an 82-kDa species that is enzymatically active in solution and represents up to 50–75% conversion of the zymogen. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to both degrade extracellular matrix and transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodelling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9 blocking monoclonal antibody.

Distribution of (Na+-K+-stimulated ATPase activity in rat intestinal mucosa

Abstract The isolation in high yield of a membrane-bound high specific activity (Na+-K+-stimulated ATPase from rat intestinal mucosal cells is descrbed. The activity is originally associated with the mitochondrial subcellular fraction but can be isolated by sucrose gradient centrifugation after aging of the crude mitochondrial fraction. The final (Na+-K+)-stimulated ATPase membrane is relatively free of brush border, mitochondria, nuclear and microsomal contamination and would appear to be plasma membrane. The activity is recovered in greater than 50% yield from the original homogenate and with a 25–35-fold increase in specific activity. A small, but consistent amount of (Na+-K+-stimulated ATPase is also found in the brush border fraction. These findings are discussed in relation to active transport in the intestine.

Viral nanoparticles as tools for intravital vascular imaging.

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 μm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.

Membrane anchored serine proteases: A rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer

Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been recognized that are directly anchored to plasma membranes. These membrane anchored serine proteases are anchored either via a carboxy-terminal transmembrane domain (Type I), a carboxy terminal hydrophobic region that functions as a signal for membrane attachment via a glycosyl-phosphatidylinositol linkage (GPI-anchored), or via an amino terminal proximal transmembrane domain (Type II or TTSP). The TTSPs also encode multiple domains in their stem regions that may function in regulatory interactions. The serine protease catalytic domains of these enzymes show high homology but also possess features indicating unique substrate specificities. It is likely that the membrane anchored serine proteases have evolved to perform complex functions in the regulation of cellular signaling events at the plasma membrane and within the extracellular matrix. Disruption or mutation of several of the genes encoding these proteases are associated with disease. Many of the membrane anchored serine proteases show restricted tissue distribution in normal cells, but their expression is widely dysregulated during tumor growth and progression. Diagnostic or therapeutic targeting of the membrane anchored serine proteases has potential as promising new approaches for the treatment of cancer and other diseases.

Human neutrophils uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis

Several lines of evidence have implicated matrix metalloproteinase 9 (MMP-9) as a protease inducing an angiogenic switch critical for tumor progression. Among MMP-9-expressing cell types, including cancer cells and tumor-associated leukocytes, inflammatory neutrophils appear to provide an important source of MMP-9 for tumor angiogenesis. However, delivery of MMP-9 by neutrophils has not been mechanistically linked to its catalytic activity at the angiogenic site. By using a modified angiogenic model, allowing for a direct analysis of exogenously added cells and their products in collagen onplants grafted on the chorioallantoic membrane of the chicken embryo, we demonstrate that intact human neutrophils and their granule contents are highly angiogenic. Furthermore, purified neutrophil MMP-9, isolated from the released granules as a zymogen (proMMP-9), constitutes a distinctly potent proangiogenic moiety inducing angiogenesis at subnanogram levels. The angiogenic response induced by neutrophil proMMP-9 required activation of the tissue inhibitor of metalloproteinases (TIMP)-free zymogen and the catalytic activity of the activated enzyme. That the high angiogenic potency of neutrophil proMMP-9 is associated with its unique TIMP-free status was confirmed when a generated and purified stoichiometric complex of neutrophil proMMP-9 with TIMP-1 failed to induce angiogenesis. Recombinant human proMMP-9, operationally free of TIMP-1, also induced angiogenesis at subnanomolar levels, but lost its proangiogenic potential when stoichiometrically complexed with TIMP-1. Similar proMMP-9/TIMP-1 complexes, but naturally produced by human monocytic U937 cells and HT-1080 fibrosarcoma cells, did not stimulate angiogenesis. These findings provide biochemical evidence that infiltrating neutrophils, in contrast to other cell types, deliver a potent proangiogenic moiety, i.e., the unencumbered TIMP-free MMP-9.

α2-macroglobulin: an evolutionarily conserved arm of the innate immune system

All animals and plants have immune systems that protect them from the diversity of pathogens that would otherwise threaten their survival. The different components of the immune system may inactivate the pathogens themselves or promote the inactivation and clearance of toxic products produced by the pathogens. An important category of virulence factors of bacterial and prokaryotic pathogens are the proteases, which act to facilitate the invasion of the pathogens and to promote their destructive growth in the host organism. The present review concentrates on the comparative biology of an evolutionarily conserved arm of the immune system, the protein, α2-macroglobulin. α2-Macroglobulin is an abundant protein of the plasma of vertebrates and members of several invertebrate phyla and functions as a broad-spectrum protease-binding protein. Protease-conjugated α2-macroglobulin is selectively bound by cells contacting the body fluids and α2-macroglobulin and its protease cargo are then internalized and degraded in secondary lysosomes of those cells. In addition to this function as an agent for protease clearance, α2-macroglobulin binds a variety of other ligands, including several peptide growth factors and modulates the activity of a lectin-dependent cytolytic pathway in arthropods.

The Inhibition of Tumor Cell Intravasation and Subsequent Metastasis via Regulation of In Vivo Tumor Cell Motility by the Tetraspanin CD151

In vivo tumor cell migration through integrin-dependent pathways is key to the metastatic behavior of malignant cells. Using quantitative in vivo assays and intravital imaging, we assessed the impact of cell migration, regulated by the integrin-associated tetraspanin CD151, on spontaneous human tumor cell metastasis. We demonstrate that promoting immobility through a CD151-specific metastasis blocking mAb prevents tumor cell dissemination by inhibiting intravasation without affecting primary tumor growth, tumor cell arrest, extravasation, or growth at the secondary site. In vivo, this loss of migration is the result of enhanced tumor cell-matrix interactions, promoted by CD151, which prevent dissociation by individual cells and leads to a subsequent inhibition of invasion and intravasation at the site of the primary tumor.

Pleiotropic Roles of Matrix Metalloproteinases in Tumor Angiogenesis: Contrasting, Overlapping and Compensatory Functions

A number of extensive reviews are available discussing the roles of MMPs in various aspects of cancer progression from benign tumor formation to overt cancer present with deadly metastases. This review will focus specifically on the evidence functionally linking the MMPs and tumor-induced angiogenesis in various in vivo models. Emphasis has been placed on the cellular origin of the MMPs in tumor tissue, the requirement of proMMP activation and the resulting proteolytic activity for the induction and progression of tumor angiogenesis, and the pleiotropic roles for some of the MMPs. The functional mechanisms of the angiogenic MMPs are discussed as well as their catalytic detection in complex biological systems. In addition, the contribution of active MMPs to metastatic spread and establishment of secondary metastasis will be discussed in view of the findings indicating that MMPs are involved in the preparation of pre-metastatic niches. Finally, the most recent evidence, indicating the pro-metastatic consequences of anti-angiogenic therapies employing MMP inhibitors will be presented as examples highlighting possible outcomes of interfering with the pleiotropic nature of the MMP functionality.