Ida K. Lund

New and Paradoxical Roles of Matrix Metalloproteinases in the Tumor Microenvironment

Processes such as cell proliferation, angiogenesis, apoptosis, or invasion are strongly influenced by the surrounding microenvironment of the tumor. Therefore, the ability to change these surroundings represents an important property through which tumor cells are able to acquire specific functions necessary for tumor growth and dissemination. Matrix metalloproteinases (MMPs) constitute key players in this process, allowing tumor cells to modify the extracellular matrix (ECM) and release cytokines, growth factors, and other cell-surface molecules, ultimately facilitating protease-dependent tumor progression. Remodeling of the ECM by collagenolytic enzymes such as MMP1, MMP8, MMP13, or the membrane-bound MT1-MMP as well as by other membrane-anchored proteases is required for invasion and recruitment of novel blood vessels. However, the multiple roles of the MMPs do not all fit into a simple pattern. Despite the pro-tumorigenic function of certain metalloproteinases, recent studies have shown that other members of these families, such as MMP8 or MMP11, have a protective role against tumor growth and metastasis in animal models. These studies have been further expanded by large-scale genomic analysis, revealing that the genes encoding metalloproteinases, such as MMP8, MMP27, ADAM7, and ADAM29, are recurrently mutated in specific tumors, while several ADAMTSs are epigenetically silenced in different cancers. The importance of these proteases in modifying the tumor microenvironment highlights the need for a deeper understanding of how stroma cells and the ECM can modulate tumor progression.

Urokinase Receptor Variants in Tissue and Body Fluids

The cellular receptor for urokinase, urokinase-type plasminogen activator receptor (uPAR) plays a central role in localizing its ligand, urokinase-type plasminogen activator (uPA) and thereby the plasminogen activation to the cell surface. uPA converts the proenzyme plasminogen to plasmin, which is involved in degradation of the extracellular matrix. In addition, uPA also cleaves uPAR, liberating the ligand-binding domain I, uPAR(I) and leaving the cleaved form, uPAR(II-III) on the cell surface. This cleavage inactivates the binding potential of uPAR toward uPA and vitronectin. uPAR can be shed from the cell surface and both intact and cleaved uPAR variants have been identified in tissue and body fluids. Identification and characterization of cleaved uPAR variants are dependent on monoclonal antibodies with known epitope specificity. Some of these have also been useful for the immunohistochemical localization of uPAR in tumor tissue. A number of immunoassays have been designed to measure uPAR and the collective amounts of all uPAR forms measured by enzyme-linked immunosorbent assay (ELISA) in tumor lysates or blood correlate to prognosis in several forms of cancer. However, the amounts of uPAR(I) and uPAR(II-III) may be directly related to the uPA activity and therefore be even stronger prognostic markers. Immunoassays measuring the individual uPAR forms have recently been designed and can be used to investigate this. This chapter is focused on the mechanism of uPAR cleavage and characterization and identification of the different uPAR forms in biological tissues and body fluids using immunologic methods. Monoclonal antibodies against uPAR are reviewed as well as different immunoassays used to investigate the prognostic potential of uPAR. Finally, an overview of the localization and prognostic significance of uPAR in different cancers and other malignancies is included.

A New Assay for Measurement of the Liberated Domain I of the Urokinase Receptor in Plasma Improves the Prediction of Survival in Colorectal Cancer

BACKGROUND The liberated domain I of the urokinase plasminogen activator receptor [uPAR(I)] is a significant prognostic marker in lung and ovarian cancer, although the uPAR(I) concentration is below the limit of quantification (LOQ) in a substantial proportion of patient samples (Lung Cancer 2005;48:349-55; Clin Cancer Res 2008;14:5785-93; APMIS 2009;117:755-61). This study was undertaken to design an immunoassay with improved functional sensitivity for measuring uPAR(I) and to evaluate the prognostic value of uPAR(I) for colorectal cancer (CRC) patients. METHODS Surface plasmon resonance analysis identified 2 monoclonal antibodies, R3 and R20, that simultaneously bind to the liberated uPAR(I) but not to intact uPAR. We used R3 for capture and Eu-labeled R20 for detection in designing a 2-site sandwich time-resolved fluorescence immunoassay (TR-FIA 4) for measuring liberated uPAR(I). TR-FIA 4 was validated for use with citrated plasma. The prognostic value of the uPAR(I) concentration was evaluated in 298 CRC patients. The Cox proportional hazards model was used for the uni- and multivariate survival analyses. RESULTS The LOQ was 0.65 pmol/L. Liberated uPAR(I) was measurable in all patient samples with TR-FIA 4. In the multivariate analysis that included sex, age, tumor stage, tumor localization, and adjuvant treatment, the uPAR(I) concentration measured with TR-FIA 4 (hazard ratio, 1.72; 95% CI, 1.15-2.57; P = 0.009), as well as the concentration of intact soluble uPAR plus the cleaved uPAR fragment containing domains II and III, tumor stage, and age were independent predictors of prognosis. CONCLUSIONS TR-FIA 4 has a functional sensitivity improved 4-fold over that of the previous uPAR(I) assay. The uPAR(I) concentration measured with TR-FIA 4 is an independent predictor of prognosis in CRC patients.

Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.

Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: inhibitory effects on receptor-mediated uPA activity in vitro and in vivo

Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.

Presence of Insulin-Like Growth Factor Binding Proteins Correlates With Tumor-Promoting Effects of Matrix Metalloproteinase 9 in Breast Cancer

The stroma of breast cancer can promote the disease’s progression, but whether its composition and functions are shared among different subtypes is poorly explored. We compared stromal components of a luminal [mouse mammary tumor virus (MMTV)–Neu] and a triple-negative/basal-like [C3(1)–Simian virus 40 large T antigen (Tag)] genetically engineered breast cancer mouse model. The types of cytokines and their expression levels were very different in the two models, as was the extent of innate immune cell infiltration; however, both models showed infiltration of innate immune cells that expressed matrix metalloproteinase 9 (MMP9), an extracellular protease linked to the progression of many types of cancer. By intercrossing with Mmp9 null mice, we found that the absence of MMP9 delayed tumor onset in the C3(1)-Tag model but had no effect on tumor onset in the MMTV-Neu model. We discovered that protein levels of insulin-like growth factor binding protein-1 (IGFBP-1), an MMP9 substrate, were increased in C3(1)-Tag;Mmp9−/− compared to C3(1)-Tag;Mmp9+/+ tumors. In contrast, IGFBP-1 protein expression was low in MMTV-Neu tumors regardless of Mmp9 status. IGFBP-1 binds and antagonizes IGFs, preventing them from activating their receptors to promote cell proliferation and survival. Tumors from C3(1)-Tag;Mmp9−/− mice had reduced IGF-1 receptor phosphorylation, consistent with slower tumor onset. Finally, gene expression analysis of human breast tumors showed that high expression of IGFBP mRNA was strongly correlated with good prognosis but not when MMP9 mRNA was also highly expressed. In conclusion, MMP9 has different effects on breast cancer progression depending on whether IGFBPs are expressed.

Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells.

Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer. No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies.

Allosteric Inactivation of a Trypsin-Like Serine Protease by An Antibody Binding to the 37- and 70-Loops

Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities for elucidating fundamental allosteric mechanisms. The monoclonal antibody mU1 has previously been shown to be able to inhibit the function of murine urokinase-type plasminogen activator in vivo. We have now mapped the epitope of mU1 to the catalytic domains 37- and 70-loops, situated about 20 Å from the S1 specificity pocket of the active site. Our data suggest that binding of mU1 destabilizes the catalytic domain and results in conformational transition into a state, in which the N-terminal amino group of Ile16 is less efficiently stabilizing the oxyanion hole and in which the active site has a reduced affinity for substrates and inhibitors. Furthermore, we found evidence for functional interactions between residues in uPAs C-terminal catalytic domain and its N-terminal A-chain, as deletion of the A-chain facilitates the mU1-induced conformational distortion. The inactive, distorted state is by several criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics.

Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies

The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.

Interconversion of active and inactive conformations of urokinase-type plasminogen activator

The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the fully active state. Both a monoclonal antibody (mU3) and a peptidic inhibitor (mupain-1--16) stabilize the active state. The epitope of mU3 is located in the 37- and 70-loops at a site homologous to exosite I of thrombin. The N-terminus((Ile16)) of muPA((16--243)) was less exposed upon binding of mU3 or mupain-1--16. In contrast, introduction of the mutations F40Y or E137A into muPA((16--243)) increased exposure of the N-terminus((Ile16)) and resulted in large changes in the thermodynamic parameters for mupain-1--16 binding. We conclude that the distorted state of muPA((16--243)) is conformationally ordered upon binding of ligands to the active site and upon binding of mU3 to the 37- and 70-loops. Our study establishes the 37- and 70-loops as a unique site for binding to compounds stabilizing the active state of serine proteases.