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Age is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer‐like phenotype, while promoting motility and invasiveness in MDA‐MB‐231 cells. Decellularized breast matrix from aged mice leads to loss of E‐cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer‐related proteins. The aged matrix upregulates cancer‐related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial‐mesenchymal transition‐related genes. Lysyl oxidase knockdown reverts the aged matrix‐induced changes to the young levels; it relocalizes E‐cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer‐like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention., Aging leads to reduced collagen XV and V, and increased stiffness, cytokine content, collagen and fiber thickness, and curvature in the breast tissue. The aged extracellular matrix, without the cellular components, predisposes the normal and cancer cells to a more invasive phenotype, by changing their gene expression profile. Lysyl oxidase knockdown reverses all the effects.

Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase, and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge and many approaches used to study other solid tumors (lung, colon, and breast, for example) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment. This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients, and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis, and chemoresistance. As these new approaches more accurately recapitulate the complex tumor microenvironment, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge.

During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multi-cellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich sub-mesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior, however the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the sub-mesothelial collagen matrix. Acquisition of Ncad by E-cadherin expressing cells (Ecad+) increased mesothelial clearance activity, but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a “leader-follower” mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad+ in pre-clinical models of EOC metastasis.

A predominant characteristic of metastatic cells is the ability to invade host tissues and establish distant metastatic foci. Release of metastatic cells from a primary tumor results from disruption of tissue architecture and requires reversible modulation of cell-matrix and cell-cell contacts, cytoskeletal rearrangement, and acquisition of enhanced proteolytic potential. Malignant cells produce a spectrum of extracellular proteinases including matrix metalloproteinases (MMPs) that process extracellular matrix components, cell surface proteins, and immune modulators. Dysregulated proteolysis has been implicated in tumor invasion and metastasis in multiple model systems. This review will focus on data that highlight the influence of cell-matrix and cell-cell interactions and their associated signal transduction pathways on proteinase regulation. These data highlight cell adhesion signaling as a mechanism for a versatile cellular proteolytic response to changing microenvironmental cues.

Udgivelsesdato: 1999-Aug-1 Ovarian carcinomas secrete single-chain urinary-type plasminogen activator (scuPA) and expression of uPA is up-regulated relative to normal ovarian epithelium, leading to an enhanced proteolytic capacity which may facilitate invasion. Furthermore, the uPA receptor (uPAR) is present on ovarian carcinoma cells and is occupied in tumour tissues. In the present study, incubation of scuPA with serum-free conditioned medium from ovarian carcinoma cells resulted in release of a 14 kDa polypeptide. N-terminal sequence analysis identified this fragment as the uPA N-terminal fragment (NTF), which contains a growth-factor and a kringle domain. NTF generation was abolished by serine-proteinase inhibitors, but not inhibitors of matrix metalloproteinases, and was not enhanced by the addition of plasminogen or plasmin. To determine whether ovarian carcinoma-cell growth is altered by uPA, the effect of exogenous scuPA or NTF on proliferation was analysed. Both NTF and scuPA induced a dose-dependent increase in proliferation, with maximal stimulation obtained at 10-20 nM. Furthermore, blocking the interaction of endogenous uPA with uPAR using anti-NTF antibodies significantly inhibited proliferation. Together these data indicate that, in addition to enhancing the invasive activity of ovarian carcinoma cells via increased pericellular proteolysis, uPA also acts as a mitogen for ovarian carcinoma cells, suggesting a biochemical mechanism whereby uPA may contribute to ovarian carcinoma progression by modulating both cell invasion and proliferation.

Tryptase is a 31 kDa, glycosylated, trypsin-like enzyme stored in and released from mast cell granules. Human tryptase exists as a tetramer, binds heparin, and has a limited substrate specificity, yet it displays remarkable resistance to inhibition by blood plasma proteinase inhibitors. In this study we have examined the cleavage of human fibrinogen by tryptase. alpha chain cleavage was shown to occur in the carboxyl terminal region at Arg572 and beta chain cleavage was found to occur at Lys21. Kinetic analyses of these reactions yielded Km values of 0.2 microM for alpha chain cleavage and 0.26 microM for beta chain cleavage, as well as kcat/KM values of 7 x 10(5) and 4.6 x 10(5) M-1 s-1 for alpha and beta chain reactions, respectively. Proteolysis at Arg572 destroyed the Arg-Gly-Asp (RGD) sequence motif recognized by cell surface alphavbeta3 integrins, and endothelial cell binding to tryptase-modified fibrinogen was significantly reduced, consistent with loss of the RGD motif. Tryptase competed with thrombin in clotting assays using pure fibrinogen with heparin or blood plasma in the absence of heparin. Thrombin failed to initiate the clotting of fibrinogen following modification by tryptase, and fibrin clotting initiated with Ancrod was stopped and partially reversed by tryptase. These data provide insight concerning the mechanism by which tryptase renders fibrinogen unclottable by thrombin and suggests a novel role for tryptase in the modulation of cellular interactions with fibrin(ogen).

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which are secreted from cells as zymogens and can be activated by treatment with organomercurial reagents or limited proteolysis. The proenzyme forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are found in complex with tissue inhibitor of metalloproteinases (designated proMMP-2/ TIMP-2 and proMMP-9/TIMP-1, respectively). The proposed mechanism of activation by mercurial compounds involves the induction of a conformational change in the zymogen which leads to propeptide autoprocessing. To investigate the possibility of conformational differences in MMPs, solute quenching of MMP intrinsic fluorescence was used to probe the relative exposure of tryptophan residues in latent and mercurial-activated MMPs. Our data demonstrate that fluorescence quenching of the proMMP-2/TIMP-2 complex by either acrylamide or iodide is significantly increased following mercurial activation. In contrast, no significant change in tryptophan accessibility accompanies mercurial treatment of either proMMP-2 or TIMP-2 alone, or mercurial-activated MMP-2 mixed with TIMP-2. To determine whether the enhanced fluorescence quenching was unique to the activated proMMP-2/TIMP-2 complex, similar experiments were performed using MMP-1, MMP-3, and MMP-9/TIMP-1 complex. In all cases, both latent and mercurialtreated MMPs exhibited similar fluorescence quenching profiles, suggesting that there are no significant conformational differences between the zymogen and activated forms of MMP-1, -2, -3, or -9/TIMP-1. The enhanced fluorescence quenching observed with mercurial-treated proMMP-2/TIMP-2 is indicative of increased exposure of a previously buried tryptophan residue(s), providing evidence for a structural rearrangement of the activated complex. These data, together with our previous biochemical observation that mercurial treatment of proMMP-2/TIMP-2 exposes the MMP-2 active site without propeptide processing (Y. Itoh et al. (1995) Biochem. J. 308, 645-651), suggest that the activated proMMP-2 in the complex may represent a transitional conformational intermediate in MMP activation.

Udgivelsesdato: 1995-May-1 The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.

Human lung mast cell tryptase is a trypsin-like serine proteinase that is stored in mast cell granules and released by activated mast cells. Here we report that mast cell tryptase is a potent activator of single-chain urinary-type plasminogen activator (scu-PA, or prourokinase), the zymogen form of urinary-type plasminogen activator (u-PA). Activation was complete within 75 min using an enzyme:substrate molar ratio of 1:50 and was accompanied by cleavage of scu-PA at Lys158-Ile159, generating active two-chain u-PA. The reaction was dependent on enzyme concentration and obeyed Michaelis-Menten kinetics. Kinetic constants calculated for scu-PA activation by mast cell tryptase are Km = 34 microM, Vmax = 3.6 pmol of u-PA/min, and kcat = 0.08 s-1. These data suggest that tryptase from tumor-associated mast cells may participate in the activation of scu-PA.

This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix.

Publisher Summary This chapter focuses on methods to assess proteolytic modification of extracellular matrix (ECM) proteins using isolated proteinases or cell-associated enzymes. Degradation of the ECM and its component proteins by proteolytic enzymes is necessary for a number of physiologic and pathologic processes—including embryonic development, tissue resorption and remodeling, angiogenesis, wound healing, and tumor invasion and metastasis. A number of proteinases from distinct mechanistic classes contribute to ECM processing. However, predominant among the matrix-degrading proteinases are the matrix metalloproteinases (MMPs) or matrixins. This family of secreted and membrane-bound zinc-dependent metalloendopeptidases is widely expressed and can cleave a variety of ECM components—including native fibrillar collagen, denatured collagen, basement membrane collagen, and noncollagenous ECM glycoproteins such as fibronectin, laminin-1, and laminin-5. Analysis of matrix proteolysis can be performed using purified enzymes, proteinase containing conditioned medium or intact cells. In general, enzyme : substrate ratios should be maintained at approximately 1:20–1:50, although this is difficult to ascertain in nonpurified systems. Furthermore, it may be speculated that supraphysiologic enzyme : substrate ratios may prevail at proteinase-rich sites of cell–matrix contact.

Via their integration of the intermediate filament cytoskeleton into the cell membrane, desmosomes facilitate the maintenance of cell shape and tissue integrity as well as intercellular communication. The transmembrane components of the desmosome, the desmogleins and desmocollins, are members of the cadherin family of cell–cell adhesion molecules. Each of these proteins exists as three distinct isoforms, which are the products of individual genes and expressed in a cell-type and differentiation-specific manner. Previous work has suggested that desmoglein 1 binds to its catenin partner, plakoglobin, in an approximately 6:1 stoichiometry. In this study, the molecular organization of complexes formed by plakoglobin and desmoglein 1, 2, or 3 are further examined through immunoprecipitation, size exclusion chromatography and sucrose density sedimentation analysis. It is shown that the complex formed between plakoglobin and desmoglein 1 has an overall molecular weight greater than that of plakoglobin/desmoglein 2 or plakoglobin/desmoglein 3; however, the stoichiometry of the plakoglobin/desmoglein 1 complex does not appear to exceed 2:1.

The initial site of melanoma cell metastasis is frequently the regional lymph nodes, and the appearance of lymph node metastasis correlates with poor prognosis. Lymph node adhesion is mediated by an interaction between the tumor cell integrin alphavbeta3 and lymph node vitronectin. In this study, we explored the relationship between adhesion and proteolysis by examining the direct effect of vitronectin receptor ligation on matrix metalloproteinase-2 (MMP-2) production by B16F1 and B16F10 melanoma cells. We report a dose-dependent increase in secretion of both MMP-2 and tissue inhibitor of metalloproteinases-2 (TIMP-2) in response to vitronectin. Cellular invasiveness was also enhanced by vitronectin, as shown by the increased ability of vitronectin-treated cells to invade a synthetic basement membrane (Matrigel). Both the vitronectin-induced MMP-2 production and vitronectin-enhanced invasion were blocked by the peptide ligand Arg-Gly-Asp-Ser (RGDS). Furthermore, neither plasmin-degraded vitronectin nor the peptide ligand RGDS stimulated MMP-2 secretion or invasiveness, indicating that a multivalent ligand-receptor interaction rather than simple receptor occupancy was required for MMP-2 induction. MMP-2 and MMP-2/TIMP-2 interaction with the plasma membrane of melanoma cells resulted in enhanced catalytic activity against 14C-labeled gelatin, suggesting that membrane association may function in posttranslational regulation of MMP-2 activity. This is supported by data showing increased cellular invasion by cells containing membrane-bound MMP-2. Binding of proMMP-2 and proMMP-2/TIMP-2 to melanoma cells was not inhibited by RGDS, and melanoma cell adhesion to vitronectin was unaffected by pro- or active MMP-2, indicating that MMP-2 did not interact with the murine vitronectin receptor. Together, these data provide evidence for a functional link between adhesion and proteolysis and suggest a potential mechanism whereby adhesion of an invasive cell to the extracellular matrix regulates subsequent invasive behavior.

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).