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Blood, 1 September 2006, Vol. 108, No. 5, pp. 1441-1450. Prepublished online as a Blood First Edition Paper on April 11, 2006; DOI 10.1182/blood-2006-02-005363.
REVIEW ARTICLES Cell-surface association between matrix metalloproteinases and integrins: role of the complexes in leukocyte migration and cancer progressionFrom the Department of Biological and Environmental Sciences, University of Helsinki, Finland.
Leukocyte motility is known to be dependent on both  2-integrins and matrix metalloproteinases MMP-2/-9 or gelatinases, which mediate leukocyte adhesion and the proteolysis needed for invasion, respectively. Gelatinases not only play an important role in cell migration, tissue remodeling, and angiogenesis during development, but are also involved in the progression and invasiveness of many cancers, including leukemias. The concept that MMPs associate with integrins, as well as their importance in some physiologic and pathologic conditions, has been advanced previously but has not been examined on leukocytes. This review will examine mainly the function of the MMP-integrin complexes in normal leukocyte migration and the effect of integrin and broad-spectrum MMP inhibitors in tumor progression.
Neutrophils, also known as polymorphonuclear leukocytes (PMNs) originate from stem cells in the bone marrow. They represent 60% to 70% of the total circulating leukocytes and are the first cells to be recruited to the sites of infection or injury within minutes to hours after maturation, forming a primary defense against infectious agents or "foreign" substances that invade our body's physical barriers. The initiation of an inflammatory response involves 3 major steps: (1) increased blood flow by dilation of capillaries; (2) escape of plasma proteins from the bloodstream; and (3) extravasation of neutrophils through the endothelium and accumulation at the site of injury. Elimination of invading microorganisms is accomplished by phagocytosis, generation of reactive oxygen metabolites, as well as through release of proteolytic enzymes and microbicidal substances, all stored in intracellular granules of mature PMNs.1
The main functions of neutrophils involve adhesion, extravasation, chemotaxis, phagocytosis, and production of oxidative agents. Like all leukocytes, these functions can be triggered by appropriate stimuli and the synergistic action of different adhesion molecules that are present on the surface of both neutrophils and activated endothelial cells.2 Interactions of neutrophils with the activated endothelium have been extensively studied either under static conditions or under physiologic conditions (flow shear forces). Neutrophil tethering and capture have been shown to be mediated by P-selectin binding to its ligand PSGL-1; neutrophil activation by chemokines, such as IL-8; and firm adhesion by ICAM-1 binding to
Structure and function of leukocyte 2-integrins
The structural characteristics and functional roles of leukocyte
Loss of heteromerization of the integrin during biosynthesis caused by mutations in the gene encoding the
Regulation of cell adhesion and migration Leukocyte migration is a complex process, controlled by a wide spectrum of leukocyte and endothelial cell adhesion molecules and by the presence of chemotactic molecules. These molecules, as well as growth factors, are responsible for the establishment of a polarized cell migration and there is enough evidence to prove that signaling from both phospholipids and proteins from the Rho family of small GTPases are also involved in directed cell motility.15 Migration of leukocytes is essential for immune responses, tissue repair, and embryonic development.
A polarized morphology of leukocytes was first described to be similar to that of a migrating amebae, with a leading edge at the front and a uropod at the rear of a migrating cell.16 T cells recognize and bind to antigen presenting cells (APCs) through their leading edge. A number of receptors are concentrated at the leading edge, including Cell-surface association of MMPs and other proteases Matrix metalloproteinases (MMPs) are a family of structurally related and highly conserved zinc-dependent endopeptidases collectively capable of degrading most components of the basement membrane and ECM.20 MMP substrates also include a wide variety of proteins, such as chemotactic molecules, adhesion molecules, proteinase inhibitors, cell-surface receptors, blood clotting factors, latent growth factors, and growth factor-binding proteins. Most human MMPs can be divided according to their sequence homology, substrate specificity, and cellular location into several subclasses: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and others. The basic multidomain structure of MMPs comprises the following: (1) an amino-terminal domain; (2) a catalytic domain; and (3) a carboxy-terminal domain. To date, there are at least 25 secreted or membrane-bound known human MMPs.21 The expression, secretion, and activity of MMPs in normal tissues are subject to tight control. Data generated from intensive studies on MMP activities in different cells and tissues, as well as studies from knock-out animals, witness the importance of these enzymes in many normal physiologic processes (eg, embryonic development, bone resorption, angiogenesis, and wound healing) and pathologic processes (rheumatoid arthritis, multiple sclerosis, periodontal disease, and tumor growth and metastasis).20,22,23
MMPs are secreted as zymogens from inside the cell to the cell surface and into the extracellular environment where they are able to degrade both ECM and non-ECM proteins. It remains unclear how these enzymes make it to the correct location at the cell surface and how the proteolytic activity is controlled at the pericellular space. However, it has been suggested that MMP binding to cell-surface proteins can have an effect on intracellular signaling, facilitate proenzyme localization and activation, mediate cell motility by disruption of cell contacts with the ECM, and promote internalization of the enzyme. For example, integrins are shown to act as receptors for several proteases, including MMPs. Such interactions have been detected in caveolae, in invadopodia, and at the leading edge of migrating cells, where directed proteolytic activity is needed. The first interaction between an integrin (
In leukocytes, uPA could bind to its receptor, uPAR, and to  M2 simultaneously, forming a trimolecular complex where M2 could serve as a signaling receptor.86 This interaction is likely to be mediated by both the kringle and proteolytic domains for uPA and the I-domain for M2. This complex plays an essential role in the migration of inflammatory cells and vascular homeostasis. The uPA/uPAR complex was also found to be associated with the 51-integrin and capable of promoting adhesion and migration of Chinese hamster ovary cells as well as intracellular signal transduction through the integrin. In addition, a cyclic peptide DDGW discovered by phage display and an MMP-9-derived peptide motif HFDDDE both inhibited proMMP-9/M2 complex formation and leukocyte migration in vitro and in vivo.47,87 However, this motif did not block leukocyte adhesion to ICAM-1 and fibrinogen, suggesting the integrin-bound MMP is essential for degradation of integrin-directed bonds to matrix proteins. Recently, proMMP-9 was found to be associated with ICAM-142 and DNA repair protein Ku44 on the surface of leukemic cells. ICAM-1 cleavage by MMP-9 resulted in tumor cell resistance to natural killer cell-mediated cytotoxicity. Also, a chaperone heat shock protein 90 (Hsp90) was found to interact with MMP-2 on the cell surface of fibrosarcoma cells, thus promoting MMP-2 activation, which is critical for tumor invasiveness.33 The binding mechanism of most of these interactions has not yet been elucidated. Several cell-surface hyaluronan receptor CD44 isoforms, RECK, TSP-1, LRP, and cell-surface collagen IV chains also serve as MMP-9-docking molecules. The CD44/MMP-9 complex was found to be associated with invasiveness of mouse mammary carcinoma and human melanoma cells in vivo,41 suggesting that CD44 helps to localize MMP-9 activity to the cell surface. The GPI-linked proteins RECK and TSP-1 were not only identified as cell-surface receptors for MMP-9 but also were found to block their enzymatic activity.46,55 Interaction of MMPs with the cell surface not only may be needed for proenzyme activation and targeting at specific sites for degradation of cell-surface substrates, but also could promote intracellular degradation via receptor-mediated endocytosis (RME). Regulation of the cell-surface activity of proteolytic enzymes that are involved in cancer progression, including MMP-2, -9, -13, tPA, and uPA by endocytosis, has led to suppression of tumor cell invasion.88
A disintegrin and a metalloproteinase (ADAMs) and ADAM with a thrombospondin motif (ADAMTS) comprise a large family of proteins capable of interacting with integrins and involved in processes such as angiogenesis, fertilization, myogenesis, neurogenesis, and inflammation. Unlike the transmembrane proteins AD-AMs, ADAMTS proteins are soluble ECM proteases consisting of a prodomain, metalloprotease, and disintegrin domains, but devoid of ADAMs' cysteine-rich, EGF-like transmembrane and cytoplasmic domains.89 ADAM2 or fertilin
Early events in tumor progression are characterized by increases in cell proliferation, insensitivity to growth-inhibitory signals, reduced ability for differentiation, as well as the ability to escape from apoptosis and immune surveillance.91 Proteinases that degrade components of the ECM and are capable of processing nonmatrix substrates (eg, growth factors and their receptors, chemokines, adhesion molecules, and apoptotic mediators) have long been considered to be important at all stages of tumorigenesis.92 The combined participation of integrins and MMPs is required for invasion of tumor cells into surrounding connective tissues, intravasation and extravasation from blood vessels, and metastasis to distant organs.93 Indeed, studies on TIMPs have shown that overexpression or administration of these inhibitors as recombinant proteins inhibited experimental invasion and metastasis.94 In most cases, the stage of tumor progression correlates with the expression levels of gelatinases, as the invasive and metastatic potential of tumor cells is strongly affected by changes in gelatinase expression in animal models. Expression of MMP-2 and MMP-9 was found to be strongly up-regulated in cancers of lung, colon, breast, skin, and prostate, which correlated with increased tumor invasiveness and metastasis.22 Inhibition of MMP-9 expression in a model of experimental metastasis reduced the number of colonies formed in the lungs of mice.95 Further evidence supporting this hypothesis came from studies on MMP-2 and -9 null mice. These mice developed fewer tumors than the wild type.21 Integrins and gelatinases in invasion and metastasis The initial step of tumor cell invasion is characterized by the breakdown of the basement membrane, a process known to be dependent on type IV collagen-degrading enzymes, mainly MMP-2 and MMP-9. Liotta et al obtained results where type IV gelatinase activity correlated with cancer metastasis.96 Endothelial cell proliferation and migration into the tumor tissue are mediated by angiogenic (eg, MMP-9, VEGF, and basic fibroblast growth factor [bFGF]) and lymphangiogenic factors that are released by tumor cells. Using DNA microarrays, primary tumor-gene expression profiles could be arranged in classes of "good" and "poor" prognosis. DNA-microarray analysis on human breast carcinoma cell lines that have metastasized to bone revealed some of the genes (eg, MMP-1, MMP-2, CXCR4, IL-11, and CTGF) responsible for the increased metastatic potential of breast cancer cells.97,98 Video-microscopy studies showed that MMPs play a significant role in tumor metastasis, as TIMP-1 and MMP inhibitor batimastat (BB-94) blocked the formation of tumors in secondary sites.99 The role of MMPs in tumor invasion and metastasis has also been studied using small-interfering RNAs and antisense technology.100,101 Gelatinases and MT-MMPs revealed a new mechanism to control metastasis by cleavage of the metastasis suppressor gene, KiSS-1.102 Finally, recent studies supporting the in vitro data from double MMP-2/MMP-9-deficient mice demonstrated that these enzymes cooperate in promoting the invasive phenotype of malignant keratinocytes in an experimental model in vivo.103
Changes in integrin expression and localization can also influence invasion and metastasis of tumor cells.104 Integrins were shown to be involved in the migration and liver metastasis of large cell lymphoma cells and angiogenesis, as Integrins and gelatinases in cancer-associated inflammation
Chronic inflammation is also associated with a variety of cancers, including breast, liver, prostate, and skin.92 In human cancer, tumor cells are not the only source of MMPs. MMPs, mainly gelatinases, are predominantly produced by stromal cells, ranging from immune (lymphocytes and dendritic cells), inflammatory (granulocytes and monocytes), and vascular cells (vascular- and lymph-endothelial cells and pericytes). MMPs have been involved in the escape of cancer cells from immune surveillance. The escape mechanism occurs through MMP-9-induced cleavage of the interleukin-2 receptor (IL-2R Chemokines play an essential role in regulating directional migration of leukocytes. Proteolytic cleavage of chemokines by MMPs can lead to enhanced or reduced leukocyte recruitment into tumors. For example, a cleaved form of MCP-3 produced by MMP-2 can bind to CC-chemokine receptors, and unlike intact MCP-3, it abrogates chemotaxis and suppresses inflammation.114 ET-1 processing by MMP-9 generates endothelin-1 (ET-1) that induces secretion of MMP-9 from neutrophils,115 suggesting that MMPs are both effectors of leukocyte migration and regulators of the inflammatory response. The importance of chemokine receptors in metastasis was demonstrated by inhibition of SDF-1 binding to its receptor. Dissociation of SDF-1/CXCR-4 complex by blocking antibodies strongly reduced breast cancer metastasis to lungs and lymph nodes in vivo.110 MMP-9 and VEGF are produced by mammary tumor-infiltrating immune cells.116 Expression of MMP-9 by tumor-infiltrating macrophages promotes angiogenesis as well as growth and invasion of xenografted ovarian cancer cells in vivo.117 Several studies show that cancer cells can promote the secretion of MMPs by stromal cells in a paracrine manner via secretion of growth factors, interleukins, and EMMPRIN.21 Recruitment of hematopoietic precursor cells is also required for tumor angiogenesis.118
Leukemia can be described as the uncontrolled proliferation of hematopoietic cells that lack the ability to differentiate into mature blood cells. The precise role of gelatinase expression in acute leukemias is not clear. So far, it is known that invasiveness of many hematologic malignancies, including myelo-monocytic leukemias, involves overexpression of proteolytic enzymes, such as MMP-2 and MMP-9.119 MMP-9 is induced and secreted in conditioned media of leukemic cell lines in response to extracellular stimuli, after pretreatment of cells with chemokines, and after cell adhesion to the ECM.119 Higher gelatinase expression levels were detected in the bone marrow plasma of patients with leukemia compared with healthy controls. After chemotherapy, the levels of TIMP-1 and TIMP-2 were significantly increased, whereas MMP-9 levels were lower in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients. Accordingly, AML patients who achieved a complete remission showed significantly lower MMP-9 levels, suggesting that MMP-9 could be a surrogate marker of leukemic status in these patients. Also, the low MMP-9 expression levels in patients with leukemia correlated with increased survival.120
Several reports have demonstrated the involvement of both MMP-2/-9 gelatinases and Increased vessel density was detected in the bone marrow of acute and chronic leukemia patients compared with normal bone marrow, and is known to be mediated by angiogenic factors such as VEGF and bFGF.128,129 Both increased plasma MMP-9 and VEGF correlated with high leukemia cell infiltration, suggesting that MMP-9 and VEGF act cooperatively in the process of leukemia cell invasion.122 Another study showed that increased vessel density was mediated by MMP-2 and MMP-9 overexpression in primary AML blasts by promoting endothelial cell migration.130 After achieving complete remission, the vessel number in AML patients was restored to normal levels. Furthermore, a gene therapy approach using a retroviral vector encoding for gelatinase inhibitors, endostatin and angiostatin, strongly inhibited bone marrow angiogenesis and leukemia tumor growth in vivo.131 These data suggest that gelatinases could be involved in leukemia progression. As a result, inhibitors of MMPs may be useful in treating hematologic malignancies.
Due to the fact that integrins and MMPs are involved in tumor cell invasion and metastasis, over the past 20 years a lot of effort has been put into designing integrin and MMP inhibitors (MMPIs). Although endogenous inhibitors, such as TIMPs, inhibited tumor growth in transgenic mouse models, their use in cancer was limited due to poor pharmacokinetics, difficulties in protein administration, and broad spectrum of inhibition. To date, several synthetic MMPIs have been developed, tested widely in clinical trials, and classified into the following pharmacologic groups: collagen peptidomimetics, nonpeptidomimetics, tetracycline derivatives, and biphosphonates.132 The efficacy of these inhibitors in clinical trials is summarized in Table 2.
The design of collagen peptidomimetic MMPIs is based on the collagen-peptide backbone with zinc-binding hydroxamate moiety that coordinates the Zn2+ ion, thus inhibiting the MMP catalytic activity. An oral MMPI, marimastat, significantly increased survival of patients with gastric carcinoma. Treatment with marimastat was well tolerated by the patients, except for some minor side effects such as musculoskeletal pain, probably because of the need of MMPs in normal remodeling of the connective tissue of tendons and joints. In patients with advanced pancreatic cancer (a phase 2 study), marimastat showed comparable therapeutic effects as conventional therapy with gemcitabine that was used.133 The survival of patients suffering from glioblastoma multiforme was also improved by using marimastat in combination with temozolomide, a cytotoxic drug.134 Several nonpeptidomimetic MMP inhibitors, including BMS-275291, AG3340, and MMI270, have also been tested in clinical trials (Table 2). Tetracyclines and biphosphonates have also been shown to block MMP activity.135 For example, a broad spectrum MMP inhibitor, metastat (or Col-3), showed increased tumor cell toxicity, reduced tumor-induced angiogenesis, as well as antimetastatic activity,136 and is currently being tested in patients with Kaposi sarcoma and brain cancer in a phase 2 clinical trial. Periostat, a tetracycline used for the treatment of periodontal diseases, is the only MMPI on the market. Of interest, compounds (TSRI265) capable of inhibiting interactions between MMPs and integrins showed promising results in animal experiments.85 Also, a cyclic peptide, CTTHWGFTLC, discovered by phage display technology as a selective gelatinase inhibitor, could block cell migration and tumor growth in a gelatinase-dependent manner.137
The involvement of integrins in tumor cell invasion and metastasis became clear after using
The failure of MMPIs in several cancer clinical trials is not surprising.146 Most MMPIs were used to treat patients with late-stage tumors, whereas most results obtained from animal experiments show the need for targeting MMPs in early stages of cancer progression. Also, these inhibitors target all MMPs, many of which are needed for the processing of antiangiogenic factors, including angiostatin and endostatin. For that, increasing the selectivity of these compounds (for example, for gelatinases involved in metastasis) could solve the problem of side effects reported so far. MMPIs are known to target also ADAMTS, enzymes capable of reducing tumor growth by blocking tumor angiogenesis.147 It should be taken into consideration that other proteases are up-regulated during tumor progression that could compensate for the loss of MMPs. These proteases should be identified and targeted along with MMPs.
Extensive effort has been made in developing small molecules, peptides, and peptidomimetics capable of inhibiting interactions that occur on the cell surface. Several linear and cyclic peptides derived from sequences of Studies on the role of MMP-9 in leukocyte migration have been controversial. For example, some reports have supported MMP-9 function in leukocyte migration,150,151 whereas others have not.152,153 These findings are not surprising as MMPs are known to have overlapping functions and other MMPs within the family could compensate for the loss of MMP-9.
The physiologic role of MMP-2 and -9 is not fully understood, but to our current knowledge they are involved in the processing of the extracellular matrix during growth and tissue differentiation, probably as critical factors for cell motility. Proteases and integrins for such a function have been expected to be colocalized at the surface of migrating leukocytes and other cells. Most MMPs, however, are secreted enzymes and the search for cell-surface receptors for MMPs has been going on for years. At the moment there are some hundred publications describing receptors, such as integrins for various MMPs, among them MMP-2 and -9. Like-wise, gelatinase activity has been found in the membrane of leukocytes, but the identification of the leukocyte integrins as gelatinase receptors is new to our knowledge47,87 and likely to extend our understanding of further mechanisms involved in leukocyte migration. Studies from knock-out models for integrins, including leukocyte
MMP-9 has been reported to cooperate with
We have recently shown that proMMP-9/
Submitted February 28, 2006; accepted March 28, 2006.
Prepublished online as Blood First Edition Paper, April 11, 2006; DOI 10.1182/blood-2006-02-005363.
Reprints: Michael Stefanidakis, Department of Biological and Environmental Sciences, POB 56 (Viikinkaari 5D), University of Helsinki, Helsinki, Finland; e-mail: stefanid{at}mappi.helsinki.fi.
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Abstract
Leukocyte adhesion and migration
R-IIIb, CD2, CD43, and CD44,