Keywords

1 Introduction

Cancer originating from mutations in genes that regulate essential pathways of cell function leading to uncontrolled outgrowth of tissue cells [1] seems to be one of the leading causes of disease and mortality worldwide [2]. The tumors are complex structures of malignant cancer cells embedded in vasculature and surrounded by a dynamic tumor stroma consisting of various nonmalignant cells, such as fibroblasts and myeloid cells. The milieu of the tumor microenvironment is akin to the inflammatory response in a healing wound, which promotes angiogenesis, turnover of the extracellular matrix (ECM), and tumor cell motility. Understanding the molecular mechanisms of this complex interplay between malignant cancer cells and the surrounding nonmalignant stroma represents one of the major challenges in cancer research. However, the past two decades of biomedical research have yielded an enormous amount of information on the molecular events that take place during carcinogenesis and the signaling pathways participating in cancer progression. Our laboratory has also marked a significant contribution to elucidate the key molecular machineries responsible for carcinogenesis along with the therapeutic approaches using dietary polyphenols [3–8].

Metastasis is a cascade of linked sequential steps involving multiple host-tumor interactions [9]. To successfully create a metastatic colony, a cell or group of tumor cells must be able to leave the primary tumor, invade the local host tissue, enter the circulation, arrest at the distant vascular bed, extravasate into the target organ interstitium and parenchyma, and proliferate as a secondary colony. During invasion, tumor cells disobey the social order of organ boundaries and cross into tissues where they do not belong. The mammalian organism is divided into a series of tissue compartments separated by the extracellular matrix unit consisting of the basement membrane and its underlying interstitial stroma [10]. The basal cells of the epithelium or organ parenchymal side of this unit are attached to the basement membrane. On the opposite side, the interstitial stroma contains stromal cells, fibroblasts, and myofibroblasts. During all types of benign tissue remodeling, proliferative disorders, and carcinoma in situ, the cell populations on either side of this connective tissue unit do not intermix. Only during the transition from in situ to invasive carcinoma do tumor cells penetrate the epithelial basement membrane and enter the underlying interstitial stroma to interact with the stromal cells. Thus, a definition of the behavior of the metastatic tumor cell is the tendency to cross tissue compartment boundaries and intermix with opposite cell types [11]. The continuous basement membrane is a dense meshwork of collagen, glycoproteins, and proteoglycans which normally does not contain any pores large enough for passive tumor cell transversal. Consequently, invasion of the basement membrane must be an active process. Once the tumor cells enter the stroma, they gain access to lymphatics and blood vessels for further dissemination.

Interactions of the tumor cell with the basement membrane can be separated into three steps: attachment, matrix dissolution and migration. The first step is binding of the tumor cell to the basement membrane surface-mediated cell-surface receptors of the integrin [12] and non-integrin [13] variety. Matrix receptors recognize glycoproteins such as laminin, type IV collagen, and fibronectin in the basement membrane. Two to eight hours after attachment, a localized zone of lysis is produced in the basement membrane at the point of tumor cell contact. Tumor cells directly secrete degradative enzymes [14] or induce the host to elaborate proteinases to degrade the matrix and its component adhesion molecules. Matrix lysis takes place in a highly localized region close to the tumor cell surface [15], where the amount of active enzyme outbalances the natural proteinase inhibitors present in the serum, those in the matrix, or that secreted by normal cells in the vicinity. Locomotion is the third step of invasion which propels the tumor cell across the basement membrane and stroma through the zone of matrix proteolysis.

During metastatic dissemination, cancer cells activate a complex molecular machinery to migrate through the surrounding extracellular matrix (ECM) and intravasate into blood or lymphatic vessels [16]. To negotiate barriers to cell migration, cancer cells secrete their own proteolytic enzymes or induce their expression in other cells through the release of cytokines (e.g., endothelial cells, tumor-infiltrating fibroblasts or leukocytes) [17]. In particular, matrix metalloproteinases (MMPs) are considered key players in tumor progression because of their ability to remodel the ECM and cleave/activate membrane-bound and matrix molecules, and cytokines that stimulate cancer cell migration and proliferation [18].

Studies conducted over more than 40 years have revealed mounting evidence supporting that extracellular matrix remodeling proteinases, such as matrix metalloproteinases (MMPs), are the principal mediators of the alterations observed in the microenvironment during cancer progression. MMPs belong to a zinc-dependent family of endopeptidases implicated in a variety of physiological processes, including wound healing, uterine involution and organogenesis, as well as in pathological conditions, such as inflammatory, vascular and auto-immune disorders, and carcinogenesis [19]. MMPs have been considered as potential diagnostic and prognostic biomarkers in many types and stages of cancer [20]. The notion of MMPs as therapeutic targets of cancer was introduced 25 years ago because the metastatic potential of various cancers was correlated with the ability of cancer cells to degrade the basement membrane. Subsequently, a growing number of MMP inhibitors (MMPIs) have been developed and evaluated in several clinical trials. Recent report from our laboratory also suggested that downregulation of MMPs like MMP-2 and MMP-9 is prerequisite for the anti-migratory effect of the flavins in breast cancer cells [21].

2 Matrix Metalloproteinases: What Is So “Mysterious” About These Enzymes?

The matrix metalloproteinases (MMPs), also called matrixins, are a group of genetically distinct but structurally related calcium-dependent zinc-containing endopeptidases that are involved in the degradation and repair of major macromolecular components of extracellular matrix (ECM), connective tissue, and cell-surface-bound molecules. They are naturally occurring proteolytic enzymes found in most mammals that are secreted especially by mesenchymal cells, macrophages, and polymorphonuclear leukocytes [22]. A large set of experimental data indicated that MMPs play essential roles in the processes of tissue remodeling and repair, morphogenesis, angiogenesis, embryonic development, apoptosis, ovulation, neural development, wound healing, chemotherapy-induced alimentary tract (AT) mucositis, cell adhesion and proliferation as well [23]. Moreover, these enzymes have frequently been detected in human tumor specimens and their production and/or misregulation has been associated with the tumor aggressiveness and poor prognosis [24, 25].

Under normal physiological conditions, the expression and activity of these enzymes are very low and strictly controlled by endogenous specific tissue inhibitors of metalloproteinases (TIMPs). Generally, there are a total of four TIMPs (TIMP-1, -2, -3 and -4) and these four protein inhibitors are able to control the proteolytic activity of all MMPs and mediate the stability of cells. That is, overexpression or high activation of MMPs has been causally linked with the pathological destruction of connective tissue and the ensuing pathological disorders characterized by the breakdown of ECM components or connective tissues [26]. These diseases include cancer, osteoarthritis (OA), rheumatoid arthritis (RA), angiogenesis, chronic periodontitis, pulmonary emphysema, skin ulceration, atherosclerosis, gingivitis, central nervous system disease, type I diabetes, myocarditis and dilated cardiomyopathy, coronary artery disease, multiple sclerosis (MS), congestive heart failure and cardiovascular disease [27]. On the basis of their primary roles in various oncologic events, the MMPs have been a highly active set of targets for the design of therapeutic agents to intervene the MMP-related pathological states, such as carcinogenesis and arthritis [28]. Here, we review the recent advances in our understanding of MMP-driven regulation of the tumor invasion.

2.1 Domain Structures of Matrix Metalloproteinases

Matrix metalloproteinases belong to a family of zinc- and calcium-dependent endopeptidases called metzincin. The endopeptidases belong to the wide metzincin group, in turn constitutes one of several metalloendopeptidase families; according to their structural characteristics, all the metzincins are mainly subdivided into astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases/matrixins, snapalysins, leishmanolysins, and pappalysins [29]. All the metzincins are mostly multidomain proteins with approximately 130–260-residue globular catalytic domains showing a common core architecture characterized by a long zinc-binding consensus motif, HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins have been characterized to participate in unspecific protein degradation such as digestion of intake proteins and tissue development, maintenance, and remodeling, but they are also involved in highly specific cleavage events to activate or inactivate themselves or other (pro)enzymes and bioactive peptides [30]. Among these proteinases, the matrix-degrading metalloenzymes are the most common enzymes, mainly named matrixins or matrix metalloproteinases (MMP). They form a multigenic family of proteolytic calcium-/zinc-dependent enzymes (expressed as 26 distinct proteins), functioning at neutral pH, secreted in their latent form (proenzymes or inactive zymogens or pro-MMPs), and requiring proteolytic activation [31].

2.2 Structural Classification of Matrix Metalloproteinases

MMPs are a family of zinc-dependent endopeptidases first described almost half a century ago [32]. They play a crucial role in various physiological processes including tissue remodeling and organ development [33], in the regulation of inflammatory processes [34], and in diseases such as cancer [35]. The general structural blueprint, shared by 23 MMPs, shows three domains that are common to almost all MMPs - the pro-peptide, the catalytic domain, and the hemopexin-like C-terminal domain that is linked to the catalytic domain via a flexible hinge region. MMPs are initially expressed in an inactive state due to the interaction of a cysteine residue of the pro-domain with the zinc ion of the catalytic site. Only after disruption of this interaction by a mechanism called cysteine switch, which is usually mediated by proteolytic removal of the pro-domain or chemical modification of the cysteine residue, does the enzyme become proteolytically active. The pro-domain contains a consensus sequence and requires proteolytic cleavage by convertases, which, depending on the sequences, occurs intracellularly by furin or extracellularly by other MMPs or serine proteinases such as plasmin [36].

Structurally related members of MMPs can be broadly classified into five subfamilies based on the variation in their primary structure and function, substrate specificity, as well as their cellular sources: collagenase group (MMP-1, -8, -13, -18), gelatinase group (MMP-2, -9), stromelysin group (MMP-3, -10, -11), membrane-type (MT)-MMP group (MMP-14, -15, -16, -17), and a nonclassified group (MMP-7, -12) [37] (Fig. 10.1). This superfamily shares a conserved structural topology comprising a catalytic domain containing three histidines that constitutes the zinc-binding site and a “methionine-turn” motif that lies beneath the active site zinc ion. The ion-binding motif reads HEBXHXHBGBXHZ, where histidine (H), glutamic acid (E), and glycine (G) are invariant, B is a bulky hydrophobic residue, X is a variable residue, and Z is a family-specific amino acid (serine in MMPs). All MMPs have an N-terminal hydrophobic signal sequence, i.e., predomain, which leads their secretion into the extracellular space after the synthesis in the endoplasmic reticulum. Predomain is followed by a 77–87 amino acid-long pro-domain that constitutes the N-terminus of the secreted enzyme and maintains it in its latent form until its removal or disruption. The prodomain keeps the enzyme inactive through a mechanism identified as “cysteine switch” where the unpaired cysteine in the highly conserved “Pro-Arg-Cys-Gly-X-Pro-Asp” sequence forms a bridge with the catalytic zinc, thereby preventing enzymatic activity. The enzyme acquires total proteolytic capacity when the prodomain becomes chemically removed by cleavage [38]. The active site is of great importance: it specifically binds to selective substrates by means of its active site cleft, through specificity subsite pockets that bind amino acids adjacent to the scissile peptide bond, and through secondary substrate binding exosites located outside the active site [39]. These domains represent the minimal structure of MMPs found in MMP-7 (matrilysin) and MMP-26 (endometase/matrylisin-2) which lack any other domain. All the other MMPs have a hinge region varying in length and composition which also influences substrate specificity, and a four-blade structure representing the hemopexin/vitronectin-like domain [40, 41]. Two metalloproteinases, MMP-2 and MMP-9 (also named gelatinase A and B), are further characterized by the presence of three head-to-tail cysteine-rich repeats within the catalytic domain [42]. This structure resembles the collagen-binding type II repeats of fibronectin and is necessary for the binding and cleavaging activities of these MMPs. Not all MMPs are secreted enzymes; membrane-type (MT) MMPs have been identified to contain a single-pass transmembrane domain and a short cytoplasmic C-terminal tail or to be anchored to the cell membrane by a glycosylphosphatidylinositol anchor [43].

Fig. 10.1
figure 1

Conserved domain structures of different groups of MMPs. SH zinc interacting thiol, Fi insert that resemble collagen-binding type II repeats of fibronectin, Fu furin cleavage site, Zn zinc, HR hinge region, CA cysteine array, Cat catalytic domain, HPX hemopexin domain, GPI glycosylphosphatidylinositol, TM single span transmembrane domain, CY short cytoplasmic domain, MMP matrix metalloproteinases, MT-MMPs membrane-type metalloproteinases, Ig immunoglobulin

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Most MMPs are secreted as latent precursors (zymogens) that are proteolytically activated in the extracellular space (Fig. 10.2), with the exception of MMP-11 and MT1-MMP, which are activated prior to secretion by Golgi-associated, furin-like proteases. The activity of MMPs in extracellular space is specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs), which bind to the highly conserved zinc-binding site of active MMPs at molar equivalence.

Fig. 10.2
figure 2

General overview of activation and inactivation mechanism of MMPs within cytoplasm and extracellular space. TIMP tissue inhibitor of metalloproteinases, ECM extracellular matrix, ROS reactive oxygen species, MMP matrix metalloproteinases

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3 Regulation of Matrix Metalloproteinases Activity in Tumor Milieu: The Mysterious Interdependence

The complexity of the tumor microenvironment allows a variety of regulatory cascades to determine the functions of the diverse MMPs expressed. Proteolytic activity of MMPs can be regulated at different levels, i.e., gene expression, compartmentalization, conversion from zymogen to active enzyme, and, finally, the presence of specific inhibitors. While judging the patho-physiological relevance of increased expression of proteinases in tumor tissues, it is important to judge whether endogenous inhibitors or activating/converting enzymes are present in the microenviornment. A key step in regulating MMP activity is the conversion of the zymogen into an active proteolytic enzyme (Fig. 10.3). There are several proteinases that mediate MMP activation, such as plasmin, furin, or active MMPs [44, 45]. The function of MMPs can also be influenced by reactive oxygen species (ROS). The inflammatory response at the tumor site creates large amounts of ROS that are produced by activated neutrophils and macrophages. These oxidants initially activate MMPs via oxidation of the pro-domain cysteine [46] but, eventually, in combination with the enzyme myeloperoxidase contributed by inflammatory cells, inactivate MMPs by modification of amino acids of the catalytic domain by hypochlorous acid [47].

Fig. 10.3
figure 3

Molecular mechanism of conversion of inactive pro-MMPs into active form of MMPs. Zn zinc, MMP matrix metalloproteinases, S sulfhydryl group

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The localization or compartmentalization of MMPs under physiological conditions often dictates their biological function. Several MMPs interact with surface receptors such as integrins or localize to specific areas of the ECM, which potentiates MMP activity by increasing their local concentration and also may interfere with accessibility to endogenous inhibitors [48]. The binding of MMP-2 to integrin αvβ3 via its hemopexin domain is crucial for mesenchymal cell invasive activity [49]. Likewise, high local concentrations of active MMP-14 on the cell membrane of metastatic cancer cells play important roles in cell migration [50]. However, there may also be additional mechanisms to concentrate extracellular proteinases in specific sites in the microenvironment.

Mechanical forces contribute to tumor progression [51], potentially by modulating proteolysis of ECM components. These forces may unwind the conformation of MMP substrate proteins, thus allowing recognition and cleavage by proteinases. For example, the ECM component fibronectin is unfolded by mechanical forces in the ECM of living cells and this unfolded fibronectin then acts as potent MMP substrates [52]. Tumor progression is frequently characterized by increased tissue stiffness, elevated interstitial fluid pressure, and altered blood flow conditions [53]. Thus, it is conceivable that similar mechanisms involving mechanical force are regulatory factors for MMP function in the tumor microenvironment.

The expression and activity of MMPs is regulated at the transcriptional level by cytokines and growth factors and after secretion by endogenous natural inhibitors. The tissue inhibitors of matrix metalloproteinases (TIMPs) provide a negative control of MMP-activity. Four various inhibitors of metalloproteinases characterized so far, are designated as TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Among them, TIMP-1 and TIMP-2 have been characterized most extensively. The TIMPs inhibit active MMPs by forming 1:1 stoichiometric non-covalent complexes with the endopeptidase [54]. TIMP-1 and TIMP-2 are capable of inhibiting the activities of all known MMPs except MT-MMPs, and play a key role in MMP-driven different physiological processes. Moreover, TIMP-1 can also complex with the precursor of MMP-9 [55], whereas TIMP-2 and TIMP-4 can bind to the zymogen form of MMP-2, a 92-kDa type IV procollagenase [56]. TIMP-3 inhibits not only the activity of MMP-1, -2, -3, -9 and -13 [57], but also the activity of MT-MMPs as well as TNF-α converting enzyme. However, the role of TIMPs is not restricted to the inhibition of MMPs. They possess growth-promoting activities for various cell types as well as having antiangiogenic and proapoptotic properties [58].

4 Matrix Metalloproteinases in Cancer Progression: The Mystery Revisited

During development of carcinogenesis, tumor cells participate in several interactions with the tumor microenvironment involving extracellular matrix (ECM), growth factors and cytokines associated with ECM, as well as surrounding cells (endothelial cells, fibroblasts, macrophages, mast cells, neutrophils, pericytes and adipocytes) [59]. Four hallmarks of cancer that include migration, invasion, metastasis and angiogenesis are dependent on the surrounding microenvironment. Critical molecules in these processes are MMPs because they degrade various cell adhesion molecules, thereby modulating cell–cell and cell–ECM interactions.

The emerging view, reflected by several studies, reveals that the expression and role of MMPs and their natural inhibitors, i.e., tissue inhibitor of metalloproteinases (TIMP), is quite diverse during cancer development. The overexpression of MMPs in the tumor microenvironment depends not only on the cancer cells, but also on the neighboring stromal cells, which are induced by the cancer cells in a bidirectional paracrine manner. Cancer cells stimulate host cells (e.g., fibroblasts) and are themselves stimulated by host cell (e.g., neutrophil) to constitute an important source of MMPs through the secretion of interleukins and growth factors and direct signaling through extracellular MMP inducer [60, 61].

Recent studies show that members of the MMP family exert different roles at different stages during cancer progression (Fig. 10.4). In particular, they may promote or inhibit cancer development depending among other factors on the tumor stage, tumor site (primary, metastasis), enzyme localization (tumor cells, stroma), and substrate profile.

Fig. 10.4
figure 4

Different stages of metastatic tumor progression (tumor growth and survival, angiogenesis, intravasation and extravasation) are positively and negatively regulated by different MMPs. EGF epidermal growth factor, IGF insulin-like growth factor, TGF β transforming growth factor-beta, bFGF basic fibroblast growth factor, FGF-R1 fibroblast growth factor receptor 1, MMP matrix metalloproteinases, VEGF vascular endothelial growth factor, FasL Fas ligand, Transglut transglutaminase

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5 Matrix Metalloproteinases in Tumor Metastasis: A Tale of the Mysterious Mediators

The extracellular matrix holds cells together and maintains the three-dimensional structure of the body. It also plays critical roles in cell growth, differentiation, survival and motility. For a tumor cell to metastasize from the primary tumor to other organs, the collagen-rich ECM and basement membrane that are the physical barriers for cell migration must be degraded. The key enzymes responsible for ECM breakdown are MMPs that actively fuel the progression of cancer from localized growth to the invasion of surrounding tissues and the development of distant organ metastasis. Also there is a cooperation between these two components, i.e., ECM and MMPs, enabling the tumor cell to reach its target organ and survive. However, the classic view that these enzymes simply provide a mechanism for the breakdown of connective tissue barriers has been challenged.

5.1 Matrix Metalloproteinases in Extracellular Matrix Degradation and Distant Metastasis

Although all five major classes (serine, aspartic, cysteine, threonine, and metalloproteinases) are involved in metastasis, a great deal of emphasis has been placed on the type IV collagenases, MMP-2 and MMP-9 [62]. Type IV collagen is a major structural protein in the basement membrane and ECM. A number of studies have linked elevated MMP-2 and MMP-9 levels with an increased metastasis. The conclusions which can be drawn thus far are that the number and the relative levels of MMPs increase with tumor progression.

Several recent studies have been done to try and characterize the phenotypic and enzymatic profiles of more aggressive tumor cell lines. Selection of progressively more invasive human lung carcinoma cells from an established CU cell line revealed that the more invasive cells had a higher expression of MMP-9. These cells had a four- to sixfold increase in invasive activity over the parental and had an increased metastatic potential in vivo. MMP-9 has also been shown to be overexpressed in advanced stage melanoma cells and in breast cancer cell lines. Other tumor models involving MMP-9 in their invasive phenotype include human non-Hodgkin’s lymphoma cells and human giant cell tumors [63]. MMP-2 has also been observed to be overexpressed in more aggressive tumor cells. The level of pro-MMP-2 vs. active MMP-2 also plays a role in determining invasive and metastatic capacity of pancreatic tumor cells towards regional lymph node [64–66]. Cell lines displaying an intermediate level of activation were the most invasive while those cells with a high level of activation were the least invasive. This is probably due to the balance required between MMPs and TIMP to create a controlled proteolytic system.

Although the major role of MMPs in metastasis has been inferred from the in vivo and in vitro data presented above to be breakdown of the ECM, recent studies have proposed additional roles for the MMP family. Most of the in vivo and in vitro assays designed to examine the role of MMPs on tumor invasion measure the end results, as in the number of micrometastases formed. The mechanism, however, remains unknown. Intravital video microscopy (IVVM) allows for the observation of the metastatic cascade by following the tumor cell through the microcirculation [67]. The results from these experiments suggest that the destruction of tumor cells in the circulation and during extravasation do not contribute as much as previously thought to the inefficiency of metastasis. Rather, the growth of the individual tumor cell once in the target organ appears to be the rate-limiting step. Tumor cells engineered to overexpress TIMP-1 were shown to extravasate at rates equal to wild-type cells but were unable to form proliferative colonies within the target organ [68]. Although these data suggest that MMPs may play a role in tumor cell growth, the studies of MMP involvement in ECM degradation and basement membrane invasion still support the core role of MMPs in metastatic invasion.

5.2 Matrix Metalloproteinases in Epithelial to Mesenchymal Transition

Epithelial–mesenchymal transformation (EMT) is a critical step in malignant transformation of epithelial cells into carcinoma [69]. Loss of the homotypic cell–cell adhesion molecule E-cadherin and nuclear translocation of β-catenin are common features of EMT and are associated with the progression of most epithelial cancers [70, 71]. Several MMPs, including MMP-3, MMP-7, and MT1-MMP, cleave E-cadherin releasing a soluble 80 kDa peptide with motility stimulatory activity, which suggest that MMPs could actively contribute to EMT [72]. Interestingly MMPs like MMP-7 and MT1-MMP are also transcriptionally upregulated by β-catenin LEF/TCF complexes [73], suggesting the existence of an MMP-dependent positive feedback mechanism by which E-cadherin degradation by MMPs also results in an increase in MMP expression.

5.3 Matrix Metalloproteinases in Invadopodia Formation

Invadopodia (podosomes) are specialized cell-surface structures that have been identified on transformed malignant cells and are composed of a meshwork of actin ring, microfilaments and metalloproteinases, involved in degradation of underlying matrix. Invadopodia utilize proteases to degrade a variety of immobilized substrates including fibronectin, laminin, type I and IV collagens and other ECM components. Several integral membrane enzymes of different classes have been identified as important functional components of invadopodia [74]. These include the serine proteases, seprase (surface-expressed protease), and dipeptidyl peptidase IV, which must form oligomeric structures for expression of proteolytic activity and also MT-MMP (Fig. 10.5). Plasma membranes shed vesicles containing densely clustered MMP-9 and MMP-2, which might facilitate directional proteolysis of the ECM during cell migration and especially during cancer invasion [75].

Fig. 10.5
figure 5

Localization of membrane-bound MMP (MT1-MMP) at invasive bodies of tumor cell (invadopodium). Arp actin-related protein, phospho phospho tyrosine, MT1-MMP membrane-type 1 matrix metalloproteinases, N-WASP neuronal Wiskott–Aldrich syndrome protein, F-actin filamentous actin

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5.4 Matrix Metalloproteinases in Cancer Dissemination

A positive correlation between tumor progression and the expression of multiple MMP family members (MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, and MT1-MMP) in tumor tissues has been demonstrated in numerous human and animal studies [76, 77]. On the basis of numerous studies, it was proposed that pharmacologic targeting of MMP activity might provide a mechanism to prevent cancer dissemination [78]. Further support for the role of MMPs in cancer dissemination came from the demonstration that TIMPs can interfere with experimental metastasis [79]. However, the role of MMPs and TIMPs in cancer is far more complicated than suggested initially. For example, increased TIMP-1 levels in human cancer tissues have been associated with poor prognoses [80]. It is uncertain whether this reflects the growth-potentiating properties of TIMPs or some other undetermined property of TIMPs [81]. Other experimental studies demonstrated that MMPs act primarily to alter the extracellular environment to allow sustained cancer cell growth in an ectopic site, as opposed to having a specific role of allowing the cells to extravasate from the blood stream [82]. Furthermore, in some experimental tumor systems, increased MMP production did not correlate with increased metastasis [83]. One potential explanation of this finding is that excess proteolysis might degrade matrix signals and receptors, thereby disrupting cell–matrix interactions and inhibiting migration [84].

5.5 Matrix Metalloproteinases Contribute to Intravasation and Extravasation

Metastasis is the most devastating event associated with cancer because it heralds an irreversible stage of progression that responds poorly if at all to current therapeutic regimens. Cancer metastasis is a complex, multistage process, which includes cell detachment from the primary tumor mass, migration through the ECM, degradation of the vascular endothelial basement membrane and penetration into the vascular lumen, survival within the circulation, proliferation on distal vascular endothelia, and finally penetration into a new host tissue microenvironment and establishment of a relationship with the local stroma that is conducive to new tumor colony outgrowth. Several if not all of these steps depend at least in part on MMP activity.

Detachment of cells from the primary tumor mass requires the downregulation of cell–cell adhesion mechanisms. Role of several MMPs in this aspect (especially E-cadherin downregulation), has been discussed before. Migration of tumor cells through the host tissue stroma requires partial degradation of the ECM and coordinated sequential attachment to and detachment from the ECM scaffold. Recent work using two-photon microscopy has provided spectacular real-time evidence that MMP proteolytic activity causes controlled degradation of collagen fibrils that are in contact with the invading tumor cell surface, leaving trail of released cell-surface molecules in the cell’s wake [14]. Interestingly, inhibition of MMPs does not result in abrogation of tumor cell migration through the collagen gel but rather transforms the crawling movement associated with collagen fibril cleavage into amoeboid movement that leaves the collagen lattice intact. This model strongly supports the notion that the MMP activity relevant to ECM degradation is associated with the tumor cell surface.

As already discussed, cleavage of ECM components by MMPs generates proteolytic fragments that enhance tumor cell migration. Thus, cleavage of laminin-5 by MMP-2 and -14 results in laminin fragments that trigger migration signals in cells [85], and cleavage of collagen IV discloses cryptic sites that are recognized by integrins and contribute to migration stimuli [86]. MMPs also cleave adhesion receptors responsible for cell–matrix interaction, thereby presumably participating in the detachment of cells from the ECM. The cell-surface hyaluronan receptor and facultative proteoglycan CD44 is cleaved by MMP-14, and its cleavage promotes migration. Expression of CD44 containing a mutation of the proteolytic cleavage site abrogates cell migration on ECM [87].

Intravasation, the process whereby tumor cells penetrate the vascular endothelial wall, has been proposed to be a rate-limiting event in metastasis. Although it is likely that a variety of MMPs may be involved in the degradation of the vascular endothelial basement membrane, MMP-9 has thus far been shown to play a potentially leading role [88].

Survival in the face of the immune response is key for the ability of tumor cells with metastatic potential to establish new colonies. Among the wide range of mechanisms that have been proposed to explain tumor cell evasion of immune surveillance, several are MMP dependent. Tumor cells typically interact with neutrophils, macrophages, cytotoxic T cells (CTLs), and natural killer (NK) cells. T cell proliferation is controlled in large part by the engagement of the interleukin-2 receptor (IL-2R) by its natural ligand IL-2. MMPs, including MMP-9, have been shown to cleave the α-chain of IL-2R [89], resulting in the inhibition of T cell proliferation. MMP-9-mediated activation of latent TGF-β may also contribute to immune suppression, since TGF-β is a potent inhibitor of T cell function [90]. Recent evidence indicates that MMP-9-mediated shedding of cell-surface ICAM-1 may block the ability of CTLs and NK cells to interact with target cells, thereby reducing the effectiveness of their cytotoxicity. Interestingly, an MMP-11 cleavage product of α1-proteinase inhibitor reduces the sensitivity of tumor cells to NK-mediated killing [91].

Recent evidence indicates that MMPs cleave a variety of chemokines in ways that can either enhance or block their function. SDF-1/CXCL12, which is inactivated by several MMPs [92], is a ligand for the CXC chemokine receptor 4 (CXCR4) on leucocytes and breast carcinoma cells. Inhibition of CXCR4 engagement by its ligand using monoclonal antibodies reduces metastasis from breast to lung and lymph nodes in vivo [93].

Extravasation was believed to be a key step in cancer metastasis. However, increasing evidence indicates that extravasation is not a rate-limiting step. This process does not appear to require the proteolytic action of MMPs but results from the mechanical disruption of blood vessels by locally growing tumor cells.

The final step in metastasis is the establishment of tumor colonies at sites distant to that of the origin, which relies on interactions between the tumor cells and host tissue stroma. Invading tumor cells may have their own repertoire of MMPs, but it is becoming increasingly clear that they direct, either by physical contact or in paracrine manner, MMP expression and secretion by stromal cells, including fibroblasts, endothelial cells, and leucocytes [94]. MMPs produced by the stroma augment the release of ECM sequestered growth factors, which may help enhance tumor survival, promote angiogenesis, and contribute to further tumor dissemination. A key question is whether reliance on MMP activity lasts throughout metastatic tumor growth or whether MMP-dependent events serve to initiate colony development, which may then proceed in the absence of further MMP-mediated proteolysis. This is an important consideration for therapeutic strategies targeted toward controlling MMP activity and one that remains to be adequately addressed.

5.6 Matrix Metalloproteinases Help Cancer Cells to Communicate with Distant Organ Cells to Form “Metastatic Niche”

Certain organs such as lung, liver, or bone are the preferential sites for the formation of metastases. MMPs and other proteinases are crucially involved in the formation of receptive environment at distant site, known as “metastatic niche”. Soluble factors released from the primary tumor appear to trigger the formation of a metastatic niche that is induced initially by the expression of embryonic-type fibronectin, which is most likely produced by fibroblasts at these sites. This event takes place before disseminated tumor cells are detectable at these distant organs, hence, these authors name this process the formation of a “premetastatic niche” [95]. Increased fibronectin production at these sites allows for the infiltration of VEGFR1-positive, bone marrow-derived progenitor cells, which then establish a metastasis-supporting microenvironment. Interestingly, the production of MMPs, namely MMP-3 and -10, is upregulated together with the angiogenic modulator angiopoietin-2 in premetastatic lung tissue even before myeloid cells are recruited to these sites [96]. These findings imply an important role of extracellular proteolysis in premetastatic niche generation.

6 Matrix Metalloproteinases and the Risk for Recurrence of Metastasis: The Mysterious Contributors

MMPs can be used as markers to predict tumor recurrence in several cancer types. High preoperative serum levels of MMP-2 or MMP-3 predicts recurrence in patients with advanced urothelial carcinoma [97]. Similarly in ovarian cancer, high expression levels of MMP-2 in tumor cells can predict tumor recurrence [98]. Kuniyasu et al. [99] found that a high ratio of gelatinase expression (MMP-2 or MMP-9) to E-cadherin expression in tumor cells can predict recurrence and death in pancreatic cancer. Similarly, expression of activated MMP-2 is related to regional lymph node and distal metastasis as well as to postresection recurrence of the same tumor [100]. The expression of certain MMPs in primary tumor can predict the risk of metastasis. Expression of MMP-1 is associated with lymph-vascular invasion and lymph node metastasis in stage IB cervical cancer [101] and peritoneal metastasis in gastric cancer [102]. Expression of MMP-2 in tumor cells can indicate increased risk of metastasis in uveal melanoma and in SCC of tongue [103]. Similarly, increased MMP-9 expression by tumor cells in colorectal cancer is associated with advanced Dukes stage and presence of distant metastases [104]. Interestingly, MMP-2 and MMP-9 expression levels are especially high in lung carcinomas and melanomas metastasizing to the spine, suggesting that they contribute to enhanced invasive properties of metastatic spinal tumors [105]. MMP determinations from patient serum have shown predictive value in estimation of metastasis risk. High serum levels of MMP-2 correlate with the presence of metastases in lung cancer or to disease progression in patients with prostate cancer [106], and a high serum MMP-9/E-cadherin ratio can predict metastasis of renal cell carcinoma [107].

7 Matrix Metalloproteinases in Cancer Stem Cell Maintenance: Escalating the Mystery

Matrix metalloproteinases (MMPs) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). MMP-10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of MMP-10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10-deficient oncospheres can be restored by addition of exogenous MMP-10 protein to the culture medium, demonstrating a direct role for MMP-10 in the proliferation of these cells [108]. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas MMP-10 deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or MMP-10/MMP-2 mice show no significant difference in tumor initiation, growth, or metastasis, demonstrating the importance of MMP-10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor MMP-10 expression and metastatic behavior in many human tumor types. Thus, MMP-10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that MMP-10 is a critical lung cancer stem cell gene product and novel therapeutic target for lung cancer stem cells.

8 Matrix Metalloproteinases as Targets for Anti-metastatic Therapy: Aiming Toward Unraveling the Mystery

The data from model systems, reviewed above, suggest that MMPs are involved in most phases of carcinogenesis from initiation to metastasis. Inhibition of these proteinases might thus lead both to prevention of cancer development and to inhibition of dissemination.

Two main types of MMP inhibitor exist: the TIMPs and low-molecular-weight synthetic inhibitors [109]. Because of their protein nature and multiplicity of actions, it is unlikely that TIMPs will be widely used as anticancer molecules. Because of this, most research in recent years has focused on the synthetic inhibitors. Many of these are peptides and are similar to the cleavage site in collagen [110]. Some of the zinc-binding groups that are currently being investigated in model systems include the hydroxamates, carboxylates, amino carboxylates, and sulfhydryls [111]. Some of these inhibitors (e.g., the hydroxamates) are presently undergoing clinical trials in patients with advanced cancers [112]. We are unaware of any studies so far in human breast cancer, however.

Although MMP inhibitors are currently being evaluated in patients with metastatic cancers, there are still many unanswered questions concerning the use of these compounds. Some of these are as follows. Is it better to use a broad-spectrum or specific matrix metalloproteinases inhibitor? In order to answer this question, it will be necessary to establish which are the MMPs whose involvement in the different phases of cancer progression is critical. If the action of MMP inhibitors is blocking of MMP activity only, these compounds may not induce the type of tumor shrinkage that is seen with the traditionally used cytotoxic agents. Conventional approaches that are used to assess tumor regression may thus not be possible. A novel approach taken to address this issue has been to monitor the rate of rise in levels of serum tumor markers. The use of these tests in phase 2 trials has shown a dose-dependent decrease in rate of rise after treatment with the MMP inhibitor marimastat [108]. Furthermore, this decreased rate of marker rise appeared to correlate with extended patient survival.

It is reported that MMPs have functional overlap with other proteases, e.g., plasmin, and arrest of invasion will require inhibition of plasmin as well as of the MMPs [113] thereby making the targeting of matrix metalloproteinases in cancer therapy even more challenging.

9 Therapeutically Targeting EMT-Promoting Matrix Metalloproteinases: The Mystery Unfolded

An obvious point for intervention in MMP-induced or -mediated EMT is the catalytic inhibition of MMPs themselves. Unfortunately, clinical trials of first- and second-generation small-molecule MMP-inhibiting drugs in breast cancer and other cancers proved disappointing [114]. A phase III trial of the MMP inhibitor marimastat in patients with metastatic breast cancer found no therapeutic benefit [115], while phase II trials of marimastat and rebimastat in patients with early-stage breast cancer concluded that large adjuvant trials with these agents were not feasible due to musculoskeletal toxicity and failure to achieve therapeutic plasma levels [116]. Many of the problems with the MMP inhibitors tested to date appear to stem in large part from a lack of specificity; the drugs employed simply target too many enzymes. This is a critical problem, because some MMPs appear to protect against tumor progression at certain stages of breast cancer development, and inhibition of these MMPs at the wrong time can lead to increased tumor aggressiveness [117]. For example, high levels of MMP-8 have been shown to suppress breast cancer metastasis [118, 119] significantly. Ribozyme-mediated knockdown of MMP-8 in a nonmetastatic, high MMP-8 breast cancer cell line conferred metastatic competence [120]. Thus, pharmacological inhibition of MMP-8 along with invasion- and metastasis-promoting MMPs would be anticipated to reduce or limit the potential benefit of the therapy.

As another consequence of poor specificity, clinical trials of MMP inhibitors were plagued by the serious side effect of musculoskeletal syndrome (MSS). This dose-limiting toxicity frequently resulted in failure to achieve targeted plasma levels, and in patients withdrawing from treatment. The specific molecular target responsible for these side effects has not been conclusively identified. Remaining candidate mediators of MSS include MT1-MMP, metalloproteinases outside of the MMP and ADAM families [121], or nonprotease metalloproteins. To minimize off-target effects, well-tolerated MMP-directed therapeutics will need to achieve selectivity for the MMP family in preference to other metalloenzymes, as well as the ability to distinguish among MMPs. In the arena of more highly selective small-molecule MMP inhibitors, slow progress is being made. These synthetic compounds typically feature a zinc-chelating group such as hydroxamate derivatized with peptidic or nonpeptidic groups designed to mimic a peptide substrate; they target the MMP active site zinc and substrate binding site [122]. Structure-based design of selective inhibitors has been hampered by the close structural homology of active sites and overlapping substrate specificities among the MMPs, and by the elastic and flexible nature of the MMP active site, which further complicates computational drug design even when high-resolution crystal structures are available [123, 124]. Current approaches to small-molecule MMP inhibitors include optimization of compounds based on an array of different zinc-binding groups to yield more selective inhibitors toward a variety of MMPs, as well as the development of non-zinc-binding inhibitors that selectively target unique aspects of the MMP-13 active site. A less conventional approach has pursued development of irreversible mechanism-based inhibitors, selective for gelatinases MMP-2 and MMP-9 that covalently modify the catalytic glutamate residue of the MMP active site. In yet another approach, several groups have attempted to exploit the selective substrate binding exosites present on MMP accessory domains to develop selective allosteric inhibitors of MMPs; while a promising concept, this approach has yet to yield highly potent and selective drug leads. Thus, the challenges are clear: while some MMPs facilitate breast cancer development and could potentially be targeted for therapeutic benefit, others are essential for basic physiological processes, interference with which can have serious negative consequences. We now need better understanding of which MMPs to target and when, as well as new generation technologies to target specific matrix metalloproteinases for regression of metastatic cancer.

10 Conclusions

MMPs are associated with multiple human cancers; hence they were early considered as drug targets to treat cancer. The first drug development programs based on the notion of blocking MMP-mediated angiogenesis and metastasis were started about 25 years ago and led to a number of small-molecule metalloproteinases inhibitor (MPI) drugs in phase III clinical trials. The effects of MPIs in these trials turned out to be disappointing as they failed to increase the survival rate of cancer patients. Possible reasons for the failure of MPIs have been extensively discussed previously. Indeed, the clinical studies were suboptimally designed with respect to the stage of cancer, so the question remains whether MPIs might have proven more effective when used in earlier stages of the disease.

Part of the rationale to use MPIs as anticancer drugs was to block interstitial migration of metastatic cancer cells. However, recent analyses have shown that cancer cells can switch to an amoeboid-like protease-independent migration mode by forming actin-rich protrusions and “squeezing” through the ECM. This would render MPIs impotent to inhibit the migratory behavior of metastatic tumor cells. Whether this alternative mode of migration is actually relevant for cancer cell migration, under in vivo conditions, in the presence of a naturally cross-linked collagen matrix, currently remains questionable.

The cytostatic potential attributed to MPIs is certainly in keeping with the numerous studies describing MMP-mediated regulation of cell growth signals, such as the activation of TGF-β by MMP-2, -9 and -14, the proteolytic release of soluble EGFR ligands, or the degradation of E-cadherin by MMP-3 or -9. Moreover, MMPs interfere with apoptosis induction, especially after chemotherapy, by cleaving Fas ligand from the surface of cancer cells as shown for MMP-7. In the clinical trials, MPIs were administered to patients with advanced cancer, which was most likely too late to exert any beneficial effect on survival.

Interfering with the tumor vasculature is regarded as one of the most promising strategies to inhibit tumor growth and has motivated the development of drugs like Bevacizumab (Avastin, anti-VEGF monoclonal antibody), which has been FDA approved for the treatment of metastatic cancers in combination with chemotherapy. Many studies also support a dominant role of MMP-9 in the angiogenic switch by regulating the bioavailability of VEGF tumors, suggesting a beneficial effect of MPI on tumor angiogenesis. However, in other cancer models, MMP-9 generates ECM fragments like tumstatin, a potent suppressor of tumor vasculature formation, resulting in increased tumor growth in MMP-9-deficient mice. This illustrates that one MMP can have opposing effects in different tumor types and highlights that the use of MPIs has to be carefully considered and evaluated for each specific kind of cancer.

Certainly, the complexity of the mode of action of MMPs has expanded considerably from proteinases that simply degrade the ECM, to specific modulators of angiogenesis as well as fine-tuners of cell signaling pathways and the inflammatory response. One of the major, recent advances in MMP research is the discovery of specific regulatory effects of MMPs on the stromal cells in the tumor microenvironment. MMPs regulate the course of the inflammatory reaction in multiple ways and facilitate the recruitment of inflammatory cells by altering the function of chemokines and the bioavailability of important proinflammatory cytokines. Regarding the link between inflammation and cancer, the interference with MMP-mediated immunoregulatory functions could prove beneficial for cancer patients. For example, given that TNF-α contributes to progression of several sorts of cancer, inhibiting TNF-α activation using MPIs might dampen the inflammatory milieu at the tumor microenvironment.

Effects of MMPs on myeloid cells may well be implicated in the generation of the premetastatic niche. In fact, MMP-2, -3 and -9 have already been shown to contribute to the establishment of metastasis-prone sites at tumor-distant organs. These insights argue for the use of MPIs at early stages of malignant disease prior to the full initiation of tumor-associated inflammation and before the soil have been primed for metastasis in distant organs.

The tumor-suppressing function of these MMPs is probably another reason for the failure of broad-spectrum MPIs as anticancer drugs. The inflammation-suppressing function of MMPs accounts for increased incidence of cancer development in MMP-8 knockout mice and for the link between MMP-8 loss-of-function mutations and melanoma in humans. Also, MMP-12 delivered by macrophages can suppress the growth of lung metastases, which appears to involve regulation of the tumor vasculature. Apart from that, some MMPs carry out biological functions other than proteolytic, mediated by specific binding to certain target molecules, for instance, via their hemopexin domain. Small-molecule MMP inhibitors as used in clinical trials are certainly ineffective to interfere with a nonproteolytic role of MMPs.

One of the major tasks for the future is the development of active site-directed inhibitors or antibodies that are specific for single MMPs and show little or no cross-reaction with other MMPs. Antibodies could also target functional noncatalytic domains of MMPs. Moreover, MMP activity can be exploited to activate cytotoxic agents such as anthrax toxin to target the tumor vasculature. New activity-based imaging probes specific for MMPs will facilitate monitoring the effect of MPIs on the function of MMPs in vivo. Imaging activity of specific MMPs in vivo will further advance our understanding of the time frame of MMP function during the progression of certain tumors. Like the development of tailor-made therapies and medications based on individual oncogenic pathway signatures in human cancers, expression patterns of MMPs in cancer patients could facilitate a fully rational decision about when and in what combination MPIs and anticancer drugs should be used in the future.