Journal of Pathology J Pathol 2015; 237: 273–281 Published online 10 September 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4586
REVIEW
Current mechanistic insights into the roles of matrix metalloproteinases in tumour invasion and metastasis Gordon T Brown and Graeme I Murray* Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK *Correspondence to: GI Murray, Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail: [email protected]
Abstract The purpose of this review is to highlight the recent mechanistic developments elucidating the role of matrix metalloproteinases (MMPs) in tumour invasion and metastasis. The ability of tumour cells to invade, migrate, and subsequently metastasize is a fundamental characteristic of cancer. Tumour invasion and metastasis are increasingly being characterized by the dynamic relationship between cancer cells and their microenvironment and developing a greater understanding of these basic pathological mechanisms is crucial. While MMPs have been strongly implicated in these processes as a result of extensive circumstantial evidence – for example, increased expression of individual MMPs in tumours and association of specific MMPs with prognosis – the underpinning mechanisms are only now being elucidated. Recent studies are now providing a mechanistic basis, highlighting and reinforcing the catalytic and non-catalytic roles of specific MMPs as key players in tumour invasion and metastasis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: extracellular matrix; matrix metalloproteinase; metastasis; microenvironment; protease; tumour invasion; tumour progression
Received 28 April 2015; Revised 3 July 2015; Accepted 8 July 2015
Conflict of interest statement: GTB declares no conflicts of interest. GIM is a scientific advisor to Vertebrate Antibodies (http://www.vertebrateantibodies.com).
Introduction At the molecular level, neoplasia is a multifactorial process driven by the combination of products of mutated oncogenes and tumour suppressor genes. There is now greater awareness of the complex and reciprocal interactions between malignant cells, non-malignant stromal cells, and extracellular matrix (ECM) of the peri-tumoural milieu or tumour microenvironment [1,2]. It has become increasingly apparent that these interactions begin at a relatively early stage of tumour progression and are important mechanisms through which a malignant neoplasm can initiate both local invasion and distant metastasis [3–6]. The structural framework of the ECM consists predominantly of individual types of collagen but also includes other matrix proteins including elastin and fibrillin [7]. Fibronectin and laminin are anchoring proteins involved in the attachment of cells to the structural skeleton, while the other major components of the ECM are proteoglycans. The ECM undergoes tightly regulated and co-ordinated remodelling during processes including wound healing, inflammation, and organogenesis [7]. An essential component of this remodelling process is proteolysis of the ECM and this breakdown is also vital but dysregulated in the early steps of local invasion of malignant cells into adjacent tissue. The Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
dynamic nature of this continuous remodelling highlights the need for detailed understanding of these cellular processes but also poses therapeutic challenges for targeting this process [8]. There are various groups of proteolytic enzymes or proteases involved in matrix degradation but the matrix metalloproteinase (MMP) group of enzymes is the most important in the context of tumour invasion and metastasis [7,9–14]. The MMPs are zinc-dependent endopeptidases that play key roles in the progression and dissemination of cancer including the modulation of tumour-associated angiogenesis [11,15,16]. The MMPs are classified by a combination of amino acid sequence, peptide domain structure, and substrate specificity (Table 1 and Figure 1A). The main groups of MMPs are the collagenases, gelatinases, stromelysins and membrane-type MMPs. Distinct from their involvement in structural protein breakdown, the MMPs can also influence a range of key cellular processes including cell proliferation, cell differentiation, apoptosis, and autophagy by activating growth factors and their receptors or by releasing cytokines from the ECM [17–20]. There is very substantial circumstantial evidence for the role of MMPs in tumour invasion and metastasis and it has been shown that the expression of specific MMPs has been associated with a poor prognosis in a range of human cancers [21–26]. J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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Figure 1. (A) An overview of the domain structure of the main groups of MMPs. (B) The role of MMPs involved in basement membrane and extracellular matrix degradation. Migration of a tumour cell invading from the primary site through the basement membrane and beyond the ECM into the systemic circulation is shown. Major constituents of the ECM include collagens, laminin, fibronectin, and fibrillin, and the MMPs shown are key proteolytic enzymes involved in ECM degradation in order to facilitate cell invasion. MT1-MMP is localized at the cell surface membrane and activates proMMP2, which initiates the MMP activation cascade. The orange arrows between the MMPs represent mutual activation. Those MMPs are then recruited by the tumour cell in order to recapitulate the process of invasion to invade through the wall of a local blood vessel and allow it to become a circulating tumour cell potentially capable of forming a metastasis. (C) The interaction of invadopodia formation and MMPs to promote invasive activity at the leading edge of an invadopodium. Loss of CDCP1 and NEDD9 inhibit invadopodia formation, whilst VANGL2 loss promotes invadopodia formation. Vav1 functions through CDC42 to activate N-WASp and Arp2/3 signalling to increase invadopodia formation. N-WASp up-regulates MT1-MMP activity with the help of F-actin, enabling proteolytic activity and proMMP2 activation to initiate the MMP activation cascade.
Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
Matrix metalloproteinases and tumour invasion
Table 1. The classification and nomenclature of human MMPs MMP
Group
Alternative names
MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10 MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP21 MMP22 MMP23 MMP24 MMP25 MMP26 MMP27 MMP28
Collagenase Gelatinase, Stromelysin Matrilysin Collagenase Gelatinase Stromelysin Stromelysin Other Collagenase Membrane type Membrane type Membrane type Membrane type Other Other Other Other Other Membrane type Membrane type Matrilysin Other Other
Interstitial collagenase Gelatinase A, 72 kDa type IV collagenase Stromelysin 1, progelatinase Matrilysin 1 Neutrophil collagenase Gelatinase B, 92 kDa type IV collagenase Stromelysin 2 Stromelysin 3 Macrophage elastase, metalloelastase Collagenase 3 MT1-MMP, membrane type 1 collagenase MT2-MMP MT3-MMP MT4-MMP RASI-1, also known as MMP18 Enamolysin Known as MMP23B MT5-MMP MT6-MMP Matrilysin 2 Epilysin
This review will highlight the key recent advances and emerging mechanistic concepts involving MMPs in relation to tumour invasion and metastasis. This will include the role of MMPs in the promotion of tumour cell motility, their contribution in facilitating tumour invasion, interactions of MMPs with other pro-invasive pathways, and the influence of MMPs within the tumour microenvironment and their role in the facilitation of epithelial–mesenchymal transition (EMT).
The roles of MMPs in modulating tumour cell motility Critical to tumour invasion and metastasis is the ability of tumour cells to move from one site to another, which in cell biology terms is expressed as cell motility. Recent mechanistic studies are now highlighting the role of MMPs in promoting tumour cell movement involving both catalytic and non-catalytic MMP functions [27]. Comparison of rounded-amoeboid (epithelioid) melanoma cells and elongated-mesenchymal (spindle cell) melanoma cells showed that there was increased secretion of several MMPs, most notably MMP9 and MMP13 [27]. This ensured that melanoma cells with an amoeboid morphology degraded type I collagen more efficiently than did melanoma cells with an elongated-mesenchymal morphology. In addition, secreted MMP9 was more effective at promoting the migration of rounded-amoeboid melanoma cells; this mechanism was non-catalytic, acting through the regulation of actomyosin contractility via MMP9 binding to the cell surface CD44 receptor [27]. The key role of MMP9 in promoting tumour invasion is also supported by a recent study of lung cancer cell Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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migration [28]. The increased movement of lung cancer cells was promoted by induction of MMP9 (by the overexpression of the transcription modulator megakarocytic leukaemia 1 factor), whereas silencing of this transcription factor resulted in loss of MMP9 expression, due to methylation of the MMP9 promoter, and this decreased invasion [28]. Further evidence for the role of MMP9 in promoting tumour invasion comes from a recent investigation of the binding of MMP9 to tumour-associated fibroblasts [29]. The interaction of tumours cells and surrounding stromal cells is important in regulating both tumour invasion and metastasis, and tumour-associated fibroblasts are central in these interactions. Tumour-associated fibroblasts, when differentiated to myofibroblasts, promote tumour progression by protease-catalysed remodelling of the stroma. MMP9 is central to matrix remodelling and the mechanism by which myofibroblasts utilize MMP9 has been demonstrated [29]. Recruitment of MMP9 by lysyl hydroxylase to the fibroblast cell surface has been shown to promote the myofibroblastic differentiation of tumour-associated fibroblasts, with binding of MMP9 to the cell surface of fibroblasts occurring via its fibronectin-like domain [29].
The role of MMPs in invadopodia formation and function The mechanism by which cancer cells are able to migrate through the ECM is now being identified and these studies have focused on invadopodia. These are specialized protrusions of the cell membrane of invasive tumour cells that have emerged recently to be key cellular structures in facilitating the movement of cancer cells through the basement membrane and hence promoting tumour invasion and metastasis [30]. Invadopodia are rich in MMPs, especially membrane-type 1 MMP (MT1-MMP; also known as MMP14), which activates proMMP2, thus initiating the MMP activation cascade (Figures 1B and 1C) [30–34]. The molecular mechanisms underlying the role of MMPs, especially MT1-MMP, in invadopodia formation and function are now being identified. The formation of invadopodia is a multifactorial process and can occur despite the absence of MT1-MMP, although MT1-MMP-deficient invadopodia lack the capacity to perform ECM degradation [31–34]. The guanine nucleotide exchange factor Vav1 has recently been identified as an inducer of invadopodia formation in pancreatic cancer cells [34]. Vav1 functions through exchange activity towards the Rho GTPase cell division cycle 42 (CDC42), which in turn promotes invasive behaviour through actin nucleation, and branching via neural Wiskott–Aldrich syndrome protein (N-WASp) and actin-related proteins 2 and 3 (Arp2/3) complex signalling. The deletion of Vav1 leads to a reduced number of actin puncta, a marker of active invadopodia and decreased MMP matrix-related degradation [35,36]. J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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Membrane-type MMPs contain a characteristic peptide sequence in the catalytic domain known as the MT-LOOP, which is critical to the function of MT1-MMP (Figure 1A) [37]. Deletion of the MT-LOOP domain has been shown to inhibit cell surface functions of MT1-MMP associated with invasion including proMMP2 activation and degradation of collagen. The mechanism through which this occurs has been identified as ineffective localization of MT1-MMP to β1-integrin-rich cell adhesion complexes at the plasma membrane [38]. However, knockdown of β1-integrin did not inhibit MT1-MMP-dependent degradation, indicating that β1-integrin is not a key component in the adhesion complex driving ECM degradation. Inhibition of MT-LOOP function through specific antibody binding resulted in an identical phenotype to that of MT-LOOP-deficient MT1-MMP, highlighting the MT-LOOP as a potential therapeutic target for MT1-MMP inhibition [38]. The catalytic domain of MT1-MMP has been therapeutically targeted. However, the catalytic domain is highly conserved and is shared by many other MMPs, and these efforts led to the non-specific inhibition of MMP activity that proved unsuccessful in combating tumour progression [39]. The hemopexin domain is another key region on the MT1-MMP molecule which when blocked through intratumour injections of a novel allosteric hemopexin inhibitor, resulted in the inhibition of breast tumour growth in a xenograft mouse model [40]. However, the catalytic function of MT1-MMP including proMMP2 activation was not affected by hemopexin domain inhibition [41].
Interactions of MMPs with pro-invasive pathways Recent studies have highlighted the mechanistic interactions of MMPs with pro-invasive pathways even at the early stages of carcinogenesis. In breast cancer, the key contribution of individual MMPs in promoting the transition from in situ malignancy (ductal carcinoma in situ) to invasive malignancy has recently been identified [42]. MT1-MMP was shown to be a key player in promoting invasion through the basement membrane and thus promoting the transformation of ductal carcinoma in situ to invasive breast cancer with the increased expression of this MMP being under the control of p63. The role of MMPs in the early stages of tumour invasion may indicate that MMP inhibition could be an effective strategy to prevent the progression of in situ malignancy to invasive malignancy. Liver cancer is another type of tumour in which MMPs are important and with the mechanisms by which MMPs interact with other key signalling pathways being identified [43]. Increased expression of MMP10 (stromelysin 2) has been identified in both human hepatocellular carcinoma and a diethylnitrosamine-induced model of hepatocarcinogenesis in MMP10-deficient mice. In human hepatocellular carcinomas, MMP10 Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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expression was associated with more aggressive tumours, while the liver tumours that developed in MMP10-deficient mice were smaller with reduced vascularity and fewer lung metastases. MMP10 expression was induced by hypoxia and the C-X-C chemokine receptor-4 ligand (stromal-derived factor 1) through the C-X-C chemokine receptor-4 stromal-derived factor 1 pathway via an activator protein 1 site on the MMP10 promoter [43]. The mechanism by which signal transducers and activators of transcription (STATs), which are frequently constitutively activated in cancer, interact with MMPs to promote tumour invasion has recently been elucidated in lung cancer [44]. STAT3 is activated in lung cancer and is associated with expression of MMP1 (interstitial collagenase). STAT3 was inducible by interleukin 6, and STAT3 activation promoted transcriptional activation of MMP1, thus providing a link between STAT3 activation, increased MMP1 expression, and increased invasiveness in lung cancer [44]. There is recent evidence providing a mechanistic understanding of the relationship of leptin and MMPs in promoting invasion of pancreatic adenocarcinoma [45]. Leptin has been shown to up-regulate MMP13 (collagenase-3) in pancreatic adenocarcinoma, also via the JAK2/STAT3 pathway. Overexpression of leptin in an orthotopic model of pancreatic adenocarcinoma was associated with increased MMP13 expression and promotion of lymph node metastasis, thus providing a potential link between obesity and tumour progression [45]. Understanding the role of MMPs in promoting metastasis is also important and MMP13 has also been identified as playing a key role in promoting the development of liver metastasis from colorectal cancer [46]. In MMP13 knockout mice, there were fewer liver metastases and this was due to loss of host MMP13 resulting in fewer tumour cells extravasating from the hepatic vasculature [46]. MMP2 expressed by stromal fibroblasts has also been shown to be important in promoting the growth of metastases in breast cancer [47]. Loss of stromal fibroblast MMP2 reduced the proliferation of metastasis and this was associated with reduced MMP2-induced transforming growth factor beta 1 activity, highlighting the interaction between MMPs and the transforming growth factor pathway [47]. In view of the central role of MT1-MMP in the invadopodium and promoting tumour invasion, this MMP has also been the focus of a number of studies to elucidate the interaction of this MMP with other pro-invasive signalling pathways. The broadly expressed actin polymerizing protein N-WASp, which has previously been implicated in hepatocellular carcinoma, was found to promote trafficking of MT1-MMP into invadopodia and to stabilize it via tethering of its cytoplasmic tail to F-actin, thus ensuring optimum MT1-MMP localization for facilitating ECM degradation [48]. Furthermore, N-WASp J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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depletion led to a 50% reduction in matrix degradation, which is consistent with the effects of N-WASp overexpression [49]. Inhibition of N-WASp resulted in a 70% decrease in the density of invadopodia present, indicating a highly important role for N-WASp in the formation of invadopodia [50]. More recent research has alluded to the precise function of N-WASp in invadopodia formation through two distinct processes: one of which is the N-WASp-dependent formation of F-actin and cortactin-positive invadopodia, while the other mechanism is dependent on the activity of the complex between Wiskott–Aldrich syndrome protein and scar homologue (WASH) [51]. Cell surface expression of MT1-MMP is in part regulated by Van-Gogh-like protein 2 (VANGL2), which has recently been proposed as a tumour suppressor [52]. Loss of VANGL2 resulted in the formation of larger invadopodia as well as increased ECM degradation, adding further support to the concept that MT1-MMP inhibition could lead to reduced ECM degradation due to a lack of invadopodia formation and reduced proteolytic activity [53]. This concept received further support from a study that inhibited MT1-MMP-mediated proMMP2 activation, whilst leaving other MT1-MMP proteolytic functions intact [53]. Antibody-mediated MT1-MMP inhibition did not affect the overall proteolytic activity of MT1-MMP, although endothelial cells treated with the antibody exhibited reduced migratory behaviour, implicating MT1-MMP-mediated proMMP2 activation in lymphangiogenesis. However, possibly of more significance is the successful demonstration of blocking a specific pathway of MMP activation for the first time, which could hold promise for future therapeutic interventions by avoiding the well-documented pitfalls associated with broad-spectrum MMP inhibitors used in previous human clinical trials [54–56]. More recently, DX-2400, another MT1-MMP function-blocking monoclonal antibody, achieved reductions in Panc-1 cell invasiveness equivalent to that from marimastat (a broad-spectrum MMP inhibitor), but at a 100-fold lower concentration [57]. This potency and specificity are desirable in potential therapeutic agents and such future therapies will require similar if not greater specificity, potentially achieved through the targeting of exosites or the administration of pro-drugs that can undergo metabolic activation [58–60]. The transmembrane glycoprotein CUB domain-containing protein 1 (CDCP1) has previously been shown to encourage ECM degradation through promotion of MMP9 secretion and has been implicated in the development of solid tumours such as colorectal, pancreatic, and lung cancer [61,62]. Knockdown of CDCP1 using siRNA in both MDA-MB-231 human breast cancer cells and RPMI 7951 human melanoma cells was shown to result in reduced ECM degradation as a consequence of inefficient localization of MT1-MMP to invadopodia, without affecting MT1-MMP expression. The mechanism underlying this is unclear, but has been proposed to be due to disruption of a positive feedback loop that promotes increased MMP activity in Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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the ECM. Loss of CDCP1 would be expected to reduce delivery of MT1-MMP into invadopodia and lead to less invasive activity [63]. In contrast, siRNA knockdown of CDCP1 in oesophageal squamous cell carcinoma cells had the opposite effect by promoting invasive behaviour and its expression level in tumours was independently associated with poor prognosis [64]. Neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) is a gene responsible for encoding a scaffolding protein found in invadopodia. It has been implicated in the development of breast and lung carcinomas and has previously been shown to promote EMT [65,66]. NEDD9 is also known to be responsible for local invasion at the primary site but not at the metastatic site. Knockdown of NEDD9 by shRNA alone in several breast cancer cell lines, including ZR75.1, resulted in reduced invasion at the primary site in vitro and in vivo and was accompanied by fewer circulating tumour cells and fewer metastatic lung tumours. This was attributed to inhibition of MT1-MMP by TIMP-2, as indicated by a 50% increase in the concentration of cell surface-bound TIMP-2. NEDD9 knockdown did not reduce the amount of MT1-MMP but blocked its function through another mechanism, potentially involving inhibition of invadopodia function, although not invadopodia formation. Reintroduction of NEDD9 restored levels of MT1-MMP on the cell surface and cells’ invasive capacity [67]. A specific microRNA, miR-181a-5p, has been identified as down-regulating MT1-MMP in breast cancer [68]. A miR-181-5p responsive element was identified within the 3’-untranslated region of MT1-MMP and this microRNA mediated a reduction in MT1-MMP, which reduced cancer cell migration, invasion, and activation of proMMP2 [68].
The role of MMPs in sensing matrix stiffness and tumour invasion The tumour microenvironment is critical to promoting tumourigenesis and there is currently significant interest in how the mechanical structure of the ECM influences tumour progression [69,70]. In particular, focus has centred on how migrating cancer cells interpret the physical stiffness of the microenvironment and then respond to mechanical cues from the ECM in a manner that contributes to their invasive behaviour. The most recent studies have established how cellular movement exerts mechanical forces or ‘traction’ upon the ECM and the consequences that this has for MMP expression. Tumour cell shape and motility are modulated by altered states of cellular contractility and ECM stiffness [71,72]. When invading MDA-MB-231 breast cancer cells approach areas that are particularly stiff and have high mechanical resistance, typical of the highly fibrillar structure of the ECM, they are more likely to shift to protease-mediated invasion methods involving the J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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formation of invadopodia and high MT1-MMP activity, which may not necessarily be required if invading a matrix that is more plastic. These findings emphasize how the physical structure of the tumour microenvironment can dictate the chosen mechanism of invasion [73]. The contractility of Panc-1 pancreatic cancer cells was mediated by myosin II and increased contractility together with increased ECM stiffness led to an increase in MT1-MMP secretion, resulting in accelerated ECM breakdown. Decreased ECM stiffness and contractility result in reduced MMP activity to a level comparable to that observed with treatment with broad-spectrum MMP inhibitors [74]. Therefore it appears that migrating cancer cells must be able to sense the stiffness of the ECM in order to regulate matrix degradation, and that changes in MMP localization, invadopodia formation, and focal adhesion through integrins and heparan sulphate proteoglycans (HSPGs) could alter matrix contractility and therefore allow a cell to adjust the secretion of MMPs accordingly. To emphasize the sophistication of these mechanisms, the migrating cells are then able to modulate MMP activity in order to use the remaining ECM as a scaffold to assist in their migration [74]. However, another recent study using MDA-MB-231 breast cancer cells and HS93.T human fibrosarcoma cells suggested that low stiffness or a ‘soft’ environment was a key promoter of invasive behaviour in 3D matrices by increased expression of MMPs, including MT1-MMP, in addition to inducing invadopodia formation [75]. It was also demonstrated that increasing matrix stiffness resulted in reduced secretion of MMPs [75]. The HSPGs are a family of glycoproteins that perform a diverse array of cellular functions and have previously been implicated in tumour development and progression. They are considered to play a role in MMP activation, due to their influence on cell-to-matrix adhesion mediated by the mechanical nature of the microenvironment [76,77]. Perlecan/HSPG2 is a key component of the basement membrane and is a candidate for MMP7-mediated proteolysis [78]. It was demonstrated that a C-terminal domain of HSPG2, Dm IV-3, directs metastatic cells to arrange themselves in clusters; however, on cleavage of HSPG2 by MMP7, this phenotype is reversed to one favouring cell dispersion and migration [78]. Of the two metastatic prostate cancer cell lines investigated, the C4-2 cells were much more susceptible to the effects of Dm IV-3 than were PC-3 cells. It was proposed that the molecular switch causing the change in cell behaviour by MMP7-mediated proteolysis of perlecan clearly favours a pro-metastatic phenotype [78]. The key role of matrix stress-induced activation of MMP7 has been shown to promote the invasion of chondrosarcoma cells in response to increased shear stress [79]. Shear stress resulted in the accumulation of cyclic AMP, which activated several intracellular signalling pathways including PI3-kinase, ERK and p38, all of which led to the increased expression of MMP7 [79]. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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The role of MMPs in induction and maintenance of EMT Several MMPs, including MMP2, 3, 9, and 13, and MT1-MMP, play important regulatory roles in modulating EMT. Whilst it is clear that the secretion of MMPs by tumour-associated fibroblasts and macrophages has a prominent role in ECM degradation, more clarity is required to understand the full extent of their involvement through secondary functions, such as how they influence epithelial cell behaviour and their interactions with other drivers of EMT [80–82]. Activation-induced cytidine deaminase (AID) plays a major role in antibody diversification [83]. Due to the ability of AID to influence DNA structure, it has recently been investigated in the context of EMT. MMP2 and MMP9 expression was confirmed in ZR75.1 breast adenocarcinoma cells that are known to exhibit invasive characteristics and readily undergo EMT [84]. Knockdown of AID by shRNA inhibited the up-regulation of both MMP2 and MMP9, coinciding with loss of EMT due to an inability to degrade and migrate through the basement membrane, demonstrating that it is essential for EMT initiation. Aberrant CpG island methylation has been implicated as a potential mechanism responsible for this loss of EMT attributed to AID knockdown. CpG islands tend to be in close proximity to promoter regions associated with abnormal CpG methylation as a mechanism for silencing tumour suppressor genes. AID knockdown resulted in an increase in CpG island methylation in the 5′ -untranslated region of the MMP2 gene, leading to reduced MMP2 expression. This was supported by use of 5-aza-2′ -deoxycytidine, a DNA demethylating agent to block CpG methylation, which led to a decrease in CpG island methylation on the MMP2 gene and a concomitant increase in MMP2 expression, demonstrating AID-induced CpG methylation as a key regulator of MMP2. However, levels of CpG methylation did not change significantly with induction of EMT [84,85]. The serine/threonine-specific protein kinase Akt has long been implicated in the development of human cancers because of its influence on cell growth and survival [86]. Overexpression of Akt was shown to stimulate the development of an EMT phenotype in HCT-116 colorectal cancer cells. Furthermore, Akt-overexpressing tumours in a xenograft mouse model showed a marked increase in growth compared with controls, which was associated with a reduction in E-cadherin expression and an increased expression and nuclear localization of β-catenin, both of which are characteristic features of tumour development [87]. MMP2 and MMP9 expression was also significantly increased in the Akt-overexpressing tumours, reinforcing abnormal Akt expression as an inducer of EMT and thought to be mediated through PI3K–Akt activation [88]. Subsequently, it was suggested that Akt could be a more beneficial therapeutic target than the molecules responsible for facilitating EMT, such as the J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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MMPs, because it plays a more upstream role in tumour progression [89]. The PI3K/Akt pathway has been a prominent focus in therapeutic targeting for cancer drug discovery over the past decade and various clinical trials are ongoing that appear to show some promise when Akt inhibitors are used as combination therapy [90,91]. The guanine nucleotide exchange factor Vav1, which functions in the up-regulation of MT1-MMP and invadopodia formation, has also been implicated in EMT [92]. Overexpression of Vav1 promoted morphological changes in human ovarian cancer SKOV3 cells, demonstrated by loss of their spheroid shape, characteristic epithelial cell alignment, and an inability to distinguish cell boundaries, which was in contrast to the behaviour of Vav1-negative cells. These changes were accompanied by F-actin re-organization and fewer intercellular adhesions, suggestive of a potential role in EMT. Increased formation of podosomes was also reported and is consistent with the role of Vav1 as an inducer of invadopodia formation. The mechanism responsible for fewer cell–cell adhesions and changes in morphology was proposed to be increasing Vav1, resulting in a decrease in levels of E-cadherin through the induction of EMT transcription factors Snail and Slug [92]. Vav1 expression was also correlated with poor prognosis in early-stage ovarian cancer in this study, albeit in a small sample size of 88 cases with Vav1 expressed in 59% of tumours. Patients with ovarian cancer who had International Federation of Gynaecology and Obstetrics (FIGO) stage I and II disease and whose tumours showed increased Vav1 expression had a poorer prognosis, and Vav1 was proposed as a prognostic tissue-based biomarker [92]. The implications for the roles that EMT and the reverse process, mesenchymal–epithelial transition play in the development of cancer remain to be fully elucidated. With regard to the pathogenic role of the MMPs in EMT, there is considerable evidence of their involvement, from being recruited to co-ordinate and conduct proteolytic activity through multiple signalling pathways to actually initiating EMT in certain types of cancer. The complexity and intricacy of intercellular communication within EMT combined with difficulties separating pathological and physiological function create significant challenges with regard to therapeutic targeting [93].
Conclusions Tumour invasion and metastasis are characterized by an array of dynamic cellular processes leading to cell migration and invasion in which MMP involvement is central. The MMPs perform multiple functions in tumour invasion and metastasis, some of which are directly related to their catalytic functions to degrade extracellular matrix components, while others are related to non-catalytic functions. Current research is starting to provide a mechanistic basis for specific Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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roles of MMPs in tumour invasion and metastasis. The enhanced mechanistic understanding of the roles of MMPs in this context also indicates that it would be timely to revisit the therapeutic opportunities of manipulating MMP activity to inhibit tumour invasion and metastasis [60,94].
Author contribution statement GTB and GIM identified and interpreted the relevant literature and wrote and edited the manuscript. Both authors approved the final version of the manuscript prior to submission.
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J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
REVIEW
Current mechanistic insights into the roles of matrix metalloproteinases in tumour invasion and metastasis Gordon T Brown and Graeme I Murray* Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen, UK *Correspondence to: GI Murray, Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail: [email protected]
Abstract The purpose of this review is to highlight the recent mechanistic developments elucidating the role of matrix metalloproteinases (MMPs) in tumour invasion and metastasis. The ability of tumour cells to invade, migrate, and subsequently metastasize is a fundamental characteristic of cancer. Tumour invasion and metastasis are increasingly being characterized by the dynamic relationship between cancer cells and their microenvironment and developing a greater understanding of these basic pathological mechanisms is crucial. While MMPs have been strongly implicated in these processes as a result of extensive circumstantial evidence – for example, increased expression of individual MMPs in tumours and association of specific MMPs with prognosis – the underpinning mechanisms are only now being elucidated. Recent studies are now providing a mechanistic basis, highlighting and reinforcing the catalytic and non-catalytic roles of specific MMPs as key players in tumour invasion and metastasis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Keywords: extracellular matrix; matrix metalloproteinase; metastasis; microenvironment; protease; tumour invasion; tumour progression
Received 28 April 2015; Revised 3 July 2015; Accepted 8 July 2015
Conflict of interest statement: GTB declares no conflicts of interest. GIM is a scientific advisor to Vertebrate Antibodies (http://www.vertebrateantibodies.com).
Introduction At the molecular level, neoplasia is a multifactorial process driven by the combination of products of mutated oncogenes and tumour suppressor genes. There is now greater awareness of the complex and reciprocal interactions between malignant cells, non-malignant stromal cells, and extracellular matrix (ECM) of the peri-tumoural milieu or tumour microenvironment [1,2]. It has become increasingly apparent that these interactions begin at a relatively early stage of tumour progression and are important mechanisms through which a malignant neoplasm can initiate both local invasion and distant metastasis [3–6]. The structural framework of the ECM consists predominantly of individual types of collagen but also includes other matrix proteins including elastin and fibrillin [7]. Fibronectin and laminin are anchoring proteins involved in the attachment of cells to the structural skeleton, while the other major components of the ECM are proteoglycans. The ECM undergoes tightly regulated and co-ordinated remodelling during processes including wound healing, inflammation, and organogenesis [7]. An essential component of this remodelling process is proteolysis of the ECM and this breakdown is also vital but dysregulated in the early steps of local invasion of malignant cells into adjacent tissue. The Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
dynamic nature of this continuous remodelling highlights the need for detailed understanding of these cellular processes but also poses therapeutic challenges for targeting this process [8]. There are various groups of proteolytic enzymes or proteases involved in matrix degradation but the matrix metalloproteinase (MMP) group of enzymes is the most important in the context of tumour invasion and metastasis [7,9–14]. The MMPs are zinc-dependent endopeptidases that play key roles in the progression and dissemination of cancer including the modulation of tumour-associated angiogenesis [11,15,16]. The MMPs are classified by a combination of amino acid sequence, peptide domain structure, and substrate specificity (Table 1 and Figure 1A). The main groups of MMPs are the collagenases, gelatinases, stromelysins and membrane-type MMPs. Distinct from their involvement in structural protein breakdown, the MMPs can also influence a range of key cellular processes including cell proliferation, cell differentiation, apoptosis, and autophagy by activating growth factors and their receptors or by releasing cytokines from the ECM [17–20]. There is very substantial circumstantial evidence for the role of MMPs in tumour invasion and metastasis and it has been shown that the expression of specific MMPs has been associated with a poor prognosis in a range of human cancers [21–26]. J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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Figure 1. (A) An overview of the domain structure of the main groups of MMPs. (B) The role of MMPs involved in basement membrane and extracellular matrix degradation. Migration of a tumour cell invading from the primary site through the basement membrane and beyond the ECM into the systemic circulation is shown. Major constituents of the ECM include collagens, laminin, fibronectin, and fibrillin, and the MMPs shown are key proteolytic enzymes involved in ECM degradation in order to facilitate cell invasion. MT1-MMP is localized at the cell surface membrane and activates proMMP2, which initiates the MMP activation cascade. The orange arrows between the MMPs represent mutual activation. Those MMPs are then recruited by the tumour cell in order to recapitulate the process of invasion to invade through the wall of a local blood vessel and allow it to become a circulating tumour cell potentially capable of forming a metastasis. (C) The interaction of invadopodia formation and MMPs to promote invasive activity at the leading edge of an invadopodium. Loss of CDCP1 and NEDD9 inhibit invadopodia formation, whilst VANGL2 loss promotes invadopodia formation. Vav1 functions through CDC42 to activate N-WASp and Arp2/3 signalling to increase invadopodia formation. N-WASp up-regulates MT1-MMP activity with the help of F-actin, enabling proteolytic activity and proMMP2 activation to initiate the MMP activation cascade.
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J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
Matrix metalloproteinases and tumour invasion
Table 1. The classification and nomenclature of human MMPs MMP
Group
Alternative names
MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10 MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP21 MMP22 MMP23 MMP24 MMP25 MMP26 MMP27 MMP28
Collagenase Gelatinase, Stromelysin Matrilysin Collagenase Gelatinase Stromelysin Stromelysin Other Collagenase Membrane type Membrane type Membrane type Membrane type Other Other Other Other Other Membrane type Membrane type Matrilysin Other Other
Interstitial collagenase Gelatinase A, 72 kDa type IV collagenase Stromelysin 1, progelatinase Matrilysin 1 Neutrophil collagenase Gelatinase B, 92 kDa type IV collagenase Stromelysin 2 Stromelysin 3 Macrophage elastase, metalloelastase Collagenase 3 MT1-MMP, membrane type 1 collagenase MT2-MMP MT3-MMP MT4-MMP RASI-1, also known as MMP18 Enamolysin Known as MMP23B MT5-MMP MT6-MMP Matrilysin 2 Epilysin
This review will highlight the key recent advances and emerging mechanistic concepts involving MMPs in relation to tumour invasion and metastasis. This will include the role of MMPs in the promotion of tumour cell motility, their contribution in facilitating tumour invasion, interactions of MMPs with other pro-invasive pathways, and the influence of MMPs within the tumour microenvironment and their role in the facilitation of epithelial–mesenchymal transition (EMT).
The roles of MMPs in modulating tumour cell motility Critical to tumour invasion and metastasis is the ability of tumour cells to move from one site to another, which in cell biology terms is expressed as cell motility. Recent mechanistic studies are now highlighting the role of MMPs in promoting tumour cell movement involving both catalytic and non-catalytic MMP functions [27]. Comparison of rounded-amoeboid (epithelioid) melanoma cells and elongated-mesenchymal (spindle cell) melanoma cells showed that there was increased secretion of several MMPs, most notably MMP9 and MMP13 [27]. This ensured that melanoma cells with an amoeboid morphology degraded type I collagen more efficiently than did melanoma cells with an elongated-mesenchymal morphology. In addition, secreted MMP9 was more effective at promoting the migration of rounded-amoeboid melanoma cells; this mechanism was non-catalytic, acting through the regulation of actomyosin contractility via MMP9 binding to the cell surface CD44 receptor [27]. The key role of MMP9 in promoting tumour invasion is also supported by a recent study of lung cancer cell Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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migration [28]. The increased movement of lung cancer cells was promoted by induction of MMP9 (by the overexpression of the transcription modulator megakarocytic leukaemia 1 factor), whereas silencing of this transcription factor resulted in loss of MMP9 expression, due to methylation of the MMP9 promoter, and this decreased invasion [28]. Further evidence for the role of MMP9 in promoting tumour invasion comes from a recent investigation of the binding of MMP9 to tumour-associated fibroblasts [29]. The interaction of tumours cells and surrounding stromal cells is important in regulating both tumour invasion and metastasis, and tumour-associated fibroblasts are central in these interactions. Tumour-associated fibroblasts, when differentiated to myofibroblasts, promote tumour progression by protease-catalysed remodelling of the stroma. MMP9 is central to matrix remodelling and the mechanism by which myofibroblasts utilize MMP9 has been demonstrated [29]. Recruitment of MMP9 by lysyl hydroxylase to the fibroblast cell surface has been shown to promote the myofibroblastic differentiation of tumour-associated fibroblasts, with binding of MMP9 to the cell surface of fibroblasts occurring via its fibronectin-like domain [29].
The role of MMPs in invadopodia formation and function The mechanism by which cancer cells are able to migrate through the ECM is now being identified and these studies have focused on invadopodia. These are specialized protrusions of the cell membrane of invasive tumour cells that have emerged recently to be key cellular structures in facilitating the movement of cancer cells through the basement membrane and hence promoting tumour invasion and metastasis [30]. Invadopodia are rich in MMPs, especially membrane-type 1 MMP (MT1-MMP; also known as MMP14), which activates proMMP2, thus initiating the MMP activation cascade (Figures 1B and 1C) [30–34]. The molecular mechanisms underlying the role of MMPs, especially MT1-MMP, in invadopodia formation and function are now being identified. The formation of invadopodia is a multifactorial process and can occur despite the absence of MT1-MMP, although MT1-MMP-deficient invadopodia lack the capacity to perform ECM degradation [31–34]. The guanine nucleotide exchange factor Vav1 has recently been identified as an inducer of invadopodia formation in pancreatic cancer cells [34]. Vav1 functions through exchange activity towards the Rho GTPase cell division cycle 42 (CDC42), which in turn promotes invasive behaviour through actin nucleation, and branching via neural Wiskott–Aldrich syndrome protein (N-WASp) and actin-related proteins 2 and 3 (Arp2/3) complex signalling. The deletion of Vav1 leads to a reduced number of actin puncta, a marker of active invadopodia and decreased MMP matrix-related degradation [35,36]. J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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Membrane-type MMPs contain a characteristic peptide sequence in the catalytic domain known as the MT-LOOP, which is critical to the function of MT1-MMP (Figure 1A) [37]. Deletion of the MT-LOOP domain has been shown to inhibit cell surface functions of MT1-MMP associated with invasion including proMMP2 activation and degradation of collagen. The mechanism through which this occurs has been identified as ineffective localization of MT1-MMP to β1-integrin-rich cell adhesion complexes at the plasma membrane [38]. However, knockdown of β1-integrin did not inhibit MT1-MMP-dependent degradation, indicating that β1-integrin is not a key component in the adhesion complex driving ECM degradation. Inhibition of MT-LOOP function through specific antibody binding resulted in an identical phenotype to that of MT-LOOP-deficient MT1-MMP, highlighting the MT-LOOP as a potential therapeutic target for MT1-MMP inhibition [38]. The catalytic domain of MT1-MMP has been therapeutically targeted. However, the catalytic domain is highly conserved and is shared by many other MMPs, and these efforts led to the non-specific inhibition of MMP activity that proved unsuccessful in combating tumour progression [39]. The hemopexin domain is another key region on the MT1-MMP molecule which when blocked through intratumour injections of a novel allosteric hemopexin inhibitor, resulted in the inhibition of breast tumour growth in a xenograft mouse model [40]. However, the catalytic function of MT1-MMP including proMMP2 activation was not affected by hemopexin domain inhibition [41].
Interactions of MMPs with pro-invasive pathways Recent studies have highlighted the mechanistic interactions of MMPs with pro-invasive pathways even at the early stages of carcinogenesis. In breast cancer, the key contribution of individual MMPs in promoting the transition from in situ malignancy (ductal carcinoma in situ) to invasive malignancy has recently been identified [42]. MT1-MMP was shown to be a key player in promoting invasion through the basement membrane and thus promoting the transformation of ductal carcinoma in situ to invasive breast cancer with the increased expression of this MMP being under the control of p63. The role of MMPs in the early stages of tumour invasion may indicate that MMP inhibition could be an effective strategy to prevent the progression of in situ malignancy to invasive malignancy. Liver cancer is another type of tumour in which MMPs are important and with the mechanisms by which MMPs interact with other key signalling pathways being identified [43]. Increased expression of MMP10 (stromelysin 2) has been identified in both human hepatocellular carcinoma and a diethylnitrosamine-induced model of hepatocarcinogenesis in MMP10-deficient mice. In human hepatocellular carcinomas, MMP10 Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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expression was associated with more aggressive tumours, while the liver tumours that developed in MMP10-deficient mice were smaller with reduced vascularity and fewer lung metastases. MMP10 expression was induced by hypoxia and the C-X-C chemokine receptor-4 ligand (stromal-derived factor 1) through the C-X-C chemokine receptor-4 stromal-derived factor 1 pathway via an activator protein 1 site on the MMP10 promoter [43]. The mechanism by which signal transducers and activators of transcription (STATs), which are frequently constitutively activated in cancer, interact with MMPs to promote tumour invasion has recently been elucidated in lung cancer [44]. STAT3 is activated in lung cancer and is associated with expression of MMP1 (interstitial collagenase). STAT3 was inducible by interleukin 6, and STAT3 activation promoted transcriptional activation of MMP1, thus providing a link between STAT3 activation, increased MMP1 expression, and increased invasiveness in lung cancer [44]. There is recent evidence providing a mechanistic understanding of the relationship of leptin and MMPs in promoting invasion of pancreatic adenocarcinoma [45]. Leptin has been shown to up-regulate MMP13 (collagenase-3) in pancreatic adenocarcinoma, also via the JAK2/STAT3 pathway. Overexpression of leptin in an orthotopic model of pancreatic adenocarcinoma was associated with increased MMP13 expression and promotion of lymph node metastasis, thus providing a potential link between obesity and tumour progression [45]. Understanding the role of MMPs in promoting metastasis is also important and MMP13 has also been identified as playing a key role in promoting the development of liver metastasis from colorectal cancer [46]. In MMP13 knockout mice, there were fewer liver metastases and this was due to loss of host MMP13 resulting in fewer tumour cells extravasating from the hepatic vasculature [46]. MMP2 expressed by stromal fibroblasts has also been shown to be important in promoting the growth of metastases in breast cancer [47]. Loss of stromal fibroblast MMP2 reduced the proliferation of metastasis and this was associated with reduced MMP2-induced transforming growth factor beta 1 activity, highlighting the interaction between MMPs and the transforming growth factor pathway [47]. In view of the central role of MT1-MMP in the invadopodium and promoting tumour invasion, this MMP has also been the focus of a number of studies to elucidate the interaction of this MMP with other pro-invasive signalling pathways. The broadly expressed actin polymerizing protein N-WASp, which has previously been implicated in hepatocellular carcinoma, was found to promote trafficking of MT1-MMP into invadopodia and to stabilize it via tethering of its cytoplasmic tail to F-actin, thus ensuring optimum MT1-MMP localization for facilitating ECM degradation [48]. Furthermore, N-WASp J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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depletion led to a 50% reduction in matrix degradation, which is consistent with the effects of N-WASp overexpression [49]. Inhibition of N-WASp resulted in a 70% decrease in the density of invadopodia present, indicating a highly important role for N-WASp in the formation of invadopodia [50]. More recent research has alluded to the precise function of N-WASp in invadopodia formation through two distinct processes: one of which is the N-WASp-dependent formation of F-actin and cortactin-positive invadopodia, while the other mechanism is dependent on the activity of the complex between Wiskott–Aldrich syndrome protein and scar homologue (WASH) [51]. Cell surface expression of MT1-MMP is in part regulated by Van-Gogh-like protein 2 (VANGL2), which has recently been proposed as a tumour suppressor [52]. Loss of VANGL2 resulted in the formation of larger invadopodia as well as increased ECM degradation, adding further support to the concept that MT1-MMP inhibition could lead to reduced ECM degradation due to a lack of invadopodia formation and reduced proteolytic activity [53]. This concept received further support from a study that inhibited MT1-MMP-mediated proMMP2 activation, whilst leaving other MT1-MMP proteolytic functions intact [53]. Antibody-mediated MT1-MMP inhibition did not affect the overall proteolytic activity of MT1-MMP, although endothelial cells treated with the antibody exhibited reduced migratory behaviour, implicating MT1-MMP-mediated proMMP2 activation in lymphangiogenesis. However, possibly of more significance is the successful demonstration of blocking a specific pathway of MMP activation for the first time, which could hold promise for future therapeutic interventions by avoiding the well-documented pitfalls associated with broad-spectrum MMP inhibitors used in previous human clinical trials [54–56]. More recently, DX-2400, another MT1-MMP function-blocking monoclonal antibody, achieved reductions in Panc-1 cell invasiveness equivalent to that from marimastat (a broad-spectrum MMP inhibitor), but at a 100-fold lower concentration [57]. This potency and specificity are desirable in potential therapeutic agents and such future therapies will require similar if not greater specificity, potentially achieved through the targeting of exosites or the administration of pro-drugs that can undergo metabolic activation [58–60]. The transmembrane glycoprotein CUB domain-containing protein 1 (CDCP1) has previously been shown to encourage ECM degradation through promotion of MMP9 secretion and has been implicated in the development of solid tumours such as colorectal, pancreatic, and lung cancer [61,62]. Knockdown of CDCP1 using siRNA in both MDA-MB-231 human breast cancer cells and RPMI 7951 human melanoma cells was shown to result in reduced ECM degradation as a consequence of inefficient localization of MT1-MMP to invadopodia, without affecting MT1-MMP expression. The mechanism underlying this is unclear, but has been proposed to be due to disruption of a positive feedback loop that promotes increased MMP activity in Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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the ECM. Loss of CDCP1 would be expected to reduce delivery of MT1-MMP into invadopodia and lead to less invasive activity [63]. In contrast, siRNA knockdown of CDCP1 in oesophageal squamous cell carcinoma cells had the opposite effect by promoting invasive behaviour and its expression level in tumours was independently associated with poor prognosis [64]. Neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) is a gene responsible for encoding a scaffolding protein found in invadopodia. It has been implicated in the development of breast and lung carcinomas and has previously been shown to promote EMT [65,66]. NEDD9 is also known to be responsible for local invasion at the primary site but not at the metastatic site. Knockdown of NEDD9 by shRNA alone in several breast cancer cell lines, including ZR75.1, resulted in reduced invasion at the primary site in vitro and in vivo and was accompanied by fewer circulating tumour cells and fewer metastatic lung tumours. This was attributed to inhibition of MT1-MMP by TIMP-2, as indicated by a 50% increase in the concentration of cell surface-bound TIMP-2. NEDD9 knockdown did not reduce the amount of MT1-MMP but blocked its function through another mechanism, potentially involving inhibition of invadopodia function, although not invadopodia formation. Reintroduction of NEDD9 restored levels of MT1-MMP on the cell surface and cells’ invasive capacity [67]. A specific microRNA, miR-181a-5p, has been identified as down-regulating MT1-MMP in breast cancer [68]. A miR-181-5p responsive element was identified within the 3’-untranslated region of MT1-MMP and this microRNA mediated a reduction in MT1-MMP, which reduced cancer cell migration, invasion, and activation of proMMP2 [68].
The role of MMPs in sensing matrix stiffness and tumour invasion The tumour microenvironment is critical to promoting tumourigenesis and there is currently significant interest in how the mechanical structure of the ECM influences tumour progression [69,70]. In particular, focus has centred on how migrating cancer cells interpret the physical stiffness of the microenvironment and then respond to mechanical cues from the ECM in a manner that contributes to their invasive behaviour. The most recent studies have established how cellular movement exerts mechanical forces or ‘traction’ upon the ECM and the consequences that this has for MMP expression. Tumour cell shape and motility are modulated by altered states of cellular contractility and ECM stiffness [71,72]. When invading MDA-MB-231 breast cancer cells approach areas that are particularly stiff and have high mechanical resistance, typical of the highly fibrillar structure of the ECM, they are more likely to shift to protease-mediated invasion methods involving the J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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formation of invadopodia and high MT1-MMP activity, which may not necessarily be required if invading a matrix that is more plastic. These findings emphasize how the physical structure of the tumour microenvironment can dictate the chosen mechanism of invasion [73]. The contractility of Panc-1 pancreatic cancer cells was mediated by myosin II and increased contractility together with increased ECM stiffness led to an increase in MT1-MMP secretion, resulting in accelerated ECM breakdown. Decreased ECM stiffness and contractility result in reduced MMP activity to a level comparable to that observed with treatment with broad-spectrum MMP inhibitors [74]. Therefore it appears that migrating cancer cells must be able to sense the stiffness of the ECM in order to regulate matrix degradation, and that changes in MMP localization, invadopodia formation, and focal adhesion through integrins and heparan sulphate proteoglycans (HSPGs) could alter matrix contractility and therefore allow a cell to adjust the secretion of MMPs accordingly. To emphasize the sophistication of these mechanisms, the migrating cells are then able to modulate MMP activity in order to use the remaining ECM as a scaffold to assist in their migration [74]. However, another recent study using MDA-MB-231 breast cancer cells and HS93.T human fibrosarcoma cells suggested that low stiffness or a ‘soft’ environment was a key promoter of invasive behaviour in 3D matrices by increased expression of MMPs, including MT1-MMP, in addition to inducing invadopodia formation [75]. It was also demonstrated that increasing matrix stiffness resulted in reduced secretion of MMPs [75]. The HSPGs are a family of glycoproteins that perform a diverse array of cellular functions and have previously been implicated in tumour development and progression. They are considered to play a role in MMP activation, due to their influence on cell-to-matrix adhesion mediated by the mechanical nature of the microenvironment [76,77]. Perlecan/HSPG2 is a key component of the basement membrane and is a candidate for MMP7-mediated proteolysis [78]. It was demonstrated that a C-terminal domain of HSPG2, Dm IV-3, directs metastatic cells to arrange themselves in clusters; however, on cleavage of HSPG2 by MMP7, this phenotype is reversed to one favouring cell dispersion and migration [78]. Of the two metastatic prostate cancer cell lines investigated, the C4-2 cells were much more susceptible to the effects of Dm IV-3 than were PC-3 cells. It was proposed that the molecular switch causing the change in cell behaviour by MMP7-mediated proteolysis of perlecan clearly favours a pro-metastatic phenotype [78]. The key role of matrix stress-induced activation of MMP7 has been shown to promote the invasion of chondrosarcoma cells in response to increased shear stress [79]. Shear stress resulted in the accumulation of cyclic AMP, which activated several intracellular signalling pathways including PI3-kinase, ERK and p38, all of which led to the increased expression of MMP7 [79]. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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The role of MMPs in induction and maintenance of EMT Several MMPs, including MMP2, 3, 9, and 13, and MT1-MMP, play important regulatory roles in modulating EMT. Whilst it is clear that the secretion of MMPs by tumour-associated fibroblasts and macrophages has a prominent role in ECM degradation, more clarity is required to understand the full extent of their involvement through secondary functions, such as how they influence epithelial cell behaviour and their interactions with other drivers of EMT [80–82]. Activation-induced cytidine deaminase (AID) plays a major role in antibody diversification [83]. Due to the ability of AID to influence DNA structure, it has recently been investigated in the context of EMT. MMP2 and MMP9 expression was confirmed in ZR75.1 breast adenocarcinoma cells that are known to exhibit invasive characteristics and readily undergo EMT [84]. Knockdown of AID by shRNA inhibited the up-regulation of both MMP2 and MMP9, coinciding with loss of EMT due to an inability to degrade and migrate through the basement membrane, demonstrating that it is essential for EMT initiation. Aberrant CpG island methylation has been implicated as a potential mechanism responsible for this loss of EMT attributed to AID knockdown. CpG islands tend to be in close proximity to promoter regions associated with abnormal CpG methylation as a mechanism for silencing tumour suppressor genes. AID knockdown resulted in an increase in CpG island methylation in the 5′ -untranslated region of the MMP2 gene, leading to reduced MMP2 expression. This was supported by use of 5-aza-2′ -deoxycytidine, a DNA demethylating agent to block CpG methylation, which led to a decrease in CpG island methylation on the MMP2 gene and a concomitant increase in MMP2 expression, demonstrating AID-induced CpG methylation as a key regulator of MMP2. However, levels of CpG methylation did not change significantly with induction of EMT [84,85]. The serine/threonine-specific protein kinase Akt has long been implicated in the development of human cancers because of its influence on cell growth and survival [86]. Overexpression of Akt was shown to stimulate the development of an EMT phenotype in HCT-116 colorectal cancer cells. Furthermore, Akt-overexpressing tumours in a xenograft mouse model showed a marked increase in growth compared with controls, which was associated with a reduction in E-cadherin expression and an increased expression and nuclear localization of β-catenin, both of which are characteristic features of tumour development [87]. MMP2 and MMP9 expression was also significantly increased in the Akt-overexpressing tumours, reinforcing abnormal Akt expression as an inducer of EMT and thought to be mediated through PI3K–Akt activation [88]. Subsequently, it was suggested that Akt could be a more beneficial therapeutic target than the molecules responsible for facilitating EMT, such as the J Pathol 2015; 237: 273–281 www.thejournalofpathology.com
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MMPs, because it plays a more upstream role in tumour progression [89]. The PI3K/Akt pathway has been a prominent focus in therapeutic targeting for cancer drug discovery over the past decade and various clinical trials are ongoing that appear to show some promise when Akt inhibitors are used as combination therapy [90,91]. The guanine nucleotide exchange factor Vav1, which functions in the up-regulation of MT1-MMP and invadopodia formation, has also been implicated in EMT [92]. Overexpression of Vav1 promoted morphological changes in human ovarian cancer SKOV3 cells, demonstrated by loss of their spheroid shape, characteristic epithelial cell alignment, and an inability to distinguish cell boundaries, which was in contrast to the behaviour of Vav1-negative cells. These changes were accompanied by F-actin re-organization and fewer intercellular adhesions, suggestive of a potential role in EMT. Increased formation of podosomes was also reported and is consistent with the role of Vav1 as an inducer of invadopodia formation. The mechanism responsible for fewer cell–cell adhesions and changes in morphology was proposed to be increasing Vav1, resulting in a decrease in levels of E-cadherin through the induction of EMT transcription factors Snail and Slug [92]. Vav1 expression was also correlated with poor prognosis in early-stage ovarian cancer in this study, albeit in a small sample size of 88 cases with Vav1 expressed in 59% of tumours. Patients with ovarian cancer who had International Federation of Gynaecology and Obstetrics (FIGO) stage I and II disease and whose tumours showed increased Vav1 expression had a poorer prognosis, and Vav1 was proposed as a prognostic tissue-based biomarker [92]. The implications for the roles that EMT and the reverse process, mesenchymal–epithelial transition play in the development of cancer remain to be fully elucidated. With regard to the pathogenic role of the MMPs in EMT, there is considerable evidence of their involvement, from being recruited to co-ordinate and conduct proteolytic activity through multiple signalling pathways to actually initiating EMT in certain types of cancer. The complexity and intricacy of intercellular communication within EMT combined with difficulties separating pathological and physiological function create significant challenges with regard to therapeutic targeting [93].
Conclusions Tumour invasion and metastasis are characterized by an array of dynamic cellular processes leading to cell migration and invasion in which MMP involvement is central. The MMPs perform multiple functions in tumour invasion and metastasis, some of which are directly related to their catalytic functions to degrade extracellular matrix components, while others are related to non-catalytic functions. Current research is starting to provide a mechanistic basis for specific Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk
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roles of MMPs in tumour invasion and metastasis. The enhanced mechanistic understanding of the roles of MMPs in this context also indicates that it would be timely to revisit the therapeutic opportunities of manipulating MMP activity to inhibit tumour invasion and metastasis [60,94].
Author contribution statement GTB and GIM identified and interpreted the relevant literature and wrote and edited the manuscript. Both authors approved the final version of the manuscript prior to submission.
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