V.-M. Regulation of matrix metalloproteinase expression

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1 Regulation of matrix metalloproteinase expression in tumor invasion JUKKA WESTERMARCK 1 AND VELI-MATTI KÄHÄRI* MediCity Research Laboratory and Department of Medical Biochemistry, University of Turku; and *Department of Dermatology, Turku University Central Hospital, FIN Turku, Finland ABSTRACT Degradation of basement membranes and stromal extracellular matrix (ECM) is crucial for invasion and metastasis of malignant cells. Degradation of ECM is initiated by proteinases secreted by different cell types participating in tumor cell invasion, and increased expression or activity of every known class of proteinases (metallo-, serine-, aspartic-, and cysteine) has been linked to malignancy and invasion of tumor cells. Studies performed over the last decade have revealed that matrix metalloproteinases (MMPs) play a crucial role in tumor invasion. Expression of MMP genes is transcriptionally regulated by a variety of extracellular factors including cytokines, growth factors, and cell contact to ECM. This review will summarize the current view on the role of MMPs in tumor growth, invasion, and survival, and focus on the role of mitogen-activated protein kinases and AP-1 and ETS transcription factors in the regulation of MMP gene expression during invasion process. Westermarck, J., Kähäri, V.-M. Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J. 13, (1999) Key Words: extracellular matrix MMPs interleukin tissue inhibitors SCC MATRIX METALLOPROTEINASES Degradation of extracellular matrix (ECM) 2 is essential in many physiological processes, e.g., during development, growth, and repair of tissues. On the other hand, excessive proteolysis plays an important role in several pathological conditions, e.g., rheumatoid arthritis, osteoarthritis, autoimmune blistering disorders of skin, dermal photoaging, and periodontitis (1 3). Tumor invasion, metastasis, and angiogenesis require controlled degradation of ECM, and increased expression of matrix metalloproteinases (MMPs) is associated with tumor invasion and metastasis of malignant tumors with different histogenetic origin (4, 5). MMPs are a family of at least 17 human zinc-dependent endopeptidases, collectively capable of degrading essentially all ECM components (see refs 1, 3, 5). According to their /99/ /$02.25 FASEB substrate specificity and structure, members of the MMP gene family can be classified into subgroups of collagenases, stromelysins, gelatinases, membranetype MMPs, and other MMPs (see Fig. 1). In general, MMPs contain a signal peptide, a propeptide, a catalytic domain with the highly conserved zinc binding site, and a hemopexin-like domain linked to the catalytic domain by a hinge region (Fig. 1). In addition, gelatinase-a (MMP-2) and gelatinase-b (MMP-9) contain fibronectin type II inserts within the catalytic domain and MT-MMPs contain a transmembrane domain in the carboxyl-terminal end of the hemopexin-like domain (Fig. 1). The hemopexin domain is absent in the smallest MMP, matrilysin (MMP-7). The substrate specificity of distinct MMPs has been determined by their ability to degrade different components of ECM in vitro. However, direct evidence for the proteolytic activity of MMPs in vivo is still limited. Collagenase-1 (MMP-1), collagenase-2 (MMP-8), and collagenase-3 (MMP-13) are the principal secreted neutral proteinases capable of initiating degradation of native fibrillar collagens of types I, II, III, and V, and apparently play a crucial role in degradation of collagenous ECM in various physiological and pathological situations (see refs 1 3). They all cleave fibrillar collagens at a specific site, resulting in generation of amino-terminal 3/4 and carboxylterminal 1/4 fragments, which then rapidly denature in body temperature and are further degraded by other MMPs, e.g., gelatinases (see refs 1 3). Expression of collagenase-1 (MMP-1) in vivo is seen in areas of rapid ECM remodeling both in physiological and 1 Correspondence: University of Turku, MediCity Research Laboratory, Tykistökatu 6, FIN Turku, Finland. jukwes@utu.fi 2 Abbreviations: ECM, extracellular matrix; IL, interleukin; MAPK, mitogen-activated protein kinase; MMPs, matrix metalloproteinases; MMP-1, collagenase-1; MMP-2, gelatinase-a; MMP-3, stromelysin-1; MMP-7, matrilysin; MMP-8, collagenase-2; MMP-9, gelatinase-b; MMP-10, stromelysin-2; MMP-11, stromelysin-; MMP-12, metalloelastase; MMP-13, collagenase-3; MMP-20, enamelysin; PKC, protein kinase C; SCC, squamous cell carcinoma; TGF, transforming growth factor; TIMPs, tissue inhibitors of metalloproteinases; TNF, tumor necrosis factor. 781

2 Figure 1. Structure of human matrix metalloproteinases. Molecular weights of active and latent forms are shown (adapted from ref 1). pathological situations. MMP-1 is expressed by several types of cells including fibroblasts, keratinocytes, chondrocytes, monocytes and macrophages, hepatocytes, and a variety of tumor cells. Substrates for MMP-1 include collagens of types I, II, III, VII, VIII, X, aggrecan, serpins, and 2-macroglobulin (1 3). Collagenase-2 is synthesized by polymorphonuclear leukocytes, stored in their secretory granules, and released in response to the appropriate stimulus. In addition, expression of MMP-8 has been observed in chondrocytes, synovial fibroblasts, and endothelial cells (6). MMP-13 was originally cloned from human breast carcinoma tissue; compared with other collagenases, its substrate specificity is exceptionally wide, including fibrillar collagens of types I, II, III, and XI, basement membrane and cartilage collagen types IV and X, collagen type IX, gelatin, laminin, tenascin, aggrecan, and fibronectin (7). Apparently due to its ability to degrade a wide range of ECM components, the physiological expression of MMP-13 is limited to situations in which rapid and effective remodeling of collagenous ECM is required, i.e., fetal bone development and postnatal bone remodeling (8, 9). On the other hand, MMP-13 is expressed at sites of excessive degradation of collagenous ECM in osteoarthritic cartilage (9), rheumatoid synovium (9, 10), chronic cutaneous ulcers (11), intestinal ulcerations (12), and periodontitis (13), as well as in malignant tumors such as breast carcinomas (14 16), squamous cell carcinomas (SCCs) of the head/neck and vulva (17 19), cutaneous basal cell carcinomas (18), and chondrosarcomas (20). The stromelysin subgroup contains stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and two other MMPs with similar substrate specificities that are less closely related structurally: MMP-7 and macrophage metalloelastase (MMP-12). MMP-3 and MMP-10 are expressed by fibroblastic cells and by normal and transformed squamous epithelial cells (18, 19). Stromelysins degrade basement membrane components, type IV collagen, nidogen, and fibronectin; both matrilysin and macrophage metalloelastase have the ability to degrade elastin (1 3). MMP-2 (72 kda gelatinase-a) is expressed by a variety of normal and transformed cells. MMP-9 (92 kda gelatinase-b) is produced by keratinocytes, monocytes, alveolar macrophages, PMN leukocytes, and a large variety of malignant cells. In addition to the capacity of MMP-2 and MMP-9 to degrade gelatin, laminin, and nidogen, MMP-2 has also been reported to degrade native type I collagen and proteolytically activate MMP-9 and MMP-13 (4, 21). The first membrane-type MMP (MT1-MMP) (MMP-14) was cloned from invasive lung cancer cells (22) and revealed a typical five-domain modular structure resembling collagenases and stromelysins; it also contains an RXXR domain susceptible to intracellular proteolytic activation by furin (23), an additional short carboxyl-terminal transmembrane domain, and intracellular domain. Three additional MT-MMPs have been cloned: MT2-MMP (MMP-15); MT3-MMP (MMP-16), and MT4-MMP (MMP-17) (1 3). Active MT1-MMP serves as a cell membrane receptor for the complex formed of latent MMP-2 (prommp-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2); at least MT1-MMP and MT2-MMP have been shown to proteolytically activate prommp-2 at the cell surface (21). In addition to activation of MMPs, MT1-MMP and MT2-MMP degrade type I, II, and III collagens, gelatin, fibronectin, laminin, vitronectin, and aggrecan. In vivo, MT1- MMP is expressed both in stromal fibroblasts adjacent to invasive tumor and in malignant epithelium (19, 24). The cdna of stromelysin-3 (MMP-11) was cloned from invasive breast cancer tissue. The predicted structure of MMP-11 resembles that of other stromelysins and collagenases. It has not been shown to degrade any ECM component, but it degrades serine proteinase inhibitors 1-proteinase inhibitor and 1-antitrypsin (1 3). Cossins et al. (25) cloned cdna of a novel MMP from a human mammary gland, 782 Vol. 13 May 1999 The FASEB Journal WESTERMARCK AND KÄHÄRI

3 which is identical to MMP-19 cloned from liver (26). The predicted protein structure of MMP-19 has the closest identity with MMP-1, -3, -10, and -11. However, the presence of a carboxyl-terminal arginine rich domain suggests that this MMP belongs to a new group of MMPs (19, 26). The ability of MMP-19 to degrade native ECM components is not known. Enamelysin (MMP-20), cloned from the odontoblastic cells, is the latest member of the human MMP family (27). The expression of enamelysin has been observed only in dental tissues, which together with its ability to degrade dental amelogenin suggests a specific role for enamelysin in the remodeling of dental enamel. Most MMPs are secreted as latent precursors (zymogens) that are proteolytically activated in the extracellular space, with the exception of MMP-11 and MT1-MMP, which are activated prior to secretion by Golgi-associated, furin-like proteases (see refs 1 3, 21). 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. The TIMP gene family consists of four structurally related members, TIMP-1, -2, -3, and -4, which show 30 to 40% identity at the amino acid level and possess 12 conserved cysteine residues (28). TIMP-1, -2, and -4 are secreted in soluble form whereas TIMP-3 is associated with ECM. TIMPs have biological effects that extend beyond their role as inhibitors of MMP activity. They induce changes in cell morphology, stimulate growth of several cell types, and are involved in steroidogenesis and germcell development of both sexes (see ref 28). TIMP-1 and -3 are antiangiogenic (28); TIMP-2 is also involved in activation of MMP-2 (see ref 21). EXPRESSION OF MMPs IN CANCER Tumor invasion is a multistage process in which cellular motility is associated with controlled proteolysis and that involves interactions between tumor cells and the ECM. During this process, malignantly transformed cells detach from the primary tumor, migrate, and cross structural barriers, including basement membranes and surrounding stromal collagenous ECM. Degradation of stromal ECM is also considered essential in tumor-induced angiogenesis. The proposed role of MMPs in tumor invasion is mainly based on the observations of high-level expression of distinct MMPs in invasive malignant tumors (for reviews, see refs 4, 5), but evidence for the activity of distinct MMPs in tumor tissues in vivo is limited. However, there is recent direct evidence that MMPs play an important role in tumor invasion and progression. For example, increased expression REGULATION OF MMP EXPRESSION IN TUMOR INVASION of TIMPs by either host or tumor cells results in reduced invasion and metastatic capacity of transformed cells (29, 30). In addition, it has been shown that MMP activity is required for increased motility of the epithelial cells (31) and for growth of metastasized tumor cells (32). Furthermore, MMPs have been shown to play an essential role in angiogenesis and tumor cell intravasation (33, 34), both of which are required for tumor cell growth and metastasis. Additional evidence for the role of MMPs in invasion and growth of tumors has recently been provided by MMP knockout mice. Mice lacking MMP-7 showed reduction in intestinal tumorigenesis (35), and MMP-2-deficient mice show reduced angiogenesis and tumor progression (36). MMP-11 knockout mice show reduced tumorigenesis in response to chemical mutagenesis (37). The expression of MMPs in tumors is regulated in a paracrine manner by growth factors and cytokines secreted by tumor infiltrating inflammatory cells as well as by tumor or stromal cells; recent studies have suggested continuous cross talk between tumor cells, stromal cells, and inflammatory cells during the invasion process (4, 5, 16, 19). Accordingly, tumor cell-derived factors that increase the expression of several MMPs in stromal cells have been purified. One of these factors, EMMPRIN, was originally identified from the cell membrane of LX-1 carcinoma cells and has been shown to induce the expression of MMP-1, MMP-3, and MMP-2 by fibroblasts (38). Furthermore, the ability of MMPs to degrade and inactivate interleukin-1 (IL-1 ) (39) and cleave the tumor necrosis factor (TNF- ) precursor to a biologically active form (40), as well as the capacity of TIMP-3 to inhibit activation of TNF- (41), indicate that besides degrading ECM components, MMPs and TIMPs may also regulate the availability and activity of inflammatory cytokines at the site of the tumor invasion. The current view about the role of stromal component in tumor cell invasion points out that stroma actually participates in tumor progression and that stromal fibroblasts can cause tumorigenic conversion of epithelial cells (42, 43). Evidence for the interplay between tumor cells and the stroma has also been provided from findings that in the early stages of epithelial malignancy, when the basement membrane is still intact, angiogenesis is observed in stroma (44). In many tumors, stromal cells also actively express ECM components, e.g., type I collagen, indicating that the surrounding stromal compartment of malignant tumors is undergoing extensive tissue remodeling and that many tumors induce formation of a specific tumor stroma present both at the primary and metastatic sites (45, 46). As mentioned above, tumor cell invasion is a multistep process that involves tumor cells, fibro- 783

4 blasts, and inflammatory cells, and all these cell types have been shown to produce ECM-degrading proteases (4, 5). In vivo expression of MMPs is localized to both tumor and stromal cells at the invading margin of the tumor, providing a mechanism for highly concerted degradation of ECM (4, 5, 15, 19). Increased expression of MMP-1 has been observed in lung carcinomas (47, 48), squamous cell carcinomas of the head and neck (17), and colorectal tumors, in which expression of MMP-1 correlates with poor prognosis (49). In all these tumors the most abundant expression of MMP-1 was observed in the stromal cells, but significant expression of MMP-1 has also been observed in stromal or intratumoral endothelial cells. Expression of MMP-1 was also observed in cancer cells located at the invasive edge of head and neck squamous cell carcinoma (SCC) cells adjacent to stromal cells, whereas the expression of MMP-13 is predominantly confined to tumor cells at the invading margin; in a subset of tumors, significant expression of MMP-13 was also observed in stromal fibroblasts (17). This is in contrast to breast carcinomas, in which MMP-13 is expressed primarily by stromal fibroblasts (15, 16). Both gelatinases MMP-2 and MMP-9 are abundantly expressed in various malignant tumors (4, 5, 15). MMP-9 is mainly expressed by malignant cells, but also by inflammatory cells, including tissue macrophages and eosinophils (15, 19, 50). Immunohistochemical analysis of the expression of MMP-2 has shown increased abundance of the immunoreactive enzyme at the neoplastic epithelium of breast, colon, and gastric adenocarcinomas; however, in contrast to the immunohistochemical results, in situ hybridizations show an increase in MMP-2 mrna mainly in the stromal component of tumors. This discrepancy may be explained by recent findings that latent MMP-2 binds to the cell surface of malignant cells by interaction with cell membrane-associated MT1- MMP (22, 23). Furthermore, activated MMP-2 binds to the cell surface of invasive melanoma and endothelial cells via v 3 integrin (51). Recent findings that both MMP-2 and MT1-MMP mrnas are also expressed by stromal fibroblasts of human vulvar, breast, lung, and head and neck carcinomas (15, 19, 52) suggest that MT1-MMP binds to and activates MMP-2 at the cell surface of fibroblasts. In SCCs, MMP-2 and MMP-9 may also be activated by MMP-13, which in turn may be activated by MT1-MMP (19, 21). These kinds of activation cascades may be required to ensure cooperation between tumor and stromal cells; they greatly enhance cell surface localized proteolytic activity in vivo and indicate that selective inhibition of one MMP may be sufficient to block activation cascade and ECM degradation during tumor invasion. In summary, enhanced expression of different MMPs in cancer tissue and experimental in vitro data indicate an essential role for MMPs in tumor cell invasion. Although the expression of MMPs in malignancies has been studied widely, the specific role of distinct MMPs in the progression of cancer may be more complex than has been assumed. For example, it has recently been shown that MMP-3, MMP-7, MMP-9, and MMP-12 can generate angiostatin from plasminogen, indicating that their expression in peritumoral area may in fact serve to limit angiogenesis and thereby inhibit tumor growth and invasion in vivo (53 55). This recent view about the role of stromal cells in the progression of cancer cell growth and metastasis is particularly interesting, and additional studies about the regulation of MMP gene expression and activity in in vivo malignancies are clearly needed to understand the role and regulation of MMPs in tumor cell invasion. Although TIMPs potently inactivate MMPs in vitro, the role of TIMPs in the regulation of MMP activity and cancer progression in vivo is unclear. Earlier studies have shown decreased expression of TIMPs at the site of tumor invasion, but a positive correlation between TIMP and MMP expression and poor prognosis of malignant tumors has recently been shown (see ref 28). The contradictory roles of TIMPs may result from the bimodal function of TIMPs as inhibitors of MMPs, but also as key players in the cell surface-targeted MMP activation cascades (28). Another aspect to the role of TIMPs and MMPs in tumor growth is the observation that overexpression of TIMP-3 can induce apoptosis in normal and malignant cells (30, 56, 57), providing evidence that MMP activity in general may be important not only for invasion, but also for the survival of malignant cells. REGULATION OF MMP GENE EXPRESSION Expression of most MMPs is normally low in tissues and is induced when remodeling of ECM is required. MMP gene expression is primarily regulated at the transcriptional level, but there is also evidence about modulation of mrna stability in response to growth factors and cytokines (see refs 5, 42, 43). The promoter regions of inducible MMP genes (MMP-1, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, MMP- 13) show remarkable conservation of regulatory elements (Fig. 2), and their expression is induced by growth factors, cytokines, and other environmental factors such as contact to ECM (1 3, 58, 59). Increased expression of other MMPs, the promoter regions of which do not contain conserved cis elements (MMP-2, MMP-11), has also been observed in malignancies, indicating overlapping mechanisms in the regulation of the expression of these genes. 784 Vol. 13 May 1999 The FASEB Journal WESTERMARCK AND KÄHÄRI

5 Figure 2. Regulatory elements of promoter regions of human MMP genes. Modified from ref 59. Boxes indicate the following cis elements: AP-1, activator protein-1; PEA3, polyoma enhancer A binding protein-3; TIE, TGF- inhibitory element; GC, Sp-1 binding site; SBE, STAT binding element; C/EBP-, CCAAT/enhancer binding protein- ; OSE-2, osteoblast-specific element-2; SPRE, stromelysin-1 PDGF-responsive element; TRF, octamer binding protein; Sil, silencer sequence; NF- B, nuclear factor- B; NF-1, nuclear factor-1; RARE, retinoic acid responsive element. Role of AP-1 transcription factors in the regulation of MMP gene expression A single AP-1 element, which binds members of the AP-1 transcription factor family, is found at approximately 70 in the promoter region of each inducible MMP gene (Fig. 2). AP-1 transcription factors are leucine zipper proteins that bind to a consensus DNA sequence (5 -TGAG/CTCA-3 ) as a dimeric complex (60, 61). Three distinct members of the Jun family (c-jun, JunB, and JunD) and four members of the Fos family (cfos, Fra-1, Fra-2, and FosB) have been characterized. Members of the Jun family can bind to DNA either as Jun/Jun homodimers or Jun/Fos heterodimers, whereas Fos proteins do not form homodimers and bind DNA in the absence of Jun (60, 61). Different AP-1 dimers bind DNA with different affinities, which is thought to be at least partly responsible for the diverse biological effects of distinct AP-1 complexes. The proximal AP-1 element located between 72 and 66 plays a major role in the transcriptional regulation of MMP-1 gene expression, as shown by the results that mutation of this element dramatically reduces the basal activity and responsiveness of the MMP-1 promoter to external stimuli (62 64). Although the importance of additional AP-1 elements found in the promoters of MMP-1, -3, and -9 is not clear, it has been shown that in the rabbit MMP-1 promoter, they bind Fos and Jun containing AP-1 dimers and partially mediate the effect of phorbol ester (59). Several cotransfection studies have shown that overexpression of Jun and Fos proteins enhance REGULATION OF MMP EXPRESSION IN TUMOR INVASION MMP-1 promoter activity and, in contrast, that expression of antisense mrnas for c-jun abrogates the induction of MMP-1 gene expression. Furthermore, it has been shown that simultaneous induction of c-jun and junb mrnas precedes induction of MMP-1 gene expression by several types of stimuli (for references, see refs 1, 58, 59). c-jun is capable of inducing the minimal MMP-1 promoter activity as a Jun/Jun homodimer, whereas induction of minimal (76 bp) MMP-1 promoter by JunB requires the presence of several AP-1 elements or simultaneous expression of c-fos (65, 66). However, in HT-1080 fibrosarcoma cells, in which c-jun expression is not induced by tumor promoter okadaic acid, JunB containing AP-1 complexes mediate the activation of the full-length (3.8 kb) MMP-1 promoter (67). We have also observed that in NIH-3T3 fibroblasts, in which overexpression of JunB alone does not stimulate minimal MMP-1 promoter, JunB is a potent activator of full-length MMP-1 promoter (64). These findings indicate that c-jun is an independent activator of MMP-1 gene expression, whereas transactivation of MMP-1 promoter by JunB, and possibly by other AP-1 transcription factors, is dependent on the interaction with other transcription factors binding to additional regulatory cis-elements in the 5 flanking region of the MMP-1 gene. Recent studies with c-fos knockout mice reveal that c-fos is required for malignant and invasive progression of skin papillomas and for induction of mouse MMP-3 and MMP-13 gene expression by platelet-derived growth factors and epidermal growth factor, but not by phorbol ester (68, 69). In 785

6 addition, overexpression of c-fos in transgenic mice under an interferon-inducible promoter was shown to induce expression of mouse MMP-13 in thymus, spleen, and predominantly in bone, revealing that the capacity of c-fos to activate mouse MMP-13 gene expression in vivo is cell type specific (70). The finding that neither mouse MMP-9, MMP-3, nor MMP-10 expression was affected by c-fos overexpression suggests that c-fos differently regulates expression of distinct MMP genes in vivo and that the AP-1 element alone does not determine the inducibility of the MMP promoter by c-fos in vivo (70). Little is known about the regulation of AP-1 activity in malignant tumors in vivo. Increased expression of AP-1 genes has been reported during growth of malignant tumors, but there is no consistent pattern of AP-1 complexes that would serve as a marker for increased invasion or malignancy. Decreased expression of c-jun, junb, and c-fos genes was observed in human lung carcinomas as compared with normal tissue (71). As the expression of different AP-1 components at sites of MMP expression during tumor invasion is not known, determination of the specific AP-1 complex pattern responsible for induction of MMP gene expression in vivo would be important in developing specific approaches to prevent tumor invasion and metastasis. ETS transcription factors in the regulation of MMP expression Conserved PEA3 elements that bind members of ETS transcription factors have also been found in all inducible MMP promoters; with the exception of the MMP-12 promoter, they are located adjacent to at least one AP-1 element (Fig. 2). ETS transcription factors are helix-turn-helix proteins that share a modular domain structure characterized by a highly conserved ETS domain, which recognizes the purine rich PEA3 element A/CGGAA/T (72). Although ETS proteins have been shown to trans-activate artificial promoter constructs containing only the PEA3 element, ETS proteins do not usually dimerize and bind to DNA alone, but prefer to form complexes with other transcription factors, e.g., AP-1, for which they function as coactivators (72, 73). Expression of ETS-1 colocalizes with several MMPs to the stromal fibroblasts adjacent to the invasive edge of several types of tumors (47, 48, 74). ETS-1 expression has also been detected during angiogenesis in human embryos, in granulation tissue, and during tumor vascularization (75). Furthermore, overexpression of the ETS-related transcription factor E1A-F, which activates MMP-1, MMP-3, and MMP-9 promoters, has been shown to induce an invasive phenotype in MCF-7 cells (76). In addition, overexpression of ETS-1, ETS-2, and PEA3 enhances the activity of full-length MMP-1, MMP-3 and MMP-9 promoters (64, 77, 78). The functional interplay between AP-1 and ETS factors in the regulation of MMP gene expression has been recently shown by us and others. Our results indicate that expression of structurally distinct ETS factors (ETS-1, ERGB/Fli-1, PU.1) differentially modulate AP-1-dependent MMP-1 promoter activation (64). ETS-1 markedly enhanced the stimulation of MMP-1 promoter by both c-jun and JunB, whereas ERGB/Fli-1 alone does not regulate MMP-1 gene expression and potentiates only the effect of c-jun. In contrast, PU.1 overexpression potently blocked activation of MMP-1 promoter by overexpression of c-jun and JunB or by stimulation by phorbol ester and okadaic acid (64). ETS factor Erg was recently shown to physically interact and cooperate with Jun/Fos in the upregulation of MMP-1 promoter activity (61). These results indicate that in vivo interactions between distinct transcription factors may modulate the response of MMP promoters in situations where simultaneous induction of the expression of both ETS and AP-1 factors occurs, such as tumor cell growth and invasion. MITOGEN-ACTIVATED PROTEIN KINASES DNA binding and trans-activation capacity of both AP-1 and ETS transcription factors are regulated by phosphorylation by mitogen-activated protein kinases (MAPKs), serine/threonine kinases that mediate signals from cell membrane receptors triggered by growth factors, cytokines, hormones, and cell-cell and cell-matrix interactions (for reviews, see refs 79 81). Three MAPK pathways have been characterized in detail (see Fig. 3). The ERK1,2 pathway (Ras- Raf- MEK1,2- ERK1,2) is activated by a large variety of mitogens and by phorbol esters, whereas the JNK/SAPK (MEKK1 4- MKK4/MKK7- JNK/SAPKs) and p38 (TAK1- MKK3/MKK6- p38s) pathways are mainly stimulated by environmental stress and inflammatory cytokines (Fig. 4). Other stress-activated MAPKs, including ERK5 and ERK6, have been characterized recently (80, 81). It is thought that the balance between distinct MAPK pathways regulates cell growth, differentiation, survival, and death. Constitutively active mutants of Raf-1 and MEK1 have been shown to transform fibroblasts in vitro (82, 83), and in vivo activation of ERK1/2 pathway has been observed in renal and breast carcinomas (84, 85). Furthermore, the full transformation capacity of oncogenic Ras has been shown to require activation of stress-activated MAPKs, indicating functional interplay between mitogen and stress-activated MAPK pathways during the transformation process (86). 786 Vol. 13 May 1999 The FASEB Journal WESTERMARCK AND KÄHÄRI

7 Figure 3. Schematic presentation of MAPK pathways. For details, see refs 80, 81. Kinases of ERK1,2, JNK/SAPK, and p38 pathways are underlined. Transcriptional activity and c-jun protein stability is increased by phosphorylation of serines 63 and 73 by JNK/SAPK, whereas phosphorylation of threonines 91 and 93 leads to enhanced c-jun DNA binding (see ref 79). Activation of ERK1,2 was recently shown to induce c-jun expression and phosphorylation, indicating cross talk between ERK1,2 and JNK/SAPK pathways in the regulation of c-jun activity (87). Although p38 MAPK does not directly phosphorylate c-jun, it contributes to enhanced AP-1 activity by activation of transcription factors (ATF-2, MEF2C, Elk-1, SAP-1) that up-regulate c-jun and c-fos promoter activities (79). Phosphorylation of c-fos by p90 rsk and ERK2 stabilizes the c-fos protein and results in increased trans-activation and transformation capacity of c-fos. Furthermore, the activity and DNA binding of ETS transcription factors SAP-1 and Elk-1, which participate in c-fos promoter activation through serum responsive element, is regulated by ERK1,2, JNK/SAPK, and p38 pathways (80, 81, 88). Activation of Ras has been shown to trans-activate ETS-1 and ETS-2, and ERK1,2 and JNK/SAPK pathways have been shown to coordinately mediate activation of ETS factor PEA3 by Ras (89). Taken together, both AP-1 and ETS transcription factors are subject to regulation by mitogen- and stress-activated MAPK signaling pathways. Recent identification of specific adapter proteins that physically connect kinases of MAPK cascades suggests the existence of highly specific MAPK cascades that are activated in specific situations (90, 91). These signal transduction cascades allow strict control of amplification, feedback, cross talk, and branching of the initial signals triggered from the cell membrane resulting in a precise regulation of gene expression in situations such as tumor cell invasion. Role of MAPKs in the regulation of MMP gene expression Figure 4. Targets for inhibiting MMP expression and activity REGULATION OF MMP EXPRESSION IN TUMOR INVASION As discussed, the role of phosphorylation in the regulation of the AP-1 and ETS transcription factors is fairly well established. However, the role of distinct phosphorylation-dependent signaling pathways in the regulation of MMP gene expression has been determined only recently. Increased serine/threonine phosphorylation as a result of inhibition of serine/threonine phosphatase PP2A by tumor promoter okadaic acid induces expression of MMP-1 and MMP-3 at transcriptional level (67, 92). Evi- 787

8 dence for the role of MAPKs in the transcriptional regulation of MMP gene expression was shown by results indicating that blocking the ERK pathway abrogated Ras, serum, and TPA-elicited induction of a minimal promoter construct containing 72 bp of MMP-1 promoter, harboring the proximal AP-1 element (93). Furthermore, overexpression of dominant negative MEK was shown to block insulinelicited induction of a reporter construct under the control of an AP-1 element from the MMP-1 promoter (94). Recently, overexpression of dominant negative Raf-1 was shown to block oncostatin M elicited induction of a promoter construct containing copies of oligonucleotides encompassing the AP-1 and an earlier unidentified STAT binding site of the MMP-1 promoter in front of a heterologous promoter (95). Protein kinase C (PKC) has been shown to activate ERK1,2 signaling pathway, and it was recently shown that selective overexpression of PKC isoforms PKC, PKC, and PKC activate the full-length MMP-1 promoter (96). Taken together, these recent results indicate that mitogen-activated ERK1,2 pathway would be the major activator of MMP-1 gene expression. In normal skin fibroblasts, enhancement of MMP-1 expression by TNF and the IL-1-induced lipid second messenger ceramide is mediated by coordinate activation of ERK1,2, JNK/SAPK, and p38 MAPK pathways (97). Our recent results also show that activity of ERK1,2, JNK/SAPK, and particularly p38, is required for induction of MMP-1 expression and promoter activity by the tumor promoter okadaic acid (98). The involvement of stressactivated MAPK pathways in the regulation of MMP gene expression is also supported by findings that inhibition of p38 activity by specific chemical inhibitor SB blocks the IL-1 elicited induction of MMP-1 and MMP-3 expression in human fibroblasts and vascular endothelial cells (99). Further evidence for the importance of stress-activated pathways in migration and invasion of fibroblasts is provided in our findings showing that induction of MMP-13 expression by contact to 3-dimensional collagen is dependent on the activity of p38 MAPK (100). The role of MAPKs in regulation of MMP expression has also been examined in malignant cells. In addition to MMP-1 and MMP-3, increased transcriptional activity of the MMP-9 promoter in Ras-transformed OVCAR-3 cells was shown to be mediated by MAP kinases, but blocking the ERK pathway was not sufficient to abrogate the signal, suggesting a role for stress-activated MAPK pathways as mediators of effect of Ras (78). On the other hand, induction of MMP-9 promoter activity by oncogenic Ras in SCC cells was abrogated by blocking either the ERK1,2 or JNK/SAPK pathways, indicating cell-specific differences in the role of distinct MAPKs in the regulation of MMP promoter activity (101). The first evidence for the role of MAPKs in the regulation of cancer cell invasion was recently demonstrated by showing that inhibition of p38 activity by SB blocks phorbol ester-elicited induction of MMP-9 expression by SCC cells as well as their invasion through Matrigel (102). Taken together, these results indicate cooperation of multiple MAPK pathways in the regulation of MMP transcription in response to different signals. In addition to growth factors and cytokines, invasive cancer cells and stromal cells at the invading edge of the tumor are under physical stress, such as hypoxia and hyperthermia. Based on the current view on the importance of stress-activated MAPKs in the regulation of MMP expression, it could be assumed that activation of AP-1 and ETS transcription factors by these MAPKs is responsible for maximal activation of MMP expression in stress conditions present in the tumor and that, in addition to its role in the gene regulation, activation of ERK1,2 pathway may be important for survival of cancer cells by protecting them from the programmed cell death caused by stress-induced JNK/SAPK activation (103, 104). It will be of great interest to determine whether invasion specific MAPK AP-1/ETS routes exists in vivo and whether inhibition of these pathways could serve as a therapeutic target to specifically prevent tumor cell invasion and metastasis. MMPs AS TARGETS TO INHIBIT TUMOR CELL INVASION As discussed, MMP expression during cancer cell invasion is regulated by multiple extracellular factors including cytokines, growth factors, and interactions with adjacent cells and ECM. Every level of regulation of MMP expression and activity can be considered as a target for therapeutic intervention (see Fig. 4). In this respect, it is important to understand the mechanisms of the regulation of MMP expression in malignant tumors in vivo. For example, it was recently shown that overexpression of transforming growth factor (TGF- ) under keratin 6 promoter inhibited formation of benign skin tumors in transgenic mice but enhanced progression to invasive carcinomas in mouse skin multistage carcinogenesis model (105), indicating that different cell populations may alter their response to factors such as TGF-. Furthermore, detailed characterization of growth factor and cytokine receptors responsible for activation of signaling pathways would help in designing receptor antagonists and neutralizing antibodies to prevent the action of these factors. For example, we have previously shown that TNF- stim- 788 Vol. 13 May 1999 The FASEB Journal WESTERMARCK AND KÄHÄRI

9 ulates MMP-1 expression in fibroblasts through 55 kda TNF receptor (106), suggesting targeting of this receptor pathway, as has been shown in organ cultures (107) Regarding inhibition of MMP expression at the level of kinase pathways, it is possible that selective chemical inhibitors for distinct signaling pathways (e.g., MAPK, PKC) will soon be available for initial clinical trials. It is therefore important to identify growth and/or invasion specific signaling cascades that could serve as targets for chemical inhibitors or dominant negative kinase mutants and antisense oligonucleotides (108). Overexpression of selective dual-specificity MAPK phosphatases shown to prevent MMP promoter activation (97, 98, 101) could also be used as a novel strategy to prevent activation of AP-1 and ETS transcription factors and MMP promoters in vivo. Interactions between members of different transcription factors increases capability of fine-tuning the transcriptional regulation of MMP promoter activity. It is possible that constitutive expression of specific transcription factors as a result of transformation may modulate the response of MMP promoter to extracellular signals. For example, overexpression of several ETS factors has been observed in hematological malignancies as a result of proviral insertion into the ETS gene or by chromosomal translocation, possibly resulting in potentiation of AP-1 induced up-regulation of MMP expression, as shown recently in vitro (64, 77). It was recently shown that peritoneally injected, double-stranded AP-1 oligomers potently prevent AP-1 dependent activation of MMP gene expression in mouse arthrosis model (109), indicating that it is possible to block transcriptional activation of MMP gene expression in vivo by eliminating binding of the transcription factors. Also, novel retinoids that potently inhibit cell proliferation have been shown to selectively abrogate AP-1 dependent gene expression (110). Besides the treatment strategies targeted to inhibit MMP promoter activation, degradation of MMP mrna by antisense RNA or ribozyme techniques may also provide efficient and specific tool to prevent tumor cell invasion as shown in vitro and in vivo (111, 112). Inactivation of MMPs in pericellular space by either overexpression of TIMPs or synthetic MMP inhibitors may prevent initiation of MMP activation cascades and excessive ECM degradation. Several small molecule MMP inhibitors are currently in phase I/II clinical trials, and a hydroxamate MMP inhibitor, marimastat, is already entering phase III trials in a number of solid tumors (113, 114). 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