Int. J. Cancer: 65,717-722 (1996) 0 1996 Wiley-Liss, Inc. q b Publicaion of the InternationalUnion Aqainst Cancer PuDlcal on de I UP on InletnationdieContre le Cancer BIOLOGY OF TUMOR CELL INVASION: INTERPLAY OF CELL ADHESION AND MATRIX DEGRADATION Jyrki HEINO~ Medic& Research Laboratoiy and Department of Medical Biochemistry, UniversiQ of Turku, FIN-20520 Turku, Finland. Invasion of malignant cells requires altered cellular interaction with extracellular matrix. Integrin-typecell adhesion receptors play an important role in this process. Integrin-related cell biological phenomena explain cancer cell migration, and recent developments in the field have made it possible to propose that integrins are also involved in the penetration through basement membranes and other molecular barriers. Finally, malignant melanoma has been used here as an example to speculate on the function of each integrin in light of information from different experimental models. 5 1996 Wiley-Liss,Inc. lNTEGRlN FAMILY OF CELL AL)HESION RECEPTORS Anchored cells use members of the integrin family for adhesion to the structural proteins of the extracellular matrix (Ruoslahti, 1991; Hynes, 1992 and references therein). Sixteen a and 8 p integrin subunits can form at least 22 different, non-covalently linked a/@type heterodimers. Fifteen of them arc rcceptors for matrix proteins (Fig. 1). P l is thc most promiscuous of all integrin subunits, participating in 10 diffcrent complexes. Integrins alp1 and a 2 p l are the major cell surface collagen receptors. Both a1 and a2 subunits contain a similar, about 200 amino acids long, ligand-binding I-domain. The I-domain in a M integrin has been isolated and its 3-dimensional structure has been determined by X-ray crystallography (LCCet al., 1995). The revealed Mg’+-depcndcnt ligand-binding site has been named MIDAS (Lee et al., 1995). Collagen binding by a l p 1 and a2pl integrins is also dependent on the presence of Mg2+ but cannot be supported by Ca2+ (Crzesiak et al., 1992). Integrin a l p 1 may prefer type IV collagen over type I collagen, and it is a receptor for laininin-1. Integrin a2pl can also bind laminin-1 and tenascin (Sriramarao et al., 1993). Integrin a3pl is a receptor for laminin-5, and in different experimental modcls it has shown binding to laminin-1, type I collagen and fibronectin. Integrins a4Pl and a5pl are fibronectin receptors. They recognize, however, different domains in fibronectin. The protein sequences they bind to can be reduced to 3 or 4 amino acids; LDVP and RGD for a 4 p l and asp1 integrins, respectively. Integrins a 6 p l and a7pl are receptors for laminin-1. Integrin a8pl is a fibronectin (Miiller et al., 1995) and a vitronectin reccptor, and rx9pl intcgrin binds to tcnascin (Yokosaki et u / . , 1994). Another integrin subunit found in many different matrix receptors is aV. Receptors containing ciV subunit can recognize the RGD sequence in ligand molecules. In complex with (31 or p3 it is a receptor for both fibronectin and vitronectin. Integrin aVp3 can also bind, e.g., bone sialoprotein and thrombospondin. Integrins ciVp.5 and aVP8 are specific for vitronectin (Nishimura et aZ., 1994) and aVP6 for fibronectin. Other intcgrintype matrix rcceptors arc a6P4, which binds members of the laminin family and is a component of the hemidesmosomes, and ci4p7, which is a similar fibronectin receptor to a4pl integrin. Integrin ciIIbp3 is an RGD-dependent platelet receptor for fibrinogen, fibronectin and vitronectin. Integrins anchor cells to their surroundings, but they are also essential for cell migration and probably for invasion of tumor cells (for review, see Heino, 1993; Giancotti and Mainiero, 1994). Intracellular domains of integrin subunits are connected to cytoskeletal proteins and signal transduction pathways. Integrin occupancy leads to receptor targeting to focal contacts and accumulation of cytoskeletal proteins, whereas receptor clustering is needed for phosphorylation of signal transduction proteins (Miyamoto et al., 1995). Activation of integrin-mediated signal transduction regulates cell behavior, differentiation and phenotype. Expression of specific genes, including matrix mctalloproteinases (MMPs), is partially regulated by integrin-related signals. Integrins expressed on the cell surface are not necessarily active ligand-binding receptors, but their action can be regulated by intracellular mechanisms. This phenomenon is called “inside-out signaling” and is thought to take place by altering the conformation of extracellular domains through the cytoplasmic domains (Ginsberg Pt a[., 1992). INTEGKINS AND MMPS MMPs are a family of zinc-dependent enzymes containing at least 11 members, including collagenases (MMP-1, MMP-8), gclatinascs (MMP-2, MMP-9) and stromelysins (MMP-3-7). Two novel family members have becn described: membranetype matrix metalloproteinase (MT-MMP) and collagenase-3 (reviewed by Birkedal-Hansen, 1995). MMPs can degrade all components of the extracellular matrix, and they are probably essential for cancer cell invasion and formation of metastasis. Many types of malignant cell can produce MMPs, but they might also induce stromal cells in different tumors to produce MMPs. The tissuc inhibitors of metalloproteinases (TIMPs) are specific, naturally occurring inhibitors of MMPs. Several lines of study suggest that the MMP/TIMP ratio is critical for cell invasion (Ray and Stetler-Stevenson, 1994). Cell morphology and the presence of certain matrix molecules can regulate the expression of MMPs. Recent studies have shown that integrin-type ccll adhesion rcccptors are mediating thc matrix-generated signals behind this phenomenon (Table I). Awidely used model to study the role of matrix is to culture cells inside a 3-dimensional collagen gel. Under these conditions, the expression of interstitial collagenase (MMP-1) is induced in most cell types (Grinnell, 1994). We have shown that in human osteogenic sarcoma-derived cell lines the induction of MMP-1 in collagen gels is mediated by a 2 p l integrin (Riikonen et at., 1995h). In vivo, during wound healing, human keratinocytes exprcss MMP-1 when in contact with type I collagen but not when located on intact basement membranes (Saarialho-Kere et al., 1993). Similarly, cell cultured keratinocytes express MMP-1 when plated on collagen (Sudbeck et al., 1994). Thus, the cell-collagen contact seems to limit the collagenase expression to take place only in the presence or its substrate. Similar integrin-mediatcd recogntion mechanisms may regulatc the exprcssion of othcr MMPs spatiotemporally. In rabbit synoviocytes, 2 fibronectin receptors, a4pl and a5pl integrins, have opposite effects on the expression of 3 distinct MMPs, MMP-1, stromelysin (MMP-3), and 92 kDa gelatinase (MMP-9). Interaction of a5pl integrin with its target sequence in fibronectin, the RCD-containing cell adhe‘To whom corrcspondence and reprint requests should be sent, at MediCity Research Lahoratory, University of Turku, Tykistokdtu 6A, FIN-20520 Turku, Finland. Fax: (358) 21-633-7000. Received: August 30, 1995 and in revised form October 25,1995 718 HEINO sion domain, stimulates expression of these MMPs (Werb et al., 1989). Integrin a4pl binds to another domain in fibronectin molecules, the CS-1 containing region. This interaction suppresses production of the same MMPs (Huhtala et al., 1995). Functional analyses of the MMP-1 gene have pointed to the Ap-1- and PEA3-responsive DNA sequences as the sites essential for d p l integrin-generated signals (Tremble et al., 1995). Treatment of human keratinocytes with anti-a3 or anti-pl monoclonal antibodies, but not with other antibodies for integrins, induces expression of MMP-9 (Larjava et al., 1993~). Integrin a 3 p l has several putative ligand molecules, and it is not known which one of them might be the natural regulator of MMP-9. In human melanoma cells 72 kDa gelatinase (MMP-2) expression can be regulated by avp3 integrin-mediated signals (Seftor et al., 1992, 1993). Plasminogen and plasminogen activators are components of another proteolytic machinery suggested to have an important function in tumor cell invasion-related matrix degradation. Much less is known about their relationship with cell adhesion receptors. Nip et al. (1995) suggest coordinated expression of aVp3 integrin and urokinase-type plasminogen activator in human melanoma cells. INTEGRINS IN MALIGNANT MELANOMA Cell lines cultured from primary tumors are often the only way to study the function of specific molecules. However, the integrin pattern in these cells sometimes varies significantly F~GURE 1- Receptor heterodimers formed by integrin subunits and acting as matrix receptors. Ligands: FN, fibronectin; VN, vitronectin; LN, laminin; COL, collagen; TN, tenascin. In addition to ligands listed here, some other matrix molecules may bind to integrins (see text for more details). from that in the original tumors. It is, therefore, obvious that all of the findings must be interpreted on the basis of in vivo observations. At the same time, it must be remembered that alterations detected in tumors are not necessarily important for malignant cell phenotype but might only reflect the altered activity of transcription factors regulating a large number of genes. Also, inflammatory cells infiltrating malignant lesions may release cytokines regulating integrin expression (Heino et al., 1989; Santala and Heino, 1991). Furthermore, integrin function is regulated by intracellular signals, and the activation or inactivation of a given receptor might be more important than its number on the cell surface. Finally, cells are often in different stages of tumor progression and only some of them might be available for migration and invasion. Melanoma was selected as an example because several studies describe integrin expression in vivo in different stages of the process leading from cutaneous nevus to malignant melanoma (reviewed by Danen et al., 1995). There is some discrepancy among the published papers, but the reports agree about certain facts. Importantly, most lesions of malignant melanoma seem to contain cells expressing a2p1, a3pl and aVP3 integrins (Albelda et al., 1990; Schadendorf et al., 1993; van Duinen et al., 1994; Danen et al., 1994), suggesting that they might be the key players in cell invasion. Studies on integrin expression in melanoma-derived cell lines have also shown the correlation of a2Pl (IUein et al., 1991a, b; Etoh et al., 1992; Danen et al., 1993) and aVp3 integrin (Gehlsen et al., 1992) expression with aggressive phenotype. Integrins a l p l , a4p1, asp1 and a6pl might also be present in in vivo tumors. Different papers give somewhat variable information about their abundance, but most lesions seem to be negative in that respect (van Duinen et al., 1994). Several studies have compared alterations in integrin expression and tumor progression. Integrin a6pl/a6p4 may be more common in nevi than in malignant melanomas (Natali et al., 1993; van Duinen et al., 1994; Danen et al., 1994), whereas the frequency of a4pl- and a5pl-positive cells might increase (Danen et al., 1994). Integrin aVp3 is not expressed in nevi, unlike a3pl integrin and probably also a2pl integrin. Expression of a2pl integrin seems to be stronger in malignant melanoma than in melanoma in situ (van Duinen et al., 1994; Danen et al., 1994). Expression of aIIbp3 integrin mRNA has been reported in melanoma cell lines (Chang et al., 1992), but the corresponding protein has not been found in vivo (Danen et al., 1994). Similarly, a7pl integrin is expressed in melanoma-derived cell lines (Kramer et al., 1991), whereas less is known about its abundance in vivo. Integrin expression in uveal melanoma seems to be very different from that in cutaneous melanoma (ten Berge el al., 1993). There, a 2 p l and aVp3 integrin-positive cells seem to be rare, whereas d P l and a6p4 integrin-positive cells are TABLE I - REGULATION OF MATKIX METAI.I.OI'KOI~IYASE GENE EXPK€SSIOK BY IKTEGRIN-MEDlhTFD SIGNALS Integrin Inducer a2pl Type I collagen a3pl Anti-a3 MAb, anti-pl MAb Fibronectin, CS-1containing region a4pl a5pl Fibronectin, RGDcontaining region aVp3 Vitronectin, antiaVB3 MAb Cell type M M P ( t or 1) sarcoma (MG-63) Reference (1995b) atinocytes fibroblasts Human melanoma cells (A375M) MMP-2 f Seftor et al. (1992) CELL ADHESION, MATRIX DEGRADATION AND INVASION frequently seen (ten Berge et al., 1993). It is an important possibility that alteration in cellular integrin pattern leads to formation of metastasis to distinct anatomical locations. This is supported by the fact that uveal melanomas metastasize primarily to the liver, unlike cutaneous melanomas. Furthermore, Vink et al. (1993) have measured in vitro binding of different melanoma cell lines on cryostat sections of different organs. They were able to show that a2pl/aVp3 integrinexpressing cells colonized only the lungs, whereas d p l / a 6 p l integrin-positive cells colonized both lungs and extrapulmonary sites. PUTATIVE ROLE OF INDIVIDUAL INTEGRINS IN MELANOMA CELL INVASION In addition to local proliferation, melanoma cells must be able to interact with basement membranes and penetrate through them. After that they migrate through connective tissue stroma to reach lymph or blood vessels. A suggested role of the different integrins in these stages has been summarized in Figure 2. Increasing evidence suggests that specific adhesion receptors might act in a similar way to the tumor suppressor genes, which are down-regulated during transformation. Integrin d p l is the major fibronectin receptor of many cell types. The disappearance of fibronectin from the cell surface is frequently observed during malignant transformation, and the phenomenon has been explained by impaired function (Akiyama et al., 1990) or reduced expression of d p l integrin (Plantefaber and Hynes, 1989). In CHO cells the forced expression of the a5 subunit inhibits the transformed phenotype (Giancotti and Ruoslahti, 1990). Qian et al. (1994) suggest that a 4 p l integrin might suppress the malignant behavior of mouse melanoma cells in vivo. Integrins a4pl and a5pl are usually not detected in nevi, and during the transformation process their expression might be induced (Danen et aL, 1994), indicating that they are not putative tumor suppressors in human melanoma. Integrin a6pl is a laminin receptor and integrin 01604is a component of A 719 hemidesmosomes in basal keratinocytes. Integrin a6 is expressed in most nevi, but according to some reports, it is seen less frequently in malignant melanomas (Natali et al., 1993; van Duinen et al., 1994; Danen et al., 1994). The significance of this observation is not clear. The conclusion is that there is no in vivo evidence that the down-regulation of some specific integrin is required for malignant growth and invasion of nevi cells. At early stages, melanoma cells are operating in the same molecular environment as keratinocytes. Thus, it is interesting to know which integrins are needed by keratinocytes in wound healing-related migration. Normal basal keratinocytes express a2p1, a 3 p l and a6p4 integrins, but when activated a5 and aV integrin subunits are also induced (Larjava et al., 1993b). Indeed, the same integrin subunits are expressed in malignant melanomas and not in nevi. Thus, expression of these 2 integrins alone might explain the increased mobility of melanoma cells. Integrin aV may be required for melanoma cell proliferation in vivo (Felding-Habermann et al., 1992u), and it rescues melanoma cells from apoptosis in 3-dimensional dermal collagen (Montgomery et al., 1994). Cell adhesion-triggered signal transduction may regulate pathways containing Ras, mitogenactivated protein kinase (MAPK, Schlaepfer et al., 1994) and cyclin A (Guadagno et al., 1993). It is not known whether the proliferation of melanoma cells is modulated by the fact that melanoma cells can use the aVP3 integrin in adhesion to, eg., vitronectin (Nip et aZ., 1992) and fibrinogen (FeldingHabermann et aZ., 1992). Furthermore, the p3 subunit might not be required for in vivo tumorigeneity and formation of metastases, but it can be replaced by pS or p6 subunits (Boukerche et al., 1994). Invasion through basement membranes is suggested to require enzymes degrading at least type IV collagen. MMP-2, MMP-3 and MMP-9 are therefore the candidate metalloproteinases to be involved in the process. Cell culture experiments show that interaction of either a561 or aVP3 integrin with a ligand might trigger signal transduction pathways activating C TRANSFORMATION MAY RINS" OTHER INTEGRINS MAY BE UPREGULATED IN DIFFERENT EXPERIMENTAL MODELS a4pl AND a5pl INTEGRINS HAVE PREVENTED MALIGNANT CELL BEHAVIOUR, WHEREAS IN MM THE NUMBER OF a4pl AND a5pl POSITIVE CELLS MAY INCREASE MOST LESIONS STAY NEGATIVE MMP-2 EXPRESSION CAN BE INDUCED BY a5pl AND aVP3 INTEGRINS MMP-1 EXPRESSION CAN BE REGULATED BY n2pl AND a S p l INTEGRINS MMP-9 EXPRESSION CAN BE INDUCED BY a3pl INTEGRIN INTEGRINS a 2 p l . a5pl AND nVP3 ARE INVOLVED IN CELL MIGRATION ON COLLAGEN AND FIBRONECTIN MOST LESIONS OF MM CONTAIN CELLS POSmVE FOR &PI. a3pl AND aVP3 INTEGRINS RGURE 2 -Putative functions of different integrins in (a) the control of malignant phenotype, (b) penetration through basement membranes and (c) invasion in connective tissue stroma. MM, malignant melanoma. 720 HEINO their expression (Werb et al., 1989; Seftor et al., 1992). In growth on laminin and collagen (Turner et al., 1989). Integrin keratinocytes, a third integrin heterodimer, a3p1, can convey a4pl is needed in the extravasation process of many inflammatory cells, and it might have a similar function in melanoma signals regulating MMP-9 expression (Larjava et al., 1993a). Cell migration in dermis might require different cell adhe- cells. Indeed, a4pl integrin is frequently expressed in the sion apparatus and the expression of different metalloprotein- metastatic lesions of melanomas (Danen et al., 1994). ases. Integrin a 2 p l is frequently present in vivo in malignant melanomas (Klein et al., 1991b). Interestingly, in skin malignanOPEN QUESTIONS cies with lower tendency for invasion, like basal cell carcinoThere seems to be significant individual variation in integrin mas, a2 integrin is down-regulated (Peltonen et al., 1989). In culture, melanoma cells use a2pl integrin in initial adhesion to expression in different tumors, even in the ones originating collagen (Montgomery et al., 1994) and in migration on from the same cell type. It is probable that different integrins replace each other in the functions essential for invasion. collagen (Etoh et al., 1992; Yoshinaga et al., 1993). Integrin may For that reason, the value of analyses of integrin pattern for a2pl is also involved in the reorganization of collagen fibrils the prognostication of individual cancers or whether integrin (Shiro et at., 1991; Riikonen et al., 1995b), seen in the pattern in cancer cells could be used to estimate the most aggressive melanoma cells as the increased ability to contract probable site of metastasis is not clear. collagenous matrices (Klein et al., 1991a). With some specific Several lines of evidence support the importance of MMPs cancer cell types, there is evidence suggesting the importance in the invasion of malignant cells. Immunohistochemical obserof the a2pl integrin for invasive phenotype; however, in many carcinomas a2 integrin is often down-regulated. In rhabdomyo- vations have shown MMP expression either in cancer cells or in sarcoma cells, the forced expression of the a 2 subunit is stromal cells, generating discussion of whether MMPs are whether they only required for the formation of metastasis in the mouse model produced by malignant cells themselves or induce other cells to produce MMPs. In vitro experiments (Chan et al., 1991). In human osteogenic sarcoma (HOS) cells, reviewed in this paper show that cell interaction with matrix transformation with both a chemical mutagen, MNNG (Dedhar can induce MMP expression. This suggests that MMP expresand Saulnier, 1990; Santala et al., 1994), and murine Kirsten sion by tumor cells might be temporally and spatially limited sarcoma virus (Santala et al., 1994) causes induction of the a 2 and that it takes place only during the short period cells are integrin. Forced expression of the a 2 integrin by cDNA attached to right extracellular ligands via the corresponding transfection in HOS cells does not make the cells tumorigenic integrin-type receptors. In vivo studies planned to find evibut induces their migration on, and invasion through, type I dence to support this hypothesis should analyze single cells in collagen (Vihinen et al., 1996). In addition to its role in cell tumors and contain simultaneous localization of specific mamigration, a2pl integrin might be an important element in the trix molecules, their receptors and MMPs. In malignant regulation of MMP-1 expression by the collagenous matrix. In melanoma or in any other type of cancer this kind of study has osteosarcoma cells, the number of a2 integrins on the cell not been done. Analyses of healing wounds has, however, surface dictates the level of MMP-1 gene expression (Riikonen revealed data supporting the idea (Saarialho-Kere et al., 1993). et al., 1995b). MMP-1 can be essential for invasion for 2 Finally, an important hypothesis is that invasion of cancer reasons: (i) Type I collagen is a major component of dermis cells can be prevented by treatment with molecules blocking and collagen fibrils might form a molecular barrier preventing integrin-mediated cellular functions. There is some experimencell movement. MMP-1, in addition to leukocyte MMP-8 and tal data supporting this possibility (Humphries et al., 1986; the newly described collagenase-3, is the only known enzyme Trikha et al., 1994). In the future, a more detailed picture degrading type I collagen. (zi) Degradation of type I collagen about the mechanisms of invasion will help to target new by MMP-1 leads also to its denaturation and reveals new molecules to critical points in the process. binding sites for other integrin heterodimers, like aVp3. Signal transduction by another integrin heterodimer, d p l , might ACKNOWLEDGEMENTS also regulate MMP-1 expression (Werb et al., 1989). The lesions of malignant melanoma may also contain cells This research has been supported by the Sigrid JusClius positive for alp1 and a4pl integrins (Schadendorf et al., 1993; Foundation, the Technology Development Centre in Finland Danen et al., 1994). The first one is a laminin and a collagen ( T E E S ) , the Academy of Finland, the Finnish Cancer receptor, though its significance for cells is not known. In Association, and the Finnish Cancer Union. I thank Dr. V.-M. cultured PC 12 cells, it plays an important role in neurite Kahari for critically reading the manuscript. 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