Membrane Type-1 Matrix Metalloproteinase Promotes Human Melanoma Invasion and Growth
2004; Elsevier BV; Volume: 122; Issue: 1 Linguagem: Inglês
10.1046/j.0022-202x.2003.22114.x
ISSN1523-1747
AutoresJoji Iida, Krista L. Wilhelmson, Matt A. Price, Christopher M. Wilson, Duanqing Pei, Leo T. Furcht, James B. McCarthy,
Tópico(s)Cell Adhesion Molecules Research
ResumoMembrane type-I metalloproteinase (MT1-MMP) is a transmembrane metalloproteinase that is critical for tumor cell invasion. MT1-MMP can degrade extracellular matrix (ECM) proteins directly and/or indirectly by activating soluble MMPs such as pro-MMP-2. Although MT1-MMP is upregulated in malignant melanoma, the biological consequences of elevated MT1-MMP expression for tumor progression are not enirely understood. In the current study, we have utilized the Bowes melanoma line for evaluating MT1-MMP in invasion and growth. Our studies extend the earlier observations to demonstrate that MT1-MMP expression in Bowes melanoma cells promotes selective invasion into matrigel but not matrices consisting of type-I collagen. Furthermore, MT1-MMP expressing melanoma cells exhibit increased migration in response to laminin 1 but not to type-I or type-IV collagen. MT1-MMP expression results in enhanced 3 dimensional growth in agarose gels and in long-term cultures within matrigel. The hydroxymate inhibitor BB94 inhibits MT1-MMP enhanced invasion and growth in 3 dimensional culture systems, but had no effect on increased motility. We demonstrated that MT1-MMP expression significantly facilitated tumorigenicity and growth by intradermal injection. The results suggest a more general role for elevated MT1-MMP in promoting both the selective invasion and increased growth of malignant melanoma in vivo. Membrane type-I metalloproteinase (MT1-MMP) is a transmembrane metalloproteinase that is critical for tumor cell invasion. MT1-MMP can degrade extracellular matrix (ECM) proteins directly and/or indirectly by activating soluble MMPs such as pro-MMP-2. Although MT1-MMP is upregulated in malignant melanoma, the biological consequences of elevated MT1-MMP expression for tumor progression are not enirely understood. In the current study, we have utilized the Bowes melanoma line for evaluating MT1-MMP in invasion and growth. Our studies extend the earlier observations to demonstrate that MT1-MMP expression in Bowes melanoma cells promotes selective invasion into matrigel but not matrices consisting of type-I collagen. Furthermore, MT1-MMP expressing melanoma cells exhibit increased migration in response to laminin 1 but not to type-I or type-IV collagen. MT1-MMP expression results in enhanced 3 dimensional growth in agarose gels and in long-term cultures within matrigel. The hydroxymate inhibitor BB94 inhibits MT1-MMP enhanced invasion and growth in 3 dimensional culture systems, but had no effect on increased motility. We demonstrated that MT1-MMP expression significantly facilitated tumorigenicity and growth by intradermal injection. The results suggest a more general role for elevated MT1-MMP in promoting both the selective invasion and increased growth of malignant melanoma in vivo. extracellular matrix matrix metalloproteinase Degradation of extracellular matrix (ECM) proteins is tightly linked to tumor invasion and metastasis (Rudolph and Matrisian, 1998Rudolph L. Matrisian L. Matrix metalloproteinases in remodeling of normal and neoplastic mammary gland.J Mammary Gland Biol Neoplasia. 1998; 3: 177-189Crossref PubMed Scopus (55) Google Scholar). Matrix metalloproteinases (MMP) are a family of zinc-dependent proteases critical for tumor progression, invasion, and metastasis (Sato et al., 1997Sato H. Okada Y. Seiki M. Membrane-type matrix metalloproteinases (MT-MMP) in cell invasion.Thromb Haemost. 1997; 78: 497-500Crossref PubMed Scopus (102) Google Scholar). MMP are subgrouped into soluble MMP and membrane-anchored MMP (membrane-type MMP, MT-MMP) (Nagase and Woessner, 1999Nagase H. Woessner Jr, J. Matrix metalloproteinases.J Biol Chem. 1999; 274: 21491-21494Crossref PubMed Scopus (3736) Google Scholar). Soluble MMP are thought to be responsible for ECM degradation over relatively broad areas within tissues, whereas MT-MMP participate in pericellular degradation of ECM, as they are tethered to the plasma membrane (Sato et al., 1997Sato H. Okada Y. Seiki M. Membrane-type matrix metalloproteinases (MT-MMP) in cell invasion.Thromb Haemost. 1997; 78: 497-500Crossref PubMed Scopus (102) Google Scholar;Nagase and Woessner, 1999Nagase H. Woessner Jr, J. Matrix metalloproteinases.J Biol Chem. 1999; 274: 21491-21494Crossref PubMed Scopus (3736) Google Scholar). Although the exact functions of these enzymes in tumor invasion and metastasis are not entirely understood, degradation of ECM proteins in the tumor microenvironment functions to provide space for growing tumors both within primary and metastatic tumors (Rudolph and Matrisian, 1998Rudolph L. Matrisian L. Matrix metalloproteinases in remodeling of normal and neoplastic mammary gland.J Mammary Gland Biol Neoplasia. 1998; 3: 177-189Crossref PubMed Scopus (55) Google Scholar). More recently, MMP activation has been linked to specific biologic mechanisms of tumor progression, including cell proliferation, resistance to apoptosis, and increased angiogenesis (McCawley and Matrisian, 2001McCawley L. Matrisian L. Matrix metalloproteinases: They're not just matrix anymore!.Curr Opin Cell Biol. 2001; 13: 534-540Crossref PubMed Scopus (1064) Google Scholar). Melanomas are malignant tumors that usually arise in the epidermis of the skin (Houghton and Polsky, 2002Houghton A.N. Polsky D. Focus on melanoma.Cancer Cell. 2002; 2: 275-278Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The invasion of melanomas from the epidermis to the dermis requires degradation of basement membrane components that are substrates for several MMP (Stetler-Stevenson et al., 1993Stetler-Stevenson W. Jiotta L. Kleiner D. Extracellular matrix 6. Role of matrix metalloproteinases in tumor invasion and metastasis.FASEB J. 1993; 7: 1434-1438Crossref PubMed Scopus (572) Google Scholar;MacDougall and Matrisian, 1995MacDougall J. Matrisian L. Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis.Cancer Metastasis Rev. 1995; 14: 351-362Crossref PubMed Scopus (392) Google Scholar;Nagase and Woessner, 1999Nagase H. Woessner Jr, J. Matrix metalloproteinases.J Biol Chem. 1999; 274: 21491-21494Crossref PubMed Scopus (3736) Google Scholar). Increased expression of MMP has been linked to progression and invasion in melanomas (Hofmann et al., 2000Hofmann U. Westphal J. van Muijen G. Ruiter D. Matrix metalloproteinases in human melanoma.J Invest Dermatol. 2000; 115: 337-344Crossref PubMed Scopus (329) Google Scholar). Elevated levels of MMP-2 and MMP-9 have been correlated with melanoma invasion both in vitro and in vivo (Hofmann et al., 1999Hofmann U. Westphal J. Waas E. Zendman A. Cornelissen I. Ruiter D. van Muijen G. Matrix metalloproteinases in human melanoma cell lines and xenografts: Increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression.Br J Cancer. 1999; 81: 774-782Crossref PubMed Scopus (128) Google Scholar;Itoh et al., 1999Itoh T. Tanioka M. Matsuda H. Nishimoto H. Yoshioka T. Suzuki R. Uehira M. Experimental metastasis is suppressed in MMP-9-deficient mice.Clin Exp Metastasis. 1999; 17: 177-181Crossref PubMed Scopus (278) Google Scholar;MacDougall et al., 1999MacDougall J.R. Bani M.R. Lin Y. Muschel R.J. Kerbel R.S. "Proteolytic switching": Opposite patterns of regulation of gelatinase B and its inhibitor TIMP-1 during human melanoma progression and consequences of gelatinase B overexpression.Br J Cancer. 1999; 80: 504-512Crossref PubMed Scopus (55) Google Scholar). Endogenous inhibitors of MMP, known as tissue inhibitors of matrix metalloproteinases (TIMP) inhibit melanoma cell growth and invasion. For example, overexpression of TIMP-1 and TIMP-3 in melanoma cells inhibits tumor formation in animal models (Khokka, 1994Khokka R. Suppression of the tumorigenic and metastatic abilities of murine B16–F10 melanoma cells in vivo by the overexpression of the tissue inhibitor of the metalloproteinases-1.J Natl Cancer Inst. 1994; 86: 299-304Crossref PubMed Scopus (202) Google Scholar;Ahonen et al., 1998Ahonen M. Baker A. Kahari V. Adenovirus-mediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells.Cancer Res. 1998; 58: 210-2315PubMed Google Scholar).Valente et al., 1998Valente P. Fassina G. Melchiori A. et al.TIMP-2 over-expression reduces invasion and angiogenesis and protects B16F10 melanoma cells from apoptosis [published erratum appears in Int J Cancer 80: 485, 1999].Int J Cancer. 1998; 75: 246-253Crossref PubMed Scopus (244) Google Scholar overexpressed TIMP-2 and inhibited both invasion and angiogenesis of melanomas in vivo. Histopathologic analysis of human tumor samples shows that MT1-MMP is expressed in melanoma cells and localizes to tumor cells at the invasive front of primary tumors (Hofmann et al., 2000Hofmann U. Westphal J. van Muijen G. Ruiter D. Matrix metalloproteinases in human melanoma.J Invest Dermatol. 2000; 115: 337-344Crossref PubMed Scopus (329) Google Scholar). Furthermore, MMP-2 is colocalized with MT1-MMP, suggesting that MT1-MMP and MMP-2 act in concert to mediate melanoma invasion in vivo (Hofmann et al., 2000Hofmann U. Westphal J. van Muijen G. Ruiter D. Matrix metalloproteinases in human melanoma.J Invest Dermatol. 2000; 115: 337-344Crossref PubMed Scopus (329) Google Scholar). MT1-MMP expression in A2058 melanoma cells has recently been linked to activation of pro-MMP2 and increased tumor formation in vivo (Sounni et al., 2002aSounni N. Baramova E. Munaut C. Maquoi E. Frankenne F. Foidart J. Noel A. Expression of membrane type I matrix metalloproteinase (MT1-MMP) in A2058 melanoma cells is associated with MMP-2 activation and increased tumor growth and vascularization.Int J Cancer. 2002; 98: 23-28Crossref PubMed Scopus (46) Google Scholar). These results collectively emphasize the importance of increased MMP expression and MT1-MMP-mediated activation of MMP in melanoma invasion and progression. Human tumor xenografts are supposed to be appropriate models for addressing specific questions related to the clinical behavior and treatment sensitivity of human tumors. Melanocytes are normally found in the dermal–epidermal junction of the skin, thus tumor growth in orthotopic sites was achieved by intradermal injection of melanoma cells. Indeed, previous studies demonstrated that different melanoma cells grown in subdermal sites retained several biologic characteristics of the donor patients' tumors, including angiogenic potential, growth, histopathologic parameters, and the organ-specific metastatic pattern (Cornil et al., 1989Cornil I. Man S. Fernandez B. Kerbel R. Enhanced tumorigenicity, melanogenesis, and metastases of a human malignant melanoma after subdermal implantation in nude mice.J Natl Cancer Inst. 1989; 81: 938-944Crossref PubMed Scopus (66) Google Scholar;Rofstad, 1994Rofstad E. Orthotopic human melanoma xenograft model systems for studies of tumor angiogenesis, pathophysiology, treatment sensitivity and metastatic pattern.Br J Cancer. 1994; 70: 804-812Crossref PubMed Scopus (109) Google Scholar). These results suggest that the intradermal injection of melanoma cells show a great promise for future studies of tumor angiogenesis, pathology, and metastatic phenotype. In this study we have characterized the in vitro invasion and growth of Bowes melanoma cells stably transfected with MT1-MMP. Expression of MT1-MMP stimulates melanoma invasion into three-dimensional matrices composed of basement membrane components (matrigel) but not native type I collagen, suggesting that the collagenolytic activity of MT1-MMP is insufficient to promote invasion through native type I collagen. Additionally, MT1-MMP expression results in increased migration to laminin-1 but not to types I and IV collagens. MT1-MMP-mediated invasion is sensitive to the MMP inhibitor BB94, whereas increased migration to laminin-1 is resistant. Furthermore, MT1-MMP expressing melanoma cells exhibit increased growth in both two-dimensional and three-dimensional cultures, which is also inhibited by BB94. Increased colony growth is also observed for MT1-MMP expressing melanoma cells embedded and cultured long-term within matrigel. We demonstrated that MT1-MMP expression significantly facilitated tumor take and growth by orthotopic injection (intradermal injection). The results suggest a more general role for elevated MT1-MMP in promoting both the selective invasion and increased growth of malignant melanoma in vivo. Bowes melanoma cells have been reported to express very low amounts of MT1-MMP and produce MMP-2 as a zymogen form as described previously (Lehti et al., 2000Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.J Biol Chem. 2000; 275: 15006-15013Crossref PubMed Scopus (138) Google Scholar). We therefore chose these cells to transfect with a plasmid harboring the coding sequence for human MT1-MMP. We generated stable cell lines as well as cells transfected with a GFP-vector alone to serve as a control. MT1-MMP was expressed in the stable transfectants as three major proteins migrating at 65 kDa, 60 kDa, and 44 kDa on SDS–PAGE Figure 1a. Two higher molecular weight immunoreactive fragments of 65 kDa and 60 kDa remained following treatment of BB94. These two higher molecular weight species represent the pro- and active-forms, respectively, of MT1-MMP and they are detected in cells either treated or not with BB94. In contrast, cultures treated overnight with BB94 lacked the lower (44 kDa) band present in nontreated cultures Figure 1a. These results agree with previous studies demonstrating that autocatalytic degradation of MT1-MMP creates a 44 kDa membrane tethered fragment that lacks catalytic activity (Lehti et al., 2000Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.J Biol Chem. 2000; 275: 15006-15013Crossref PubMed Scopus (138) Google Scholar). In contrast, the expression of the 60 kDa active form of MT1-MMP is facilitated by intracellular furin-dependent mechanisms and is resistant to hydroxymate inhibitors (Lehti et al., 2000Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.J Biol Chem. 2000; 275: 15006-15013Crossref PubMed Scopus (138) Google Scholar). We analyzed the activation status of endogenous pro-MMP-2 using gelatin zymography to evaluate the catalytic activity of MT1-MMP Figure 1b. As expected, MT1-MMP expression caused a corresponding activation of MMP-2 to yield both intermediate (i) and active (a) forms of the gelatinase. Production of these forms was inhibited in the presence of BB94, indicating the requirement for MMP activity in the processing of pro-MMP-2. Thus, these results indicate that MT1-MMP transfected into melanoma cells is expressed and functionally active on the cell surface, which is consistent with previous studies demonstrating that MT1-MMP activates pro-MMP-2 in various cell types (Lehti et al., 2000Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.J Biol Chem. 2000; 275: 15006-15013Crossref PubMed Scopus (138) Google Scholar;Yana and Weiss, 2000Yana I. Weiss S. Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases.Mol Biol Cell. 2000; 11: 2387-2401Crossref PubMed Scopus (258) Google Scholar;Sounni et al., 2002aSounni N. Baramova E. Munaut C. Maquoi E. Frankenne F. Foidart J. Noel A. Expression of membrane type I matrix metalloproteinase (MT1-MMP) in A2058 melanoma cells is associated with MMP-2 activation and increased tumor growth and vascularization.Int J Cancer. 2002; 98: 23-28Crossref PubMed Scopus (46) Google Scholar). Recent studies have also suggested that serine proteases such as plasmin can activate MMP-2 in the presence of MT1-MMP (Monea et al., 2002Monea S. Lehti K. Keski-Oja J. Mignatti P. Plasmin activates pro-matrix metalloproteinase-2 with a membrane-type I matrix metalloproteinase-dependent mechanisms.J Cell Physiol. 2002; 192: 160-170Crossref PubMed Scopus (125) Google Scholar;Sounni et al., 2002aSounni N. Baramova E. Munaut C. Maquoi E. Frankenne F. Foidart J. Noel A. Expression of membrane type I matrix metalloproteinase (MT1-MMP) in A2058 melanoma cells is associated with MMP-2 activation and increased tumor growth and vascularization.Int J Cancer. 2002; 98: 23-28Crossref PubMed Scopus (46) Google Scholar); however, incubation of MT1-MMP expressing melanomas with either aprotonin or ε-amino-n-caproic acid failed to inhibit the activation of MMP-2 in these cells Figure 1b. These results led us to conclude that pro-MMP-2 activation is not dependent on plasmin activation by these cells, but rather is dependent on the catalytic activity of MT1-MMP on the cell surface. In order to characterize further the importance of MT1-MMP in melanoma invasion, melanoma cells were cultured on top of matrigel and invaded cells were counted as a function of the depth of their invasion. The number of invading cells was determined by quantifying the number of cells at successive 20 μm (1 cell diameter) depths in six randomly selected fields (Faassen et al., 1992Faassen A. Schrager J. Klein D. Oegema T. Couchman J. McCarthy J. A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion.J Cell Biol. 1992; 116: 521-531Crossref PubMed Scopus (229) Google Scholar). As demonstrated in Figure 2(a), almost all cells (95%) in mock-transfected melanoma cells remained on the top or within the first 20 μm deep layer of the gels. Only 5% of cells invaded to a level of 40 μm depth in these gels. In contrast, almost half (48%) of the MT1-MMP expressing cells invaded into the gel at a depth of more than 40 μm and 10% of cells invaded to depths as far as 80 to 100 μm beneath the gel surface Figure 2a. Invasion of MT1-MMP expressing melanoma cells was significantly inhibited in the presence of BB94 Figure 2a. These results are consistent with the previous studies in which expression of MT1-MMP promotes invasion of multiple tumor cell types by a mechanism dependent on the catalytic function of the protease (Sato et al., 1994Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells.Nature (London). 1994; 370: 61-65Crossref PubMed Scopus (2315) Google Scholar;Nakahara et al., 1997Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y.Y. Chen W.T. Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloproteinase docking to invadopodia is required for cell invasion.Proc Natl Acad Sci USA. 1997; 94: 7959-7964Crossref PubMed Scopus (351) Google Scholar;Lehti et al., 2000Lehti K. Valtanen H. Wickstrom S. Lohi J. Keski-Oja J. Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain.J Biol Chem. 2000; 275: 15006-15013Crossref PubMed Scopus (138) Google Scholar;Sounni et al., 2002aSounni N. Baramova E. Munaut C. Maquoi E. Frankenne F. Foidart J. Noel A. Expression of membrane type I matrix metalloproteinase (MT1-MMP) in A2058 melanoma cells is associated with MMP-2 activation and increased tumor growth and vascularization.Int J Cancer. 2002; 98: 23-28Crossref PubMed Scopus (46) Google Scholar). Type I collagen is enriched in dermis and melanoma cells must penetrate this structure as they progress and exit the epidermis (Benbow et al., 1999Benbow U. Schoenermark M.P. Mitchell T.I. Rutter J.L. Shimokawa K. Nagase H. Brinckerhoff C.E. A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.J Biol Chem. 1999; 274: 25371-25378Crossref PubMed Scopus (94) Google Scholar). Previous studies demonstrate MT1-MMP expression and collagenolytic activity of this protease is essential for promoting the invasion of various cell types into type I collagen gels (Hotary et al., 2000Hotary K. Allen E. Punturrieri A. Yana I. Weiss S. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinase.J Cell Biol. 2000; 149: 1309-1323Crossref PubMed Scopus (501) Google Scholar;Aznavoorian et al., 2001Aznavoorian S. Moore B.A. Alexander-Lister L.D. Hallit S.L. Windsor L.J. Engler J.A. Membrane type 1-matrix metalloproteinase-mediated degradation of type I collagen by oral squamous cell carcinoma cells.Cancer Res. 2001; 61: 6264-6275PubMed Google Scholar;Ellerbroek et al., 2001Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. Functional interplay between type I collagen and cell surface matrix metalloproteinase activity.J Biol Chem. 2001; 276: 24833-24842Crossref PubMed Scopus (141) Google Scholar;Zhuge and Xu, 2001Zhuge Y. Xu J. Rac1 mediates type I collagen-dependent MMP-2 activation.J Biol Chem. 2001; 276: 16248-16256Crossref PubMed Scopus (154) Google Scholar). Under our experimental conditions, however, we did not observe invasion of MT1-MMP transfectants into type I collagen gels Figure 2b, suggesting that there are cell type specific differences in the importance of MT1-MMP in mediating invasion into type I collagen. Laminins are a major component of basement membranes and play an active part in tumor metastasis, cell attachment and migration, and angiogenesis (Patarroyo et al., 2002Patarroyo M. Tryggavson K. Virtanen I. Laminin isoforms in tumor invasion, angiogenesis and metastasis.Semin Cancer Biol. 2002; 12: 197-207Crossref PubMed Scopus (276) Google Scholar;Yamamura et al., 1993Yamamura K. Kibbey M. June S. Kleinman H. Effect of matrigel and laminin peptide YIGSR on growth and metastasis.Semin Cancer Biol. 1993: 259-265Google Scholar;Snow et al., 2002Snow D. Smith J. Gurwell J. Binding characteristics of chondroitin sulfate proteoglycans and laminin-1, and correlative neurite outgrowth behaviors in a standard tissue culture choice assay.J Neurobiol. 2002; 51: 285-301Crossref PubMed Scopus (30) Google Scholar). Invasion of MT1-MMP expressing melanoma cells into matrigel was significantly inhibited by specific neutralizing antibodies against α6 integrin but not with anti-α2 or α3 integrin antibodies (not shown), suggesting that laminin-1/α6β1 interactions play a key part in promoting melanoma invasion into matrigel. We therefore subjected melanoma cells to migration assays in which laminin-1 was used as an attractant. Although mock-transfected melanoma cells showed relatively low migratory responses to laminin-1, MT1-MMP expressing melanoma cells exhibit a significant increase (4–5-fold) in migration Figure 3a. Transfection of MT1-MMP into Bowes melanoma cells did not alter expressions of α2β1, α3β1, and α6β1 integrins (not shown). As expected, migration of mock-transfected and MT1-MMP-transfected melanoma cells was inhibited by the addition of anti-α6 or anti-β1 integrin antibody but not by the addition of anti-α2 integrin antibody Figure 3b. These results suggest that increased invasion into matrigel is also dependent on the interaction of α6β1 with laminin-1, which is a major component of matrigel. It is of interest that BB94 failed to inhibit migration to laminin-1 by MT1-MMP expressing melanoma cells Figure 3a, although it did inhibit the invasion of these cells into matrigel. These results indicate that invasion into matrigel depends on both proteolytic and adhesion-related mechanisms. The results also suggest that MT1-MMP may stimulate motility by directly altering signal transduction pathways within melanoma cells, as the catalytic activity of the protease is not required for increased motility. The results also suggest some level of specificity in this response, as enhanced migration to type I Figure 3c or type IV collagens Figure 3d is not enhanced by the expression of MT1-MMP. We next tested the ability of MT1-MMP expressing cells to grow in culture, as increased growth is associated with progression and invasion (Freedman and Shin, 1974Freedman V. Shin S. Cellular tumorigenicity in nude mice: Correlation with cell growth in semi-solid medium.Cell. 1974; 3: 355-359Abstract Full Text PDF PubMed Scopus (708) Google Scholar;Cifone, 1982Cifone M. In vitro growth characteristics associated with benign and metastatic variants of tumor cells.Cancer Metastasis Rev. 1982; 1: 335-347Crossref PubMed Scopus (32) Google Scholar). MT1-MMP expressing Bowes melanoma cells plated in two-dimensional culture conditions exhibited significant increases in growth relative to cells containing the vector alone Figure 4a. The cells exhibited equal plating efficiencies as judged in part by the equal number of cells observed in all cultures within the first 3 d of plating. Growth of MT1-MMP expressing cells was consistently elevated by 20% or greater relative to vector controls when analyzed on days 5, 7, and 10 following plating Figure 4a. The addition of BB94 to these cultures inhibited the accelerated growth of the tumor cells to levels observed in the vector control cultures Figure 4a. BB94 treated cultures did not show decreased via-bility relative to nontreated cultures (not shown), suggesting that MMP catalytic activity in the cultures is required for increased growth of the cells. We also performed growth assays in semisolid media (i.e., agarose). Melanoma cells cultured in this medium form tightly packed colonies after 10 d in culture (not shown). MT1-MMP expressing cells exhibit highly significant (p<0.001) increases in colony numbers when compared with vector-transfected controls Figure 4b. Importantly this increased propensity to form colonies in semisolid medium was also inhibited by the addition of BB94 Figure 4b. These results indicate that MT1-MMP expression leads to increased colony forming potential of these cells, which is a correlate of tumorigenic potential. Furthermore, the increased colony forming potential of MT1-MMP expressing cells is also dependent on MMP catalytic activity within the cultures, which is similar to what was observed in two-dimensional cultures. Many studies indicate that basement membranes may serve as barriers for preventing tumor cell entry into adjacent tissues (Yamamura et al., 1993Yamamura K. Kibbey M. June S. Kleinman H. Effect of matrigel and laminin peptide YIGSR on growth and metastasis.Semin Cancer Biol. 1993: 259-265Google Scholar;Durko et al., 1997Durko M. Navab R. Shibata H.R. Brodt P. Suppression of basement membrane type IV collagen degradation and cell invasion in human melanoma cells expressing an antisense RNA for MMP-1.Biochim Biophys Acta. 1997; 1356: 271-280Crossref PubMed Scopus (59) Google Scholar). We therefore next evaluated the growth of the MT1-MMP expressing melanomas, cultured in matrigel for 7 d, to determine if increased growth is also observed in a more complex microenvironment that is rich in basement membrane components. Under these experimental conditions, the colony numbers between mock and MT1-MMP expressing melanoma cells were similar (not shown), however, the MT1-MMP expressing cells exhibited much larger colonies than the vector control cells Figure 5. These results suggest that MT1-MMP expression in melanoma cells may facilitate growth of melanoma cells at tissue boundaries, accelerating entry of tumor cells into adjacent tissues via a growth-related mechanism. Intradermal (i.d.) injections in mice were performed with mock-transfected or MT1-MMP expressing melanoma cells. Tumor sizes were monitored up to 40 d as described in Materials and Methods. Under these experimental conditions, MT1-MMP expressing melanoma cells formed palpable tumors in 65% of total injected sites within 19 d following injection Figure 6. By contrast, mock-transfected melanoma cells formed tumors at a much slower rate. Only 30% of total injected sites formed a detectable tumor within 19 d following injection. The number of sites with tumors increased slowly over the next 26 d, finally reaching a maximum (18 of 20) at day 40 Figure 6. The size of the tumors formed within 40 d by the MT1-MMP expressing cells was also significantly larger than those formed by the mock-transfected cells Figure 7. The average estimated volume of the MT1-MMP expressing tumors was 160±60 (mean ±SD), whereas the size of the mock-transfected tumors was 96±76 (mean±SD). These results suggest that elevated MT1-MMP expression may enhance the growth of tumors in vivo within sites harboring primary melanoma tumors.Figure 7Tumor growth by melanoma cells expressing MT1-MMP. Melanoma tumor growth was measured using calipers, and the volume of the tumors was estimated by the calculation described in the Materials and Methods section. Shown are the calculated volumes of tumors at 40 d postinjection. Each dot represents tumor volume at a distinct injection site (two sites per mouse), and the horizontal line depicts the mean calculated value for the entire group. The experimental group injected with MT1-MMP expressing tumors contained three less values than the control (mock-transfected group) (17 vs 20) because three of the tumors in the MT1-MMP group were exceedingly large and judged to be outliers based on the use of box-plot analysis (Williamson et al., 1989Williamson D. Parker R. Kendrick J. The box-plot: A simple visual method to interpret data.Ann Intern Med. 1989; 110: 916-921Crossref PubMed Scopus (345) Google Scholar;Tukey, 1990Tukey J. Data-based graphics: Visual display in the decades to come.Stat Sci. 1990; 5: 327-339Crossref Scopus (81) Google Scholar). Statistical significance between the means of the two groups of tumors was determined by the Student's t-test.View Large Image Figure ViewerD
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