A Mechanism for Regulation of Melanoma Invasion

Invasion of LOX human melanoma cells involves extracellular matrix (ECM) degradation and formation of cell surface invadopodia. Here we show that the ligation of α6β1 by two peptides derived from the COOH-terminal globular domain of laminin-1 α1 chain (laminin G peptides), designated AG-10 (NPWHSIYITRFG) and AG-32 (TWYKIAFQRNRK), and antibodies against α6 and β1 integrins promoted invasiveness. AG-10 and AG-32 inhibited cell adhesion on laminin, and the antibodies blocked cell adhesion on immobilized AG-10 and AG-32, suggesting that the peptides interact primarily with α6β1 integrin. These soluble peptides and integrin antibodies induced invasiveness by causing an 2-3-fold increase in ECM degradation and invadopodial activity independently of adhesion activity of integrins that were prebound to ECM. The induced ECM degradation and invasion was associated with an increased surface expression of the 170-kDa membrane-bound gelatinase, seprase, as well as its intense localization at invadopodia but not at focal adhesions. However, the total expression levels of seprase, gelatinase A and β1 integrins were not altered. We suggest that laminin G peptides act on the α6β1 integrin signaling of invasion by stimulating invadopodial activities, which is distinct from their direct effects on cell adhesion on immobilized ECM. Invasion of LOX human melanoma cells involves extracellular matrix (ECM) degradation and formation of cell surface invadopodia. Here we show that the ligation of α6β1 by two peptides derived from the COOH-terminal globular domain of laminin-1 α1 chain (laminin G peptides), designated AG-10 (NPWHSIYITRFG) and AG-32 (TWYKIAFQRNRK), and antibodies against α6 and β1 integrins promoted invasiveness. AG-10 and AG-32 inhibited cell adhesion on laminin, and the antibodies blocked cell adhesion on immobilized AG-10 and AG-32, suggesting that the peptides interact primarily with α6β1 integrin. These soluble peptides and integrin antibodies induced invasiveness by causing an 2-3-fold increase in ECM degradation and invadopodial activity independently of adhesion activity of integrins that were prebound to ECM. The induced ECM degradation and invasion was associated with an increased surface expression of the 170-kDa membrane-bound gelatinase, seprase, as well as its intense localization at invadopodia but not at focal adhesions. However, the total expression levels of seprase, gelatinase A and β1 integrins were not altered. We suggest that laminin G peptides act on the α6β1 integrin signaling of invasion by stimulating invadopodial activities, which is distinct from their direct effects on cell adhesion on immobilized ECM. INTRODUCTIONCell invasion through the extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixELISAenzyme linked immunosorbent assaymAbmonoclonal antibodyTBSTris-HCl-buffered salinePBSphosphate-buffered saline. is an essential process driving tissue development and cancer metastasis (1Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 2Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar). Invading cells possess integrated surface activities of ECM degradation and adhesion that appear at unique surface structures, invadopodia (3Chen W.-T. Lee C.C. Goldstein L. Bernier S. Liu C.H. Lin C.Y. Yeh Y. Monsky W.L. Kelly T. Dai M. Zhou J.-Y. Mueller S.C. Breast Cancer Res. Treat. 1994; 31: 217-226Crossref PubMed Scopus (47) Google Scholar). The factor that promotes cell invasiveness is not known. However, increasing evidence indicates that laminin, a major basement membrane glycoprotein, and its peptide fragments are able to promote cell invasion. One active peptide, IKVAV, is located on the COOH terminus of the long arm of the laminin α1 chain and promotes cell attachment, migration, tumor growth, metastasis (4Kanemoto T. Reich R. Royce L. Greatorex D. Adler S.H. Shiraishi N. Martin G.R. Yamada Y. Kleinman H.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2279-2283Crossref PubMed Scopus (158) Google Scholar, 5Mackay A.R. Gomez D.E. Nason A.M. Thorgeirsson U.P. Lab. Invest. 1994; 70: 800-806PubMed Google Scholar, 6Stack M.S. Gray R.D. Pizzo S.V. Cancer Res. 1993; 53: 1998-2004PubMed Google Scholar, 7Yamamura K. Kibbey M.C. Kleinman H.K. Cancer Res. 1993; 53: 423-428PubMed Google Scholar), and production of gelatinase A/type IV collagenase (matrix metalloprotease-2) (4Kanemoto T. Reich R. Royce L. Greatorex D. Adler S.H. Shiraishi N. Martin G.R. Yamada Y. Kleinman H.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2279-2283Crossref PubMed Scopus (158) Google Scholar, 5Mackay A.R. Gomez D.E. Nason A.M. Thorgeirsson U.P. Lab. Invest. 1994; 70: 800-806PubMed Google Scholar, 8Turpeenniemi-Hujanen T. Thorgeirsson U.P. Rao C.N. Liotta L.A. J. Biol. Chem. 1986; 261: 1883-1889Abstract Full Text PDF PubMed Google Scholar). A 140-kDa matrix protein, ladsin, which appears to be identical to the laminin β2 chain, has been shown to promote scattering of carcinoma cells, stimulate cell migration, and bind to α3β1 integrin (9Miyazaki K. Kikkawa Y. Nakamura A. Yasumitsu H. Umeda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11767-11771Crossref PubMed Scopus (151) Google Scholar, 10Kikkawa Y. Umeda M. Miyazaki K. J. Biochem. (Tokyo). 1994; 116: 862-869Crossref PubMed Scopus (105) Google Scholar). Thus, it is possible that laminin/integrin interactions function not only in cell adhesion but also to transduce biochemical signals that modulate surface activities of degradation and invasion (11Ramos D.M. Berston E.D. Kramer R.H. Cancer Res. 1990; 50: 728-734PubMed Google Scholar, 12Seftor R.E.B. Seftor E.A. Stetler-Stevenson W.G. Hendrix M.J.C. Cancer Res. 1993; 53: 3411-3415PubMed Google Scholar, 13Kornberg L. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar).Previously, we showed that a membrane-bound, 170-kDa gelatinase, seprase, and secreted, metallo-type gelatinase A were localized on invadopodia of LOX human melanoma cells during degradation/invasion of fibronectin-coated cross-linked gelatin films (14Aoyama A. Chen W.-T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8296-8300Crossref PubMed Scopus (104) Google Scholar, 15Monsky W.L. Lin C.-Y. Aoyama A. Kelly T. Mueller S.C. Akiyama S.K. Chen W.-T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar). Further analysis using fibronectin, laminin, type IV collagen, type I collagen, and Matrigel substrata demonstrated that degradation of these ECM components by transformed and tumor cells occurred at invadopodia, suggesting membrane association and activation of latent proteases during invasion (15Monsky W.L. Lin C.-Y. Aoyama A. Kelly T. Mueller S.C. Akiyama S.K. Chen W.-T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar, 16Kelly T. Mueller S.C. Yeh Y. Chen W.-T. J. Cell Physiol. 1994; 158: 299-308Crossref PubMed Scopus (86) Google Scholar). However, no specific membrane-associated or secreted protease has yet been associated with an induced invasion. Recently, a systematic screening for cell binding sites with 113 overlapping synthetic peptide beads covering the laminin α1 chain carboxyl-terminal globular domain (G domain amino acid residues 2111-3060) resulted in the identification of 19 potential adhesion-active sequences (17Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Here, we identified the peptides designated AG-10 and AG-32 that ligated α6β1 integrin in a similar manner as anti-β1 and α6 integrin antibodies promote melanoma invasiveness independently of the adhesion function of integrin receptors. These soluble peptides and antibodies could signal cell invasion at sites of ECM contact. We found that, accompanying the induced invasiveness, there was an increased organization of seprase on invadopodia, but the expression levels of seprase, gelatinase A, and β1 integrins were not altered.DISCUSSIONWe have shown that soluble AG-10 and AG-32 laminin G peptides and anti-integrin β1 and α6 mAbs can increase LOX human melanoma cell invasion by stimulating invadopodial activity. The peptides ligated predominantly the α6β1 integrin receptor that induced the subsequent recruitment of seprase to invadopodia found on the leading edge of invading cells, along with a concomitant increase of melanoma cell invasiveness. Our data suggest a mechanism for melanoma cell invasion in which cell invasiveness can be triggered when β1 integrins, particularly α6β1, on ECM-free surfaces of the cell are ligated by laminin G peptides or antibodies.The biochemical mechanisms by which laminin peptides exert their effect on invasion are less understood. In this report, we showed that binding of laminin peptides to the available integrin triggers a signal necessary for expression of cell invasiveness. Clustering and ligand occupancy of cell surface integrins are both involved in the activation of intracellular signaling (26Schwartz M.A. Lechene C. Ingber D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7849-7853Crossref PubMed Scopus (323) Google Scholar, 27Kornberg L.J. Earp H.S. Turner C.E. Prockop C. Juliano R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8392-8396Crossref PubMed Scopus (630) Google Scholar, 28Akiyama S.K. Yamada S.S. Yamada K.M. La Flamme S.E. J. Biol Chem. 1994; 269: 15961-15964Abstract Full Text PDF PubMed Google Scholar, 29Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar, 30Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Crossref PubMed Scopus (788) Google Scholar). Furthermore, fragments of laminin and fibronectin and antibodies against integrin stimulate cellular production of collagenases (8Turpeenniemi-Hujanen T. Thorgeirsson U.P. Rao C.N. Liotta L.A. J. Biol. Chem. 1986; 261: 1883-1889Abstract Full Text PDF PubMed Google Scholar, 24Werb Z. Tremble P.M. Behrendtsen O. Crowley E. Damsky C.H. J. Cell Biol. 1989; 109: 877-889Crossref PubMed Scopus (901) Google Scholar). Ligation and perturbation of the αvβ3 or α5β1 integrins of human melanoma cells by an antibody or soluble adhesion protein promote secretion of Gelatinase A and cell migration/invasion regardless of whether the adhesion function of integrins was affected (12Seftor R.E.B. Seftor E.A. Stetler-Stevenson W.G. Hendrix M.J.C. Cancer Res. 1993; 53: 3411-3415PubMed Google Scholar, 25Seftor R.E. Seftor E.A. Gehlsen K.R. Stetler-Stevenson W.G. Brown P.D. Ruoslahti E. Hendrix M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1557-1561Crossref PubMed Scopus (432) Google Scholar).None of the laminin peptides tested had any detectable effect on the adhesion of cells when added in soluble form to LOX melanoma cells that have already been adhered to gelatin substrates. In addition, there were no detectable changes in the expression level of either β1 integrins or membrane proteases, or their localization in focal contact sites, suggesting that the laminin peptides were functioning specifically to signal the recruitment of proteolytic molecules to invadopodia. Thus, the observed effects of the laminin peptides are independent of possible effects on cell adhesion or gene expression.Tumor cell invasiveness has been linked with either an increased production of ECM-degrading enzymes or surface association and activation of latent proteases. Using long term (72 h) Boyden chamber migration/invasion assays, laminin SIKVAV peptide was shown to stimulate invasive melanoma cells to either produce or activate gelatinase A (4Kanemoto T. Reich R. Royce L. Greatorex D. Adler S.H. Shiraishi N. Martin G.R. Yamada Y. Kleinman H.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2279-2283Crossref PubMed Scopus (158) Google Scholar, 5Mackay A.R. Gomez D.E. Nason A.M. Thorgeirsson U.P. Lab. Invest. 1994; 70: 800-806PubMed Google Scholar, 8Turpeenniemi-Hujanen T. Thorgeirsson U.P. Rao C.N. Liotta L.A. J. Biol. Chem. 1986; 261: 1883-1889Abstract Full Text PDF PubMed Google Scholar) and tissue plasminogen activator-catalyzed plasminogen system (6Stack M.S. Gray R.D. Pizzo S.V. Cancer Res. 1993; 53: 1998-2004PubMed Google Scholar). In addition, ligation of the α5β1 integrin of invasive C8161 human melanoma cells and the αvβ3 integrin of A375 human melanoma cells promotes invasion and a concurrent increase in secretion of gelatinase A (12Seftor R.E.B. Seftor E.A. Stetler-Stevenson W.G. Hendrix M.J.C. Cancer Res. 1993; 53: 3411-3415PubMed Google Scholar, 25Seftor R.E. Seftor E.A. Gehlsen K.R. Stetler-Stevenson W.G. Brown P.D. Ruoslahti E. Hendrix M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1557-1561Crossref PubMed Scopus (432) Google Scholar). In contrast, we show here the involvement of AG-10 and AG-32 peptides via the α6β1 integrin in shorter term assays (within 5 h) that resulted in re-organization of seprase at invadopodia, increase in local fibronectin degradation, and invasion but not protein expression. We suggest that receptor binding or clustering of available integrins of adherent melanoma cells affected the invasive process via re-organization of cell surface seprase, that leads to enzyme activation (15Monsky W.L. Lin C.-Y. Aoyama A. Kelly T. Mueller S.C. Akiyama S.K. Chen W.-T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar). INTRODUCTIONCell invasion through the extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixELISAenzyme linked immunosorbent assaymAbmonoclonal antibodyTBSTris-HCl-buffered salinePBSphosphate-buffered saline. is an essential process driving tissue development and cancer metastasis (1Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 2Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar). Invading cells possess integrated surface activities of ECM degradation and adhesion that appear at unique surface structures, invadopodia (3Chen W.-T. Lee C.C. Goldstein L. Bernier S. Liu C.H. Lin C.Y. Yeh Y. Monsky W.L. Kelly T. Dai M. Zhou J.-Y. Mueller S.C. Breast Cancer Res. Treat. 1994; 31: 217-226Crossref PubMed Scopus (47) Google Scholar). The factor that promotes cell invasiveness is not known. However, increasing evidence indicates that laminin, a major basement membrane glycoprotein, and its peptide fragments are able to promote cell invasion. One active peptide, IKVAV, is located on the COOH terminus of the long arm of the laminin α1 chain and promotes cell attachment, migration, tumor growth, metastasis (4Kanemoto T. Reich R. Royce L. Greatorex D. Adler S.H. Shiraishi N. Martin G.R. Yamada Y. Kleinman H.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2279-2283Crossref PubMed Scopus (158) Google Scholar, 5Mackay A.R. Gomez D.E. Nason A.M. Thorgeirsson U.P. Lab. Invest. 1994; 70: 800-806PubMed Google Scholar, 6Stack M.S. Gray R.D. Pizzo S.V. Cancer Res. 1993; 53: 1998-2004PubMed Google Scholar, 7Yamamura K. Kibbey M.C. Kleinman H.K. Cancer Res. 1993; 53: 423-428PubMed Google Scholar), and production of gelatinase A/type IV collagenase (matrix metalloprotease-2) (4Kanemoto T. Reich R. Royce L. Greatorex D. Adler S.H. Shiraishi N. Martin G.R. Yamada Y. Kleinman H.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2279-2283Crossref PubMed Scopus (158) Google Scholar, 5Mackay A.R. Gomez D.E. Nason A.M. Thorgeirsson U.P. Lab. Invest. 1994; 70: 800-806PubMed Google Scholar, 8Turpeenniemi-Hujanen T. Thorgeirsson U.P. Rao C.N. Liotta L.A. J. Biol. Chem. 1986; 261: 1883-1889Abstract Full Text PDF PubMed Google Scholar). A 140-kDa matrix protein, ladsin, which appears to be identical to the laminin β2 chain, has been shown to promote scattering of carcinoma cells, stimulate cell migration, and bind to α3β1 integrin (9Miyazaki K. Kikkawa Y. Nakamura A. Yasumitsu H. Umeda M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11767-11771Crossref PubMed Scopus (151) Google Scholar, 10Kikkawa Y. Umeda M. Miyazaki K. J. Biochem. (Tokyo). 1994; 116: 862-869Crossref PubMed Scopus (105) Google Scholar). Thus, it is possible that laminin/integrin interactions function not only in cell adhesion but also to transduce biochemical signals that modulate surface activities of degradation and invasion (11Ramos D.M. Berston E.D. Kramer R.H. Cancer Res. 1990; 50: 728-734PubMed Google Scholar, 12Seftor R.E.B. Seftor E.A. Stetler-Stevenson W.G. Hendrix M.J.C. Cancer Res. 1993; 53: 3411-3415PubMed Google Scholar, 13Kornberg L. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar).Previously, we showed that a membrane-bound, 170-kDa gelatinase, seprase, and secreted, metallo-type gelatinase A were localized on invadopodia of LOX human melanoma cells during degradation/invasion of fibronectin-coated cross-linked gelatin films (14Aoyama A. Chen W.-T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8296-8300Crossref PubMed Scopus (104) Google Scholar, 15Monsky W.L. Lin C.-Y. Aoyama A. Kelly T. Mueller S.C. Akiyama S.K. Chen W.-T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar). Further analysis using fibronectin, laminin, type IV collagen, type I collagen, and Matrigel substrata demonstrated that degradation of these ECM components by transformed and tumor cells occurred at invadopodia, suggesting membrane association and activation of latent proteases during invasion (15Monsky W.L. Lin C.-Y. Aoyama A. Kelly T. Mueller S.C. Akiyama S.K. Chen W.-T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar, 16Kelly T. Mueller S.C. Yeh Y. Chen W.-T. J. Cell Physiol. 1994; 158: 299-308Crossref PubMed Scopus (86) Google Scholar). However, no specific membrane-associated or secreted protease has yet been associated with an induced invasion. Recently, a systematic screening for cell binding sites with 113 overlapping synthetic peptide beads covering the laminin α1 chain carboxyl-terminal globular domain (G domain amino acid residues 2111-3060) resulted in the identification of 19 potential adhesion-active sequences (17Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Here, we identified the peptides designated AG-10 and AG-32 that ligated α6β1 integrin in a similar manner as anti-β1 and α6 integrin antibodies promote melanoma invasiveness independently of the adhesion function of integrin receptors. These soluble peptides and antibodies could signal cell invasion at sites of ECM contact. We found that, accompanying the induced invasiveness, there was an increased organization of seprase on invadopodia, but the expression levels of seprase, gelatinase A, and β1 integrins were not altered.