1991;51:5054s-5059s. Cancer Res

Lance A. Liotta and William G. Stetler-Stevenson

Negative RegulationTumor Invasion and Metastasis: An Imbalance of Positive and

Updated version

http://cancerres.aacrjournals.org/content/51/18_Supplement/5054sAccess the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

.pubs@aacr.orgDepartment atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

.permissions@aacr.orgDepartment atTo request permission to re-use all or part of this article, contact the AACR Publications

on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

(CANCER RESEARCH (SUPPL.) 51. 5054s-5059s, September 15. 1991]

Tumor Invasion and Metastasis: An Imbalance of Positive and Negative Regulation1Lance A. Liotta and William G. Stetler-StevensonLaboratory of Pathology, National Cancer Institute, N1H, Bethesda, Maryland 20892

Abstract

A group of coordinated cellular processes, not just one gene product,is responsible for invasion and metastasis, the most life-threateningaspect of cancer. It is now recognized that negative factors may be justas important as positive elements. Genetic changes causing an imbalanceof growth regulation lead to uncontrolled proliferation necessary for bothprimary tumor and metastasis expansion. However, unrestrained growthdoes not, by itself, cause invasion and metastasis. This phenotype mayrequire additional genetic changes. Thus, tumorigenicity and metastaticpotential have both overlapping and separate features. Invasion andmetastasis can be facilitated by proteins which stimulate tumor cellattachment to host cellular or extracellular matrix determinants, tumorcell proteolysis of host barriers, such as the basement membrane, tumorcell locomotion, and tumor cell colony formation in the target organ formetastasis. Facilitory proteins may act at many levels both intracellulari)or extracellularly but are counterbalanced by factors which can blocktheir production, regulation, or action. A common theme has emerged. Inaddition to loss of growth control, an imbalanced regulation of motilityand proteolysis appears to be required for invasion and metastasis.

Metastatic Cells Can Dominate the Primary TumorPopulation

Metastasis is a cascade of linked sequential steps involvingmultiple host-tumor interactions (1-3). To successfully createa metastatic colony, a cell or group of tumor cells must be ableto leave the primary tumor, invade the local host tissue, enterthe circulation, arrest at the distant vascular bed, extravasateinto the target organ interstitium and parenchyma, and proliferate as a secondary colony. Angiogenesis is required for theexpansion of the primary tumor mass, and new blood vesselspenetrating the tumor are frequent sites for tumor cell entryinto the circulation (4, 5). Angiogenesis is also required forexpansion of the metastatic colony. At any stage, tumor cellsmust overcome host immune cell killing (2). A very smallpercentage (

TUMOR INVASION AND METASTASIS

to 8 h after attachment, a localized zone of lysis is produced inthe basement membrane at the point of tumor cell contact.Tumor cells directly secrete degradative enzymes (17) or inducethe host to elaborate proteinases to degrade the matrix and itscomponent adhesion molecules. Matrix lysis takes place in ahighly localized region close to the tumor cell surface (18),where the amount of active enzyme outbalances the naturalproteinase inhibitors present in the serum, those in the matrix,or that secreted by normal cells in the vicinity.

Locomotion is the third step of invasion which propels thetumor cell across the basement membrane and stroma throughthe zone of matrix proteolysis. An early step in locomotion ispseudopodial protrusion at the leading edge of the migratingcell (19, 20). The induction of pseudopodia is directional, isregulated by cell surface ligand binding, and involves a coordinated mobilization of cytoskeletal elements which interact withthe inner membrane surface. The cellular machinery of tumorcell locomotion is a fertile topic for future study and willundoubtably benefit from insights learned from studies of immune cells (2) and nonmammalian organisms (20). It is nowrecognized that random tumor cell motility can be regulated bytumor cell cytokines ("autocrine motility factors" and "scatterfactors") (19, 21, 22). Autocrine motility factors act through areceptor-activated G protein susceptible to inhibition by pertussis toxin. Augmented random motility by tumor cells causesdispersion at the primary site. In addition, the direction andsite of the tumor cell locomotion may be influenced by hostorgan-derived chemoattractants. Such chemoattracts could playa role in the organ-selective homing of metastasis. This couldcomplement other mechanisms of organ homing which includepreferential adhesion to organ-specific endothelium and preferential growth in selected organs due to local growth factors(23).

Proteinases: from Correlation to Causality

A general aspect of malignant neoplasms may be an imbalance of proteolysis which favors invasion. However, proteolysisof tissue barriers is not a property unique to tumor cells. It isutilized, for example, during trophoblast implantation, embryomorphogenesis, tissue remodeling, parasitic and bacterial invasion, and angiogenesis. Furthermore, the defect in the tumorcell cannot be simply unbridled production of degradative enzymes. This is because cell migration during invasion requiresattachment and detachment of the cell as it moves forward.Lysis of all matrix components around the tumor cell wouldsimply remove the substratum necessary for proper cell traction.Thus, it is probable that the invading tumor cell uses proteolysisin a highly organized manner both spatially and temporallywhich does not differ functionally from the operating mode ofnormal cells which migrate through tissue barriers. The difference is that tumor cells couple proteolysis with motility toachieve invasion at times and places which would be inappropriate for normal cells.

The metastasis field has progressed from establishing a correlation between proteolysis and malignant progression to thefinding that the actual blockade of certain proteinases willprevent invasion and metastasis. A positive association withtumor aggressiveness has been noted for a variety of classes ofdegradative enzymes including heparanases (24, 25), serine(26), thiol (27, 28), and metal-dependent enzymes (29-32).Indeed, a cascade including all these enzymes is probably involved in the invasive process, and more than one enzyme is

necessary but not sufficient. This conclusion is justified by thefinding in multiple laboratories that inhibitors for metallopro-teinases or inhibitors of serine proteinases can each block tumorcell invasion of native or reconstituted connective tissue barriersin vitro (33-36). Thus, the enzymes involved in tumor invasionand metastasis may well resemble the proteolytic cascadesinvolved in blood coagulation.

Plasminogen activator, specifically uPA,2 has been closelylinked to the metastatic phenotype (37, 38). Anti-uPA antibodies block human HEP-3 cell invasion in the chick chorioallan-toic membrane assay and murine B16F10 melanoma cell metastasis following tail vein injection (39, 49). Overexpression ofuPA in Ha-ra--transformed 3T3 cells enhanced lung invasionand experimental metastasis formation (41). Serine proteinaseinhibitors also block tumor cell invasion through human am-niotic membranes (35).

Among the list of enzymes involved in cancer, a large bodyof information has been accumulated concerning the matrixmetalloproteinase gene family (Fig. 1). These enzymes havebeen subgrouped into three broad categories based on substratepreference: interstitial collagenases; type IV collagenases(gelatinases), and stromelysins (42). The interstitial collagenaseis the best characterized and specifically degrades type I collagen (32). Neutrophil collagenase, which has recently beencloned, appears very similar in substrate specificity (43). Thestromelysins are three related gene products, stromelysin, stro-melysin 2, and PUMP-1, which degrade a variety of matrixcomponents including proteoglycans and noncollagenous gly-coproteins such as laminin and fibronectin, as well as thenoncollagenous domains of type IV collagen. The role of stromelysin in squamous progression has recently been recognized(44). The type IV collagenases are named for their selectiveability to cleave type IV collagen in a pepsin-resistant triple-helical domain thus generating characteristic one-fourth amino-terminal and three-fourths carboxyl-terminal fragments (45).Both a 72-kDa and 92-kDa type IV collagenase exist andcomplementary DNA cloning has demonstrated that each is aunique gene product (46, 47).

All classes of matrix metalloproteinases are secreted as inactive zymogens and enzyme activation is an important controlstep in proteolysis. A new model for matrix metalloproteinaseproenzyme activation has been proposed (48-51 ). The essentialfeature of this model is that the latent form of the matrixmetalloproteinase enzymes all have a metal atom sulfhydrylside chain interaction that results in a catalytically inert activecenter. The sulfhydryl group in this interaction is donated bythe CYS-73 residue which is contained in a highly conservedpeptide present in all known members of this metalloproteinasefamily. The metal atom is presumably the zinc atom of theactive site. Disruption of this interaction results in conforma-tional rearrangement and rapid attainment of protease activity.The implication of this model is that mutations in the CYS-73residue would result in intrinsic enzyme activation and that thiscould play a role in tumor progression. The in vivo mechanismof normal metalloproteinase activation is unknown but mayinvolve the action of other proteinases either in solution or viaa cell surface-dependent mechanism (18, 52, 53). The latterwould allow for precise cellular control at the point of matrixinteraction.

Among the matrix metalloproteinase family members, anaccumulating body of evidence supports a positive correlation

2The abbreviations used are: uPA. urokinase-type plasminogen activator:TIMP. tissue inhibitor of metalloproteinases; NDP. nucleoside diphosphate.

5055s

on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TUMOR INVASION AND METASTASIS

72 kD-Type IV ProCollagenase 3.2kbglallnbinding domainCCCCCCCCCCCC

Active Type IV Collagenase

CCCCCCCCCCCC C C C C

MBDipi i* 2 (V)

92 kD-Typc IV ProCollagenase 2.8kb

MBD ProCollagenaseI 2.0kb

ijijijjjjjiliiijjjil MBD V//////////A Sagenas, 3.3kb

PfStromelysin i 9kb

!!;!!!ii!|MBD ProStromeiysin-2 1.7M

PUMP-1

I MBD | VAAHEFGHAMGLEHS

H PRCGVPNPD

Fig. 1. Matrix metalloproteinase family. Type IV procollagenase (72 kDa and92 kDa forms), interstitial procollagenase, neutrophil procollagenase, prostro-melysin, prostromelysin-2, and PUMP-1. are represented diagrammatically andaligned to show regions of protein sequence homology. MBD, active site metalion-binding domain. Type IV procollagenases contain a cysteine-rich, substrate-binding domain which shows homology to fibronectin but is absent from theother matrix metalloproteinases. Upon treatment with organomercurial compounds m vitro, all seven enzymes are activated with the concomitant removal ofan amino-terminal segment of the latent enzyme. The removed segment containsan unpaired cysteine residue within the conserved amino acid sequencePRCGVPDV located immediately adjacent to the proenzyme cleavage site. Site-directed mutagenesis studies have shown that alterations in this sequence resultin spontaneous activation of transin, the rat homologue of stromelysin. In thelatent proenzyme, this sequence interacts with the metal ion through the unpairedcysteinyl residue to block activity. Perturbation of this interaction affects activation. A/I.kilobases.

between type IV collagenase activity and tumor cell invasion(24, 31, 54, 55). Augmented type IV collagenase activity isassociated with the genetic induction of a metastatic phenotype(56-59). Furthermore, use of agents which specifically inhibittype IV collagenase activity or block collagenase secretionprevents tumor cell invasion in vitro (26, 60). Immunohisto-chemical studies using affinity purified anti-72-kDa type IVcollagenase antibodies (48, 49) demonstrate that low levels ofthis enzyme are produced by normal, nontumorigenic, non-metastatic cells such as the myoepithelial cells of the humanbreast (61). Benign proliferative lesions of the breast, benignpolyps of the colon, and normal colorectal and gastric mucosaall show negligible immunoreactivity for 72-kDa type IV collagenase. In contrast, almost all invasive colonie and gastricadenocarcinomas are positive for this antigen (62, 63). Down-regulation of type IV collagenotypic activity by retinoic acidtreatment of human melanoma cells has been correlated with aloss of the invasive phenotype (54). Studies of the mechanismof this effect reveal that retinoic acid treatment of these humanmelanoma cells results in a reduction of the steady state level

of the 72-kDa type IV collagenase mRNA and loss of theinvasive capacity (64). These results suggest that the 72-kDatype IV collagenase enzyme is a normal cell component that isdramatically overexpressed in many invasive and metastatichuman cancers.

Natural Proteinase Inhibitors Suppress Invasion

The secretion and activation of metalloproteinases are notenough to ensure that they will degrade the target matrixsubstrate (17, 65). This is because natural inhibitor proteins,produced either by the host or by the tumor cell itself, can blockthe latent or the active metalloproteinases (17). Natural pro-teinase inhibitor proteins, such as TIMPs (66) plasminogenactivator inhibitors may therefore function as metastasis suppressor proteins which act to inhibit tumor cell invasion of theextracellular matrix. TIMP-1, the original member of the TIMPfamily (67), is a glycoprotein with an apparent molecular sizeof 28.5 kDa which forms a complex of 1:1 stoichiometry withactivated interstitial collagenase, activated stromelysin, and the92-kDa type IV collagenase. It has been reported that transfec-tion of antisense RNA which blocks TIMP-1 expression enhances the malignant phenotype (68). One explanation for thisresult is that the antisense RNA blocked the production ofTIMP-1 which normally prevented the malignant phenotype.In animal models, administration of recombinant TIMP-1blocks metastasis (69, 70).

Recently, Stetler-Stevenson et al. (49, 71) have isolated,purified, determined the complete primary structure, and clonedthe first new member of the TIMP family, TIMP-2. An identicalinhibitor was isolated from endothelial cells by DeClerk et al.(72, 73). TIMP-1 and TIMP-2 are regulated independently andoppositely by 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor ,and other cytokines. TIMP-2 is a 21-kDa protein which selectively forms a complex with the latentproenzyme form of the 72-kDa type IV collagenase. The secreted protein has 194 amino acid residues and is not glycosy-lated. TIMP-2 shows 37% identity and overall 65.6% homologyto TIMP-1 at the deduced amino acid sequence level. Thepositions of the 12 cysteine residues in TIMP-2 are conservedwith respect to those present in TIMP-1, as are 3 of the 4tryptophan residues; yet the 2 proteins are immunologicallydistinct. TIMP-2 inhibits at a 1:1 ratio the type IV collageno-lytic activity and the gelatinolytic activity associated with the72-kDa enzyme. Unlike TIMP-1, TIMP-2 is capable of bindingto both the latent and activated forms of the 72-kDa type IVcollagenase and will abolish the hydrolytic activity of all members of the metalloproteinase family (49, 74). The 92-kDa typeIV procollagenase can be found as a complex with TIMP-1(46). Activation of either the latent 72-kDa or 92-kDa type IVcollagenase-TIMP complex can be reversed by binding of asecond role of TIMP-1 or TIMP-2. This suggests that on theseenzymes there are two separate TIMP-binding sites and thatbinding of TIMP-1 or TIMP-2 to the latent proenzymes servesa different function than the inactivation that occurs followingbinding to the active species. The areas of the two proteinswhich differ in homology may contain the regions responsiblefor the functional differences (71). The net 72-kDa type IVcollagenase activity consequently depends upon the balancebetween the levels of activated enzyme and TIMP-2. TIMP-2is a potent inhibitor of cancer cell invasion through reconstituted extracellular matrix (75). TIMP-2 produced by the sametumor cells which make collagenase, therefore, exists as anatural suppressor of invasion.

5056s

on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TUMOR INVASION AND METASTASIS

Metastasis and Tumorigenicity Can Be under SeparateGenetic Control

Five years ago investigators in the metastasis field were surethat the new developments in oncogenes were relevant only totumorigenicity and that separate genes would be found whichevoke the metastatic process. At the time, biological assays ofoncogenes were restricted to scoring tumorigenicity. Little attention had been paid to metastasis formation. Nevertheless,when the proper studies were done, it was found that transfec-tion of certain oncogenes in the correct recipient cell couldinduce the complete phenotype of invasion and metastasis.Although the search for specific metastasis-inducing genes goeson, oncogene transfection has provided a model to switch onthe effector processes which are required for the cell to carryout invasion and metastasis. These models have revealed thatsome of the metastasis effector genes can be regulated independently from those which confer tumorigenicity.

The incidence of aberrant gene expression and genetic alterations of the ras and myc gene families have been shown to beimportant in progression of human cancers and may be usefulas prognostic indicators (76). Thorgeirsson et al. (77) were thefirst to report that the activated (mutated) ras oncogene sequences, when transfected into mouse embryo-derived fibro-blasts (NIH-3T3 cells), produced numerous mtastases.Theresultant highly metastatic cells were not more resistant to hostimmune cell lysis (macrophage or natural killer cells) comparedto control cells, indicating that the ras oncogene had augmentedthe intrinsic aggressiveness of the NIH-3T3 cells. Transfectionof H-ras-family oncogenes has been shown to induce metastasisin fibroblasts and epithelial cells of rodent and human origin(56, 57, 59, 78-81). However, H-ras is not the only oncogenewhich can induce metastatic potential. At lower efficiency, theserine-threonine kinases \-mos, v-raf, and A-ra/(79, 82); tyro-sine kinases v-src, \-fes, and \-fms (79); and the mutated phos-phoprotein p53 (83) have been demonstrated to induce themetastatic phenotype in the appropriate recipient cell.

Experimental evidence indicates that invasion and metastasisrequire activation of a set of effector genes over and above thosewhich are required for unstrained growth alone. The downstream pathways used in ras induction of tumorigenicity andmetastasis have dissimilar features: (a) the adenovirus 2 Eiagene has been demonstrated to suppress ras induction of metastatic potential with no inhibition of soft agar colony formation or tumorigenicity (84); and (b) cells are capable of beingtransformed by ras but do not metastasize (56, 85). The failureof ras to induce metastasis in certain experimental systemsprobably reflects a deficiency in, or a suppression of, some ofthese effector proteins. Several candidate effector proteins havebeen associated with metastasis in ras transfection models, suchas proteinases including type IV collagenase (46, 57, 59, 77),cathepsin L (86), and motility-associated cytokines (21). Thus,in these ras transfection models certain effector genes areactivated, or suppressed, possibly in coordinated manner, toinduce metastasis formation. Studies have revealed that severaloncogenes such as \-src and ras, tumor-promoting phorbolesters and growth factors such as epidermal growth factor andplatelet-derived growth factor will induce transin (rat homologue of human stromelysin) mRNA transcript levels. Theobservation that these agents all induce a rapid stimulation ofc-fos that precedes the induction of transin mRNA and theknowledge that protein synthesis is required for this inductionsuggests that c-fos may act as a "third messenger" and may

directly modulate transin gene transcription (44, 87). Some ofthese oncogene-associated effector genes may regulate cell mo-tility and proteolysis, and this forms a common thread withseparate work on proteinases, motility, and angiogenesis.

Several metastasis suppressor genes have been reported intransfection experiments. The Adenovirus 2 Eia gene, previously discussed, suppressed c-Ha-ras induction of metastaticbehavior of rat embryo fibroblasts, as assayed by tail veininjections (84). In the same model system Gattoni-Celli reported that ras and Eia cotransfected rat embryo fibroblastsexpressed higher levels of major histocompatibility complexclass I genes (88). When the H-2Kb major histocompatibilitycomplex gene was transfected into rat embryo fibroblasts, previously transfected with ras, reduced rates of tumorigenesis andmetastasis were observed upon injection into triple-deficientmice (89). The data suggest the involvement of H-2Kb in armsof the immune response, such as macrophage-mediated cyto-toxicity, or in the suppression of nonimmunological aspects ofthe tumor metastatic process.

TIMP-1 was demonstrated to suppress the metastatic potential of Swiss 3T3 cell using antisense transfection. The antisenseTIMP-1 construct reduced TIMP activity by 47-68% in thetransfected cells and increased invasiveness in an in vitro am-nion assay, as well as tumorigenicity and metastatic potentialin vivo (68).

The nm23 gene was identified on the basis of its reducedsteady state RNA levels in five highly metastatic K-1735 melanoma cell lines, as compared to two related, low metastaticpotential k-1735 melanoma cell lines (90). In human breastcancer reduced nm23 RNA levels have been associated with thepresence of lymph node mtastasesat surgery (91) as well asdecreased patient disease-free and overall survival (92). In othercancer cell types, such as colorectal carcinoma, no significantassociation of nm23 RNA levels with metastatic progressionwas observeed (93). Transfection of the murine nm23-\ complementary DNA into highly metastatic murine K-1735 TKmelanoma cells resulted in a reduced incidence of primarytumor formation, significant reductions in tumor metastaticpotential, and altered tumor cell responsiveness to the cytokinetransforming growth factor in vitro (94).

An important clue to nm23 function(s) came from its virtualidentity with the Drosophila awd gene product (95-96). Mutations which result in reduced awd expression or the productionof a mutated protein do not significantly alter embryonic development but do alter the development of multiple tissuespostmetamorphosis, when presumptive adult tissue in the imaginai discs begins to divide and differentiate. These abnormalities include altered morphology of the wing discs, larval brainand proventriculus; aberrant differentiation of the wing, leg,and eye-antennae imaginai discs and ovaries; and cell necrosis,predominantly in the wing discs. nm23/awd may contribute tothe normal development of tissues, which may include signaltransduction of cell to cell communication. Loss of nm23/a\vdexpression may lead to a disordered state, favoring aberrantdevelopment or tumor progression to the metastatic state.

References1. Fidler, I. J., and Hart. 1. R. Biologic diversity in metastatic neoplasms

origins and implications. Science (Washington DC). 217: 998-1001, 1982.2. Schirrmacher, V. Experimental approaches, theoretical concepts, and im

pacts for treatment strategies. Adv. Cancer Res., 43: 1-32, 1985.3. Liotta, L. A., Rao, C. N., and Barsky, S. H. Tumor invasion and the

extracellular matrix. Lab. Invest., 49: 363, 1983.4. Folkman, J., and Klagsbrun, M. Angiogenic factors. Science (Washington

DC), 235: 442-447, 1987.5057s

on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TUMOR INVASION AND METASTASIS

5. Folkman, J.. Watson, K.. Ingber, D., and Hanahan, D. Induction of angio- 34.genesis during the transition from hyperplasia to neoplasia. Nature (Lond.),J9: 58-61. 1981.

6. Kerbel. R. S. Growth dominance of the metastatic cancer cell: cellular and 35.molecular aspects. Adv. Cancer Res.. 55: 87-131. 1990.

7. McGuire. W. L., Tandon. A. K.. Allred. D. C.. Chamness. G. C, and Clark.G. M. How to use prognostic factors in axillary node-negative breast cancer 36.patients. J. Nati. Cancer Inst.. 2:1006-1015," 1990.

8. Slamon. D. J.. Godolphin. W., Jones. L. A.. Holt. J. A.. Wong, S. G.,Keith, D. E., Levin, W. E., Stuart. S. G., Udove. J.. Ullrich. A., and Press. 37.M. F. Studies of the HER-2/neu proto-oncogene in human breast andovarian cancer. Science (Washington DC). 244: 707. 1989. 38.

9. Lidereau, R.. Callahan. R., Dickson. C., Peters, G., Escot, C., and Ali. I.U. Amplification of the int-2 gene in primary human breast tumors. Oncogene Res., 2.-285. 1988. 39.

10. Tandon. A. K.. Clark. G. M., Chamness, G. C., et al. Cathepsin D andprognosis in breast cancer. N. Engl. J. Med., 322: 297. 1990. 40.

11. Yurchenco. P. D.. and Schittny, J. C. Molecular architecture of basementmembranes. FASEB J.. 4: 1577-1590. 1990.

12. Barsky, S. H., Siegal. G. P., Jannotta. F., tal.Loss of basement membrane 41.components by invasive tumors but not by their benign counterparts. Lab.Invest., 49: 140. 1983.

13. Hynes. R. O. Integrins: a family of cell surface receptors. Cell, 48: 549. 42.1987.

14. Hymphries. M. J., Olden. K., and Vamada, K. M. A synthetic peptide fromfibronectin inhibits experimental metastasis of murine melanoma cells.Science (Washington DC). 233:467. 1986. 43.

15. Rao. C.. Castronovo. V., Schmitt. M. C.. Wewer, U., Claysmith, A., Liotta,L. A., and Sobel, M. Evidence for a precursor of the high-affinity metastasisassociated murine latninin receptor. Biochemistry. 28: 7476-7486, 1989. 44.

16. Aznavoorian, S., Stracke, M. L., Krutzsch. H., et al. Signal transductionfor chemotaxis and haptotaxis by matrix molecules in tumor cells. J. Cell 45.Biol., 110: 1427, 1990.

17. Gottesman, M. The role of proteases in cancer. Semin. Cancer Biol., /: 97-160, 1990. 46.

18. Brown. P. D., Levy. A. T., Margulies. I., Liotta, L.. and Stetler-Stevenson.W. G. Independent expression and cellular processing of the 72-kDa typeIV collagenase and interstitial collagenase in human tumorigenic cell lines.Cancer Res.. 50: 6184-6191,1990. 47.

19. Guirguis, R., Margulies, I. M. K., Taraboletti, G., et al. Cytokine-inducedpseudopodial protrusion is coupled to tumor cell migration. Nature (Lond.),329:2f>\, 1987.

20. Luna. E. J.. Wuestehube. L. J., Ingalls. H. M.. and Chia, C. P. The 48.Dictyostelium discoideum plasma membrane: a model system for the studyof actin-membrane interactions. Adv. Cell Biol., 3: 1-33, 1989.

21. Liotta, L. A., Mandler, R., Murano, G., Katz, D. A., Gordon, R. K., Chiang. 49.P. K., and Schiffmann, E. Tumor cell autocrine motility factor. Proc. Nati.Acad. Sci. USA, S3: 3302-3306, 1986.

22. Gherardi, E., Gray. J., Stoker, M., Perryman. M.. and Furlong. Purification 50.of scatter factor, a fibroblast derived basic protein that modulates epithelialinteractions and movement. Proc. Nati. Acad. Sci. USA. 86: 5844-5848, 51.1989.

23. Nicolson, G. L. Organ specificity of tumor metastasis: role of preferentialadhesion, invasion, and growth of malignant cells at specific secondary sites.Cancer Metastasis Rev.. 7: 143-188, 1988. 52.

24. Nakajima, M., Welch, D., Belloni, P. N., and Nicolson. G. L. Degradationof basement membrane type IV collagen and lung subendothelia! matrix byrat mammary adenocarcinoma cell clones of differing metastatic potentials.Cancer Res.. 47:4869-4876. 1987. 53.

25. Nakajima, M., Morikawa. K.. Fabra, A., Bucana, C. D., and Fidler, I. J.Influence of organ environment on extracellular matrix degradative activityand metastasis of human colon carcinoma cells. J. Nati. Cancer Inst., inpress. 1991. 54.

26. Reich. R., Thompson. E., Iwamoto, Y.. Martin, G. R., Deason. J. R., Fuller.G. C., and Miskin, R. Effects of inhibitors of plasminogen activator, serineproteinases and collagenase IV on the invasion of basement membranes bymetastatic cells. Cancer Res., 48: 3307-3312. 1988.

27. Recklies, A. D., Poole, A. R., and Mort, J. S. A cysteine proteinase secreted 55.from human breast tumours is immunologically related to cathepsin B.Biochem. J., 207: 633, 1982.

28. Sloane, B. R.. and Honn, K. V. Cysteine proteinases and metastasis. CancerMetastasis Rev., 3: 249. 1984. 56.

29. Ostrowski, L. E., Rinch, J., Kreig, P., and Matrisian, L. Expression patternof a gene for a secreted metalloproteinase during late stages of tumorprogression. Mol. Carcinog., /: 13-19, 1988. 57.

30. Liotta, L. A., Abe, S.. Robey, P., and Martin. G. Preferential digestion ofbasement membrane collagen by an enzyme derived from a metastaticmurine tumor. Proc. Nati. Acad. Sci. USA, 76: 2268-2272, 1979. 58.

31. Liotta. L. A., Tryggvason, K., Garbisa, S., et al. Metastatic potentialcorrelates with enzymatic degradation of basement membrane collagen.Nature (Lond.). 284: 67-68, 1980.

32. Templeton. N. S., Brown, P. D., Levy. A. T., Margulies, I. M. K., Liotta, 59.L. A., and Stetler-Stevenson. W. G. Cloning and characterization of humantumor cell interstitial collagenase. Cancer Res., 50: 5431-5437, 1990.

33. Thorgeirsson, U. P., Liotta, L. A., Kalebic. T.. et al. Effect of naturalprotease inhibitors and a chemoattractant on tumor cell invasion in vitro. J.Nati. Cancer Inst., 69: 1049, 1982. 60.

5058s

Wang, M., and Stearns, M. E. Blocking of collagenase secretion by esta-mustine during in vitro tumor cell invasion. Cancer Res., 48: 6262-6271.1988.Mignatti. P.. Robbins. E., and Rifkin, D. Tumor invasion through thehuman amniotic membrane: requirement for a proteinase cascade. Cell, 47:487-498. 1986.Mignatti. P.. Tsuboi, R., Robbins. E.. and Rifkin. D. B. In vitroangiogenesison the human amniotic membrane: requirement for basic fibroblast growthfactor-induced proteinases. J. Cell Biol., 108: 671-682, 1989.Dao,K., Andreasen, P. A., Grondahl-Hansen, J., et al. Plasminogenactivators, tissue degradation and cancer. Adv. Cancer Res., 44: 139, 1985.Sappino, A-P., Busso, N., Belin, D., and Vassali. J-D. Increase of urokinasetype plasminogen activator gene expression in human lung and breastcarcinomas. Cancer Res.. 47: 4043, 1987.Ossowski, L., and Reich, E. Antibodies to plasminogen activator inhibithuman tumor metastasis. Call, 35: 611-619, 1983.Escheicher, A., Wohlwend, A., Belin. D., and Vassalli. J. D. Characterization of the cellular binding site for the urokinase type plasminogen activator.J. Biol. Chem.. 264: 1180-1189. 1989.Axelrod. J. H.. Reich. R.. and Mishkin, R. Expression of human recombinant plasminogen activators enhance invasion and experimental metastasisof Ha-ras transformed NIH 3T3 cells. Mol. Cell. Biol., 9:2133-2141, 1989.Wilhelm, S., Collier, I., Kronberg, A., Eisen. A., Marmer. B.. Grant, G., etal. Human skin fibroblast stromelysin: structure, glycosylation, substratespecificity, and differential expression in normal and tumorigenic cells.Proc. Nati. Acad. Sci. USA. 84: 6725-6729. 1987.Hasty. K. A.. Pourmotabbed. T. F., Goldberg. G. 1., Thompson, J. P.,Spinella, D. G., Stevens, R. M., and Mainardi. C. M. Human neutrophilcollagenase. J. Biol. Chem., 265: 11421-11424. 1990.Matrisian, L., and Bowden, T. Stromelysin/transin and tumor progression.Semin. Cancer Biol., /: 107-115, 1990.Fessier, L., Duncan. K., and Tryggvason, K. Identification of the procollagenIV cleavage products produced by a specific tumor collagenase. J. Biol.Chem.. 259: 9783-9789. 1984.Collier. I. E.. Wilhelm. S. M., Eisen, A. Z., et al. H-ras oncogene transformed human bronchial epithelial cells (TBE-1) secrete a single metalloproteinase capable of degrading basement membrane collagen. J. Biol.Chem., 263: 6579-6587. 1989.Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A.,and Goldberg, G. I. SV40-transformed human lung fibroblasts secrete a 92-kDa type-I V collagenase which is identical to that secreted by normal humanmacrophages. J. Biol. Chem., 264: 17213-17221. 1989.Stetler-Stevenson, W. G., Krutzsch, H. C., Wacher, M. P., Margulies, I. M.K., and Liotta. L. A. The activation of human type IV collagenase proen-zyme. J. Biol. Chem., 264: 1353-1356, 1989a.Stetler-Stevenson, W. G., Krutzsch, H. C, and Liotta, L. A. TIMP-2, anew member of the metalloproteinase inhibitor family. J. Biol. Chem., 264:17374-17378, 1989b.Vallee, B. L., and Auld. D. S. Zinc coordination, function and structure ofzinc enzymes and other proteins. Biochemistry. 29: 5647-5659, 1990.Van Wart, H. E., and Birkedal-Hansen, H. The cysteine switch: a principleof regulation of metalloproteinase activity with potential applicability to theentire matrix metalloproteinase gene family. Proc. Nati. Acad. Sci. USA,7:5578-5582, 1990.Herrn. G. S., Banda, M. J., Clark, E. J., Gavrilovic, J., and Werb, Z.Secretion of metalloproteinases by stimulated capillary endothelial cells. II.Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J. Biol. Chem.. 261: 2814-2818. 1986.He, C., Wilhelm, S. M., Pentland, A. P., Marmer. B. L.. Grant, G. A.Eisen. A. Z., and Goldberg, G. I. Tissue cooperation in a proteolytic cascadeactivating human interstitial collagenase. Proc. Nati. Acad. Sci. USA, 86:2632-2636. 1989.Nakajima, M.. Lotan, D.. Baig, M. M., Carralero, R. M., Wood, W. R.,Hendrix, M. J. C., and Lotan, R. Inhibition of retinoic acid of type IVcollagenolysis and invasion through reconstituted basement membrane bymetastatic rat mammary adenocarcinoma cells. Cancer Res.. 49: 1698-17061989.Turpeenniemi-Hujanen. T., Thorgeirsson. U. P., Hart, 1. R.. et al. Expression of collagenase IV (basement membrane collagenase) activity in murinetumor cell hybrids that differ in metastatic potential. J. Nati. Cancer Inst.,75:99-108. 1985.Muschel, R. J.. Williams, J. E., Lowy. D. R., and Liotta, L. A. Harvey rasinduction of metastatic potential depends upon oncogene activation and thetype of recipient cell. Am. J. Pathol., 121: 1-8, 1985.Garbisa, S., Pozzatti, R., Muschel. R., et al. Secretion of type IV collagen-olytic protease and metastatic phenotype: induction by transfection with c-Haras but not C-Ha-ras plus Ad2-Ela. Cancer Res., 47: 1523-1528, 1987.Bonfil. D. R., Reddel, R. R., Ura, H.. Reich, R., Fridman, R.. Harris, C.C., and Klein-szanto. A. J. P. Invassive and metastatic potential of a v-Ha-ras-transformed human bronchial epithelial cell line. J. Nati. Cancer Inst..81: 587-594, 1989.Ura. H.. Bonfil, R. D., Reich, R., Reddel. R.. Pfeifer. A., Harris, C. C., andKlein, A. J. P. Expression of type IV collagenase and procollagen genes andits correlation with the tumorigenic. invasive, and metastatic abilities ofoncogene-transformed human bronchial epithelial cells. Cancer Res., 49:4615-4621. 1989.Wang. B. S., McLouglin, G. A.. Richie. J. P., and Mannick, J. A. Correla-

on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TUMOR INVASION AND METASTASIS

tion of the production of plasminogen activator with tumor metastasis inB16 mouse melonoma cell lines. Cancer Res., 40: 288, 1980.

61. Monteagudo, C., Merino, M.. San-Juan. J.. Liotta, L., and Stetler-Steven-son. VV.Immunohistologic distribution of type IV collagenase in normal,benign and malignant breast tissue. Am. J. Pathol., 136: 585-592, 1990.

62. D'Errico. A.. Garbisa, S., Liotta. L. A., Castronovo, V., Stetler-Stevenson,W. C.. and Griogioni. W. G. Augmentation of type IV collagenase, lamininreceptor, and K67proliferation antigen associated with human colon,gastric and breast carcinoma progression. Mod. Pathol.. in press, 1991.

63. Levy, A., Cioce, V., Sobel, M. E., Garbisa, S., Griogioni, W. F.. Liotta, L.A., and Stetler-Stevenson, W. G. Increased expression of the 72 kDa typeIV collagenase in human colonie adenocarcinoma. Cancer Res., 51: 439-444. 1991.

64. Hendrix. M., Wood, R.. Seftor, E., Lotan, D., Nakajima. M.. Misiorowski.R., Seftor. R. E. B., Steler-Stevenson, W. G., Bevacqua, S. J., Liotta, L. A.,Sobel. M. E., Raz, A., and Lotan. R. Retinoic acid inhibition of humanmelanoma cell invasion through a reconstituted basement membrane andits relation to decreases in the expression of proteolytic enzymes and motilityfactor receptor. Cancer Res.. 50: 4121-4130, 1990.

65. Levin. E. G., and Santell. L. Association of a plasminogen activator inhibitor(PAI-1) with the growth substratum and a membrane of human endothelialcells. J. Cell Biol., 105: 2543-2649, 1987.

66. Carmichael, D. F., Sommer, A., Thomson, R., Anderson, D. C., Smith, C.G., Welgus, H. G., and Snick Iin. G. P. Primary structure and cDNA cloningof human fibroblast collagenase inhibitor. Proc. Nati. Acad. Sci. USA, 83:2407-2411. 1986.

67. Murphy. G.. Cawston. T.. and Reynolds, J. An inhibitor of collagenasefrom human amniotic fluid. Purification, characterization and action onmetalloproteinases. Biochem. J.. 795: 167-170, 1981.

68. Khokha, R., Waterhouse. P.. Yagel. S.. Lala, P. K.. Overall. C. M., Norton,G.. and Denhardt. D. T. Antisense RNAinduced reduction in metallopro-teinase inhibitor causes mouse 3G3 cells to become tumorigenic. Science(Washington DC), 243: 947-950, 1989.

69. Schultz. R. M., Silberman, S., Persky, B., Bajowski, A. S., and Carmichael,D. F. Inhibition by recombinant tissue inhibitor of metalloproteinases ofhuman amnion invasion and lung colonization by murine B16-F10 melanoma cells. Cancer Res.. 48: 5539-5545. 1988.

70. Alvarez, O. A., Carmichael, D. F., and DeClerck, Y. A. Inhibition ofcollagenolytic activity and metastasis of tumor cells by a recombinant humantissue inhibitor of metalloproteinases. J. Nati. Cancer Inst., 82: 589-595.1990.

71. Stetler-Stevenson, W., Brown, P.. Onisto, M.. Levy, A., and Liotta, L.Tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression intumor cell lines and human tumor tissues. J. Biol. Chem., 265: 13933-13938, 1990.

72. DeClerck, Y., Yean. T., Ratzkin. B.. Lu, H., and Langley, K. Purificationand characterization of two related but distinct metalloproteinase inhibitorssecreted by bovine aortic endothelial cells. J. Biol. Chem., 264: 17445-17453, 1989.

73. Boone. T., Johnson, M., DeClerck. Y., and Langley, K. cDNA cloning andexpression of a metalloproteinase inhibitor related to tissue inhibitor ofmetalloproteinases. Proc. Nati. Acad. Sci. USA, 87: 2800-2804. 1990.

74. Goldberg, G. 1., Marmer, B. L., Grant, G. A., Eisen, A. Z. Wilhelm, S.,and He, C. Human 72-kDa type IV collagenase forms a complex with atissue inhibitor of metalloproteinase inhibitor. Proc. Nati. Acad. Sci. USA,6:8207-8211, 1989.

75. Albini, A., Melchiori, A., Parodi, S.. Liotta, L. A., Brown, P. D., andStetler-Stevenson, W. G. TIMP-2 inhibits tumor cell invasion. J. Nati.Cancer Inst., 83: 775-779, 1991.

76. Field, J. K., and Spandidos. D. A. The role of ras and myc oncogenes inhuman solid tumours and their relevance in diagnosis and prognosis (Review). Anticancer Res., 10:1-22, 1990.

77. Thorgeirsson, U. P., Turpeenniemi-Hujanan, T., Williams, J. E., Westin,E. H., Heilman, C. A., Talmadge, J. E.. and Liotta. L. A. NIH 3T3 cellstransfected with human tumor DNA containing activated ras oncogenesexpress the metastatic phenotype in nude mice. Mol. Cell. Biol., 5: 259-262, 1985.

78. Hill. S. A., Wilson, A., and Chambers, A. F. Clonal heterogeneity, experimental metastatic ability and p21 expression in H-rai-transformed NIH3T3 cells. J. Nati. Cancer Inst.. 80: 484-490, 1988.

79. Greenberg. A. H.. Eagen. S. E., and Wright, J. A. Oncogenes and metastaticprogression. Invasion and Metastasis, 9: 360-378, 1989.

80. Theodorescu, D., Cornil, I., Fernandez, B., and Kerbel, R. S. Overexpressionof normal and mutated forms of C-Ha-Aaj induce orthotopic bladderinvasion in a human transitional cell carcinoma. Proc. Nati. Acad. Sci.USA. in press, 1991.

81. Nicolson, G. L. Tumor cell instability, diversification and progression tothe metastatic phenotype: from oncogene to oncofetal expression. CancerRes., 47: 1473, 1987.

82. Egan, S. E., Wright, J. A., Jarolim, L., Yanagihara, K., Bassim, R. H., andGreenberg, A. H. Transformation by oncogenes encoding protein kinasesinduces the metastatic phenotype. Science (Washington DC), 238: 202,1987.

83. Pohl, J.. Goldfinger, N., Rader-Pohl, A., Roller, V.. and Schirrmacher, V.p53 increases experimental metastatic capacity of murine carcinoma cells.Mol. Cell. Biol., 8: 2078, 1988.

84. Pozzatti, R. P., Muschel, R. J., Williams, J. R., Howard, B., Liotta, L. A.,and Khoury, G. Primary rat embryo cells transformed by one or twooncogenes show different metastatic potentials. Science (Washington DC).252:223-227, 1986.

85. Tuck. A. B., Wilson, S. M., and Chambers, A. F. ras transfection andexpression does not induce progression from tumorigenicity to metastaticability in mouse LTA cells. Clin. Exp. Metastasis, in press, 1990.

86. Mason, R. W.. Gal, S., and Gottesman, M. M. The identification of themajor excreted protein (MEP) from a transformed mouse fibroblast cell lineas a catalytically active precurson form of cathepsin L. Biochem. J., 248:449, 1987.

87. Kerr, L. D.. Holt. J. T., and Matrisian. L. M. Growth factors regulatetransin gene expression by c-/oj-dependent and c-yos-independent pathways.Science (Washington DC), 242: 1424-1427, 1988.

88. Gattoni-Celli, S., Strauss, R. M., Willet, C. G., Pozzatti, R., and Isselbacher,K. J. Modulation of the transformed and neoplastic phenotypes of ratfibroblasta by MHC-1 expression. Cancer Res.. 49: 3392-3395, 1989.

89. Cattoni-Celli, S., Marazzi. A.. Timpale. R., Kirsch. K., Isselbacher, K. J.Partial suppression of metastatic potential of malignant cells in immuno-deficient mice caused by transfection of H-2Kb gene. J. Nati. Cancer Inst.,2:960-963, 1990.

90. Steeg. P. S., Bevilacqua, G.. Kopper, L., Thorgeirsson. U. P., Talmadge, J.E., Liotta, L. A., and Sobel, M. E. Evidence for a novel gene associatedwith low tumor metastatic potential. J. Nati. Cancer Inst., 80: 200, 1988.

91. Bevilacqua, G., Sobel, M. E., Liotta, L. A., and Steeg, P. S. Association oflow nm23 RNA levels in human primary infiltrating ductal breast carcinomas with lymph node involvement and other histopathological indicators ofhigh metastatic potential. Cancer Res., 49: 5185-5190, 1989.

92. Hennessy, C., Henry, J. A., May, F. E. B., Westely, B. Angus, B., Lennard,T. W. J. Expression of the antimetastatic gene nm23 in human breastcancer: An association with good prognosis. J. Nati. Cancer Inst. 83: 281-285, 1991.

93. Haut, M., Steeg, P. S., Willson, J. K. V., Markowitz, S. D. Induction ofnm23 gene expression in human colonie neoplasms and equal expression incolon tumors of high and low metastatic potential. J. Nati. Cancer Inst., 83:712-716, 1991.

94. Leone, A., Flatow, U., King, C. R., Sandeen, M. A., Margulies, I. M. K.,Liotta, L. A., and Steeg, P. S. Reduced tumor incidence metastatic potential,and cytokine responsiveness of nm2J-transfected melanoma cells. Cell, 65:25-35. 1991.

95. Rosengard, A. M., Krutzsch, H. C., Shearn, A., Biggs, J. R., Barker, E.,Margulies, I. M. K., King. C. R.. Liotta. L. A., and Steeg, P. S. Reducednm23/awd protein in tumor metastasis and aberrant Drosophila development. Nature (Lond.). 342: 177, 1989.

96. Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Vernon, M., andLacombe, M. A. Dictyostelium nucleoside diphosphate kinase highly homologous to nm23 and awd proteins involved in mammalian tumor metastasis and Drosophila development. J. Nati. Cancer Inst., /:1199-1202,1990.

5059s

on June 3, 2014. 1991 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from