Tumor Invasion and Metastasis: an Imbalance of Positive and Negative Regulation1

(CANCER RESEARCH (SUPPL.) 51. 5054s-5059s, September 15. 1991] Tumor Invasion and Metastasis: An Imbalance of Positive and Negative Regulation1

Lance A. Liotta and William G. Stetler-Stevenson

Laboratory of Pathology, National Cancer Institute, N1H, Bethesda, Maryland 20892

Abstract ular marker in a primary tumor sample is likely to reflect the general metastatic propensity of the entire tumor. Indeed, this A group of coordinated cellular processes, not just one gene product, is responsible for invasion and metastasis, the most life-threatening has been borne out by a number of clinical studies indicating that the average level of specific protein markers or amplified aspect of cancer. It is now recognized that negative factors may be just oncogenes measured in the primary' tumor can be correlated as important as positive elements. Genetic changes causing an imbalance of growth regulation lead to uncontrolled proliferation necessary for both with clinical aggressiveness parameters of metastasis and recur primary tumor and metastasis expansion. However, unrestrained growth rence (7-10). does not, by itself, cause invasion and metastasis. This phenotype may require additional genetic changes. Thus, tumorigenicity and metastatic Tumor Cell Interaction with the Extracellular Matrix potential have both overlapping and separate features. Invasion and metastasis can be facilitated by proteins which stimulate tumor cell During the development of invasive tumors, tumor cells attachment to host cellular or extracellular matrix determinants, tumor disobey the social order of organ boundaries and cross into cell proteolysis of host barriers, such as the basement membrane, tumor cell locomotion, and tumor cell colony formation in the target organ for tissues where they do not belong. The mammalian organism is metastasis. Facilitory proteins may act at many levels both intracellulari) divided into a series of tissue compartments separated by the or extracellularly but are counterbalanced by factors which can block extracellular matrix unit consisting of the basement membrane their production, regulation, or action. A common theme has emerged. In and its underlying interstitial stroma (3, 11). The basal cells of addition to loss of growth control, an imbalanced regulation of motility the epithelium or organ parenchymal side of this unit are and proteolysis appears to be required for invasion and metastasis. attached to the basement membrane. On the opposite side, the interstitial stroma contains stromal cells, fibroblasts, and my- Metastatic Cells Can Dominate the Primary Tumor ofibroblasts. The nervous system, muscle cells, and blood ves Population sels are also surrounded by a continuous basement membrane. During all types of benign tissue remodeling, proliferative dis Metastasis is a cascade of linked sequential steps involving orders, and carcinoma in situ, the cell populations on either multiple host-tumor interactions (1-3). To successfully create side of this connective tissue unit do not intermix. Only during a metastatic colony, a cell or group of tumor cells must be able the transition from in situ to invasive carcinoma do tumor cells to leave the primary tumor, invade the local host tissue, enter penetrate the epithelial basement membrane and enter the the circulation, arrest at the distant vascular bed, extravasate underlying interstitial stroma to interact with the stromal cells. into the target organ interstitium and parenchyma, and prolif Thus, a definition of the behavior of the metastatic tumor cell erate as a secondary colony. Angiogenesis is required for the is the tendency to cross tissue compartment boundaries and expansion of the primary tumor mass, and new blood vessels intermix with opposite cell types (3, 12). penetrating the tumor are frequent sites for tumor cell entry The continuous basement membrane is a dense meshwork of into the circulation (4, 5). Angiogenesis is also required for collagen, glycoproteins, and proteoglycans which normally does expansion of the metastatic colony. At any stage, tumor cells not contain any pores large enough for passive tumor cell must overcome host immune cell killing (2). A very small transversal. Consequently, invasion of the basement membrane percentage (<0.01%) of circulating tumor cells ultimately ini must be an active process. Once the tumor cells enter the tiate successful metastatic colonies. Consequently, metastasis stroma, they gain access to lymphatics and blood vessels for has been viewed originally by Fidler and Hart (1) as a highly further dissemination. Tumor cells must cross basement mem selective competition favoring the survival of a subpopulation branes to invade nerves and muscle. During intravasation or of metastatic tumor cells that preexist within the heterogeneous extravasation, the tumor cells of any histolÃ³gica! origin must primary tumor. penetrate the subendothelial basement membrane. As a general Fidler's metastatic subpopulation concept is well accepted, feature of all types of carcinomas (12), defects in the basement but the relative size of this subpopulation in the primary tumor membrane are associated with invasion. In contrast, benign may vary with time and between tumors. Estimating the size of proliferative disorders are all characterized by a continuous the metastatic subpopulation is of clinical significance since a basement membrane separating the epithelium from the prognostic assay based on a sample of the primary tumor would stroma. The general observation of defective basement mem be highly inaccurate if the aggressive subpopulation was only a branes associated with cancer invasion and progression indi very small proportion of the total number of tumor cells being cates that aggressive tumor cells may interact with basement sampled. Recently, Kerbel (6) has addressed the subpopulation membranes in a manner fundamentally different from that of question experimentally using genetic markers. He determined normal cells. This general feature of malignant tumors provides that the metastatic subpopulation dominates the primary tumor the foundation for investigation of molecular mechanisms. mass early in its growth. The dominance may be due to selective Interactions of the tumor cell with the basement membrane growth of the metastatic subpopulation in response to local can be separated into three steps: attachment; matrix dissolu cytokines. Thus, measurement of the average level of a molec- tion; and migration. The first step is binding of the tumor cell to the basement membrane surface-mediated cell surface recep ' Presented at the Symposium "Discoveries and Opportunities in Cancer tors of the integrin (13, 14) and non-integrin (15, 16) variety. Research: A Celebration of the 50th Anniversar) of the Journal Cancer Research." May IS. 1991, during the 82nd Annual Meeting of the American Association for Matrix receptors recognize glycoproteins such as laminin, type Cancer Research, Houston, TX. IV collagen, and fibronectin in the basement membrane. Two 5054s

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1991 American Association for Cancer Research. TUMOR INVASION AND METASTASIS to 8 h after attachment, a localized zone of lysis is produced in necessary but not sufficient. This conclusion is justified by the the basement membrane at the point of tumor cell contact. finding in multiple laboratories that inhibitors for metallopro- Tumor cells directly secrete degradative enzymes (17) or induce teinases or inhibitors of serine proteinases can each block tumor the host to elaborate proteinases to degrade the matrix and its cell invasion of native or reconstituted connective tissue barriers component adhesion molecules. Matrix lysis takes place in a in vitro (33-36). Thus, the enzymes involved in tumor invasion highly localized region close to the tumor cell surface (18), and metastasis may well resemble the proteolytic cascades where the amount of active enzyme outbalances the natural involved in blood coagulation. proteinase inhibitors present in the serum, those in the matrix, Plasminogen activator, specifically uPA,2 has been closely or that secreted by normal cells in the vicinity. linked to the metastatic phenotype (37, 38). Anti-uPA antibod Locomotion is the third step of invasion which propels the ies block human HEP-3 cell invasion in the chick chorioallan- tumor cell across the basement membrane and stroma through toic membrane assay and murine B16F10 melanoma cell me the zone of matrix proteolysis. An early step in locomotion is tastasis following tail vein injection (39, 49). Overexpression of pseudopodial protrusion at the leading edge of the migrating uPA in Ha-raÃ-transformed 3T3 cells enhanced lung invasion cell (19, 20). The induction of pseudopodia is directional, is and experimental metastasis formation (41). Serine proteinase regulated by cell surface ligand binding, and involves a coordi inhibitors also block tumor cell invasion through human am- nated mobilization of cytoskeletal elements which interact with niotic membranes (35). the inner membrane surface. The cellular machinery of tumor Among the list of enzymes involved in cancer, a large body cell locomotion is a fertile topic for future study and will of information has been accumulated concerning the matrix undoubtably benefit from insights learned from studies of im metalloproteinase gene family (Fig. 1). These enzymes have mune cells (2) and nonmammalian organisms (20). It is now been subgrouped into three broad categories based on substrate recognized that random tumor cell motility can be regulated by preference: interstitial collagenases; type IV collagenases tumor cell cytokines ("autocrine motility factors" and "scatter (gelatinases), and stromelysins (42). The interstitial collagenase factors") (19, 21, 22). Autocrine motility factors act through a is the best characterized and specifically degrades type I colla receptor-activated G protein susceptible to inhibition by pertus gen (32). Neutrophil collagenase, which has recently been sis toxin. Augmented random motility by tumor cells causes cloned, appears very similar in substrate specificity (43). The dispersion at the primary site. In addition, the direction and stromelysins are three related gene products, stromelysin, stro- site of the tumor cell locomotion may be influenced by host melysin 2, and PUMP-1, which degrade a variety of matrix organ-derived chemoattractants. Such chemoattracts could play components including proteoglycans and noncollagenous gly- a role in the organ-selective homing of metastasis. This could coproteins such as laminin and fibronectin, as well as the complement other mechanisms of organ homing which include noncollagenous domains of type IV collagen. The role of stro preferential adhesion to organ-specific endothelium and pref melysin in squamous progression has recently been recognized erential growth in selected organs due to local growth factors (44). The type IV collagenases are named for their selective (23). ability 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). Proteinases: from Correlation to Causality Both a 72-kDa and 92-kDa type IV collagenase exist and A general aspect of malignant neoplasms may be an imbal complementary DNA cloning has demonstrated that each is a ance of proteolysis which favors invasion. However, proteolysis unique gene product (46, 47). of tissue barriers is not a property unique to tumor cells. It is All classes of matrix metalloproteinases are secreted as in utilized, for example, during trophoblast implantation, embryo active zymogens and enzyme activation is an important control morphogenesis, tissue remodeling, parasitic and bacterial in step in proteolysis. A new model for matrix metalloproteinase vasion, and angiogenesis. Furthermore, the defect in the tumor proenzyme activation has been proposed (48-51 ). The essential cell cannot be simply unbridled production of degradative en feature of this model is that the latent form of the matrix zymes. This is because cell migration during invasion requires metalloproteinase enzymes all have a metal atom sulfhydryl attachment and detachment of the cell as it moves forward. side chain interaction that results in a catalytically inert active Lysis of all matrix components around the tumor cell would center. The sulfhydryl group in this interaction is donated by simply remove the substratum necessary for proper cell traction. the CYS-73 residue which is contained in a highly conserved Thus, it is probable that the invading tumor cell uses proteolysis peptide present in all known members of this metalloproteinase in a highly organized manner both spatially and temporally family. The metal atom is presumably the zinc atom of the which does not differ functionally from the operating mode of active site. Disruption of this interaction results in conforma- normal cells which migrate through tissue barriers. The differ tional rearrangement and rapid attainment of protease activity. ence is that tumor cells couple proteolysis with motility to The implication of this model is that mutations in the CYS-73 achieve invasion at times and places which would be inappro residue would result in intrinsic enzyme activation and that this priate for normal cells. could play a role in tumor progression. The in vivo mechanism The metastasis field has progressed from establishing a cor of normal metalloproteinase activation is unknown but may relation between proteolysis and malignant progression to the involve the action of other proteinases either in solution or via a cell surface-dependent mechanism (18, 52, 53). The latter finding that the actual blockade of certain proteinases will prevent invasion and metastasis. A positive association with would allow for precise cellular control at the point of matrix tumor aggressiveness has been noted for a variety of classes of interaction. degradative enzymes including heparanases (24, 25), serine Among the matrix metalloproteinase family members, an (26), thiol (27, 28), and metal-dependent enzymes (29-32). accumulating body of evidence supports a positive correlation Indeed, a cascade including all these enzymes is probably in 2The abbreviations used are: uPA. urokinase-type plasminogen activator: volved in the invasive process, and more than one enzyme is TIMP. tissue inhibitor of metalloproteinases; NDP. nucleoside diphosphate. 5055s

72 kD-Type IV ProCollagenase 3.2kb of the 72-kDa type IV collagenase mRNA and loss of the gÂ«lallnbinding domain invasive capacity (64). These results suggest that the 72-kDa CCCCCCCCCCCC type IV collagenase enzyme is a normal cell component that is dramatically overexpressed in many invasive and metastatic Active Type IV Collagenase human cancers.

Natural Proteinase Inhibitors Suppress Invasion CCCCCCCCCCCC CCC C The secretion and activation of metalloproteinases are not MBD Â»ipii* 2 (V) enough to ensure that they will degrade the target matrix 92 kD-Typc IV ProCollagenase 2.8kb substrate (17, 65). This is because natural inhibitor proteins, produced either by the host or by the tumor cell itself, can block the latent or the active metalloproteinases (17). Natural pro- ProCollagenaseI 2.0kb MBD teinase inhibitor proteins, such as TIMPs (66) plasminogen activator inhibitors may therefore function as metastasis sup pressor proteins which act to inhibit tumor cell invasion of the ijijijjjjjiliiijjjil MBD V//////////A SagenasÂ«, 3.3kb extracellular matrix. TIMP-1, the original member of the TIMP family (67), is a glycoprotein with an apparent molecular size

PfStromelysin i 9kb of 28.5 kDa which forms a complex of 1:1 stoichiometry with activated interstitial collagenase, activated stromelysin, and the 92-kDa type IV collagenase. It has been reported that transfection of antisense RNA which blocks TIMP-1 expression en ÃŒÃŒ!ÃŒ!ÃŒ;ÃŒ!ÃŒ!ÃŒ!iiÃŒÃŒÃŒ!ÃŒ|MBD ProStromeiysin-2 1 .7MÂ» hances the malignant phenotype (68). One explanation for this result is that the antisense RNA blocked the production of TIMP-1 which normally prevented the malignant phenotype. PUMP-1 In animal models, administration of recombinant TIMP-1 blocks metastasis (69, 70). I MBD | VAAHEFGHAMGLEHS Recently, Stetler-Stevenson et al. (49, 71) have isolated, purified, determined the complete primary structure, and cloned H PRCGVPNPD the first new member of the TIMP family, TIMP-2. An identical Fig. 1. Matrix metalloproteinase family. Type IV procollagenase (72 kDa and inhibitor was isolated from endothelial cells by DeClerk et al. 92 kDa forms), interstitial procollagenase, neutrophil procollagenase, prostro- (72, 73). TIMP-1 and TIMP-2 are regulated independently and melysin, prostromelysin-2, and PUMP-1. are represented diagrammatically and aligned to show regions of protein sequence homology. MBD, active site metal oppositely by 12-O-tetradecanoylphorbol-13-acetate, trans ion-binding domain. Type IV procollagenases contain a cysteine-rich, substrate- forming growth factor ÃŸ,andother cytokines. TIMP-2 is a 21- binding domain which shows homology to fibronectin but is absent from the other matrix metalloproteinases. Upon treatment with organomercurial com kDa protein which selectively forms a complex with the latent pounds m vitro, all seven enzymes are activated with the concomitant removal of proenzyme form of the 72-kDa type IV collagenase. The se an amino-terminal segment of the latent enzyme. The removed segment contains creted protein has 194 amino acid residues and is not glycosy- an unpaired cysteine residue within the conserved amino acid sequence PRCGVPDV located immediately adjacent to the proenzyme cleavage site. Site- lated. TIMP-2 shows 37% identity and overall 65.6% homology directed mutagenesis studies have shown that alterations in this sequence result to TIMP-1 at the deduced amino acid sequence level. The in spontaneous activation of transin, the rat homologue of stromelysin. In the positions of the 12 cysteine residues in TIMP-2 are conserved latent proenzyme, this sequence interacts with the metal ion through the unpaired cysteinyl residue to block activity. Perturbation of this interaction affects activa with respect to those present in TIMP-1, as are 3 of the 4 tion. A/I.kilobases. tryptophan residues; yet the 2 proteins are immunologically distinct. TIMP-2 inhibits at a 1:1 ratio the type IV collagenolytic activity and the gelatinolytic activity associated with the between type IV collagenase activity and tumor cell invasion 72-kDa enzyme. Unlike TIMP-1, TIMP-2 is capable of binding (24, 31, 54, 55). Augmented type IV collagenase activity is to both the latent and activated forms of the 72-kDa type IV associated with the genetic induction of a metastatic phenotype collagenase and will abolish the hydrolytic activity of all mem (56-59). Furthermore, use of agents which specifically inhibit bers of the metalloproteinase family (49, 74). The 92-kDa type type IV collagenase activity or block collagenase secretion IV procollagenase can be found as a complex with TIMP-1 prevents tumor cell invasion in vitro (26, 60). Immunohisto- (46). Activation of either the latent 72-kDa or 92-kDa type IV chemical studies using affinity purified anti-72-kDa type IV collagenase-TIMP complex can be reversed by binding of a collagenase antibodies (48, 49) demonstrate that low levels of second role of TIMP-1 or TIMP-2. This suggests that on these this enzyme are produced by normal, nontumorigenic, non- enzymes there are two separate TIMP-binding sites and that metastatic cells such as the myoepithelial cells of the human binding of TIMP-1 or TIMP-2 to the latent proenzymes serves breast (61). Benign proliferative lesions of the breast, benign a different function than the inactivation that occurs following polyps of the colon, and normal colorectal and gastric mucosa binding to the active species. The areas of the two proteins all show negligible immunoreactivity for 72-kDa type IV col which differ in homology may contain the regions responsible lagenase. In contrast, almost all invasive colonie and gastric for the functional differences (71). The net 72-kDa type IV adenocarcinomas are positive for this antigen (62, 63). Down- collagenase activity consequently depends upon the balance regulation of type IV collagenotypic activity by retinoic acid between the levels of activated enzyme and TIMP-2. TIMP-2 treatment of human melanoma cells has been correlated with a is a potent inhibitor of cancer cell invasion through reconsti loss of the invasive phenotype (54). Studies of the mechanism tuted extracellular matrix (75). TIMP-2 produced by the same of this effect reveal that retinoic acid treatment of these human tumor cells which make collagenase, therefore, exists as a melanoma cells results in a reduction of the steady state level natural suppressor of invasion. 5056s

Metastasis and Tumorigenicity Can Be under Separate directly modulate transin gene transcription (44, 87). Some of Genetic Control these oncogene-associated effector genes may regulate cell motility and proteolysis, and this forms a common thread with Five years ago investigators in the metastasis field were sure separate work on proteinases, motility, and angiogenesis. that the new developments in oncogenes were relevant only to Several metastasis suppressor genes have been reported in tumorigenicity and that separate genes would be found which transfection experiments. The Adenovirus 2 Eia gene, previ evoke the metastatic process. At the time, biological assays of ously discussed, suppressed c-Ha-ras induction of metastatic oncogenes were restricted to scoring tumorigenicity. Little at behavior of rat embryo fibroblasts, as assayed by tail vein tention had been paid to metastasis formation. Nevertheless, injections (84). In the same model system Gattoni-Celli re when the proper studies were done, it was found that transfec- ported that ras and Eia cotransfected rat embryo fibroblasts tion of certain oncogenes in the correct recipient cell could expressed higher levels of major histocompatibility complex induce the complete phenotype of invasion and metastasis. class I genes (88). When the H-2Kb major histocompatibility Although the search for specific metastasis-inducing genes goes complex gene was transfected into rat embryo fibroblasts, pre on, oncogene transfection has provided a model to switch on viously transfected with ras, reduced rates of tumorigenesis and the effector processes which are required for the cell to carry metastasis were observed upon injection into triple-deficient out invasion and metastasis. These models have revealed that mice (89). The data suggest the involvement of H-2Kb in arms some of the metastasis effector genes can be regulated inde of the immune response, such as macrophage-mediated cyto- pendently from those which confer tumorigenicity. toxicity, or in the suppression of nonimmunological aspects of The incidence of aberrant gene expression and genetic alter the tumor metastatic process. ations of the ras and myc gene families have been shown to be TIMP-1 was demonstrated to suppress the metastatic poten important in progression of human cancers and may be useful tial of Swiss 3T3 cell using antisense transfection. The antisense as prognostic indicators (76). Thorgeirsson et al. (77) were the TIMP-1 construct reduced TIMP activity by 47-68% in the first to report that the activated (mutated) ras oncogene se transfected cells and increased invasiveness in an in vitro am- quences, when transfected into mouse embryo-derived fibro- nion assay, as well as tumorigenicity and metastatic potential blasts (NIH-3T3 cells), produced numerous mÃ©tastases.The in vivo (68). resultant highly metastatic cells were not more resistant to host The nm23 gene was identified on the basis of its reduced immune cell lysis (macrophage or natural killer cells) compared steady state RNA levels in five highly metastatic K-1735 mel to control cells, indicating that the ras oncogene had augmented anoma cell lines, as compared to two related, low metastatic the intrinsic aggressiveness of the NIH-3T3 cells. Transfection potential k-1735 melanoma cell lines (90). In human breast of H-ras-family oncogenes has been shown to induce metastasis cancer reduced nm23 RNA levels have been associated with the in fibroblasts and epithelial cells of rodent and human origin presence of lymph node mÃ©tastasesat surgery (91) as well as (56, 57, 59, 78-81). However, H-ras is not the only oncogene decreased patient disease-free and overall survival (92). In other which can induce metastatic potential. At lower efficiency, the cancer cell types, such as colorectal carcinoma, no significant serine-threonine kinases \-mos, v-raf, and A-ra/(79, 82); tyro- association of nm23 RNA levels with metastatic progression sine kinases v-src, \-fes, and \-fms (79); and the mutated phos- was observeed (93). Transfection of the murine nm23-\ com phoprotein p53 (83) have been demonstrated to induce the plementary DNA into highly metastatic murine K-1735 TK metastatic phenotype in the appropriate recipient cell. melanoma cells resulted in a reduced incidence of primary Experimental evidence indicates that invasion and metastasis tumor formation, significant reductions in tumor metastatic require activation of a set of effector genes over and above those potential, and altered tumor cell responsiveness to the cytokine which are required for unstrained growth alone. The down transforming growth factor ÃŸinvitro (94). stream pathways used in ras induction of tumorigenicity and An important clue to nm23 function(s) came from its virtual identity with the Drosophila awd gene product (95-96). Muta metastasis have dissimilar features: (a) the adenovirus 2 Eia gene has been demonstrated to suppress ras induction of met tions which result in reduced awd expression or the production astatic potential with no inhibition of soft agar colony forma of a mutated protein do not significantly alter embryonic de tion or tumorigenicity (84); and (b) cells are capable of being velopment but do alter the development of multiple tissues transformed by ras but do not metastasize (56, 85). The failure postmetamorphosis, when presumptive adult tissue in the im of ras to induce metastasis in certain experimental systems aginai discs begins to divide and differentiate. These abnormal ities include altered morphology of the wing discs, larval brain probably reflects a deficiency in, or a suppression of, some of and proventriculus; aberrant differentiation of the wing, leg, these effector proteins. Several candidate effector proteins have and eye-antennae imaginai discs and ovaries; and cell necrosis, been associated with metastasis in ras transfection models, such predominantly in the wing discs. nm23/awd may contribute to as proteinases including type IV collagenase (46, 57, 59, 77), cathepsin L (86), and motility-associated cytokines (21). Thus, the normal development of tissues, which may include signal transduction of cell to cell communication. Loss of nm23/a\vd in these ras transfection models certain effector genes are expression may lead to a disordered state, favoring aberrant activated, or suppressed, possibly in coordinated manner, to development or tumor progression to the metastatic state. induce metastasis formation. Studies have revealed that several oncogenes such as \-src and ras, tumor-promoting phorbol esters and growth factors such as epidermal growth factor and References platelet-derived growth factor will induce transin (rat homo 1. Fidler, I. 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5059s

Lance A. Liotta and William G. Stetler-Stevenson

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

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