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1991;51:5054s-5059s. Cancer Res
Lance A. Liotta and William G. Stetler-Stevenson
Negative RegulationTumor Invasion and Metastasis: An Imbalance
of Positive and
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(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 (
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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.
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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.
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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.
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