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Metastatic Cancer CellMarina Bacac and Ivan Stamenkovic

Experimental Pathology Unit, Department of Pathology, University of Lausanne,Switzerland; email: [email protected], [email protected]

Annu. Rev. Pathol. Mech. Dis. 2008. 3:22147

First published online as a Review in Advance onSeptember 17, 2007

The Annual Review of Pathology: Mechanisms ofDisease is online at pathmechdis.annualreviews.org

This articles doi:10.1146/annurev.pathmechdis.3.121806.151523

Copyright c 2008 by Annual Reviews.All rights reserved

1553-4006/08/0228-0221$20.00

Key Words

tumor-host interactions, invasion, adhesion, proteolysis

Abstract

Metastasis is the result of cancer cell adaptation to a tissue microen-vironment at a distance from the primary tumor. Metastatic cance

cells require properties that allow them not only to adapt to a for-eign microenvironment but to subvert it in a way that is conducive

to their continued proliferation and survival. Recent conceptual andtechnological advances have contributed to our understanding o

the role of the host tissue stroma in promoting tumor cell growthand dissemination and have provided new insight into the genetic

makeup of cancers with high metastatic proclivity.

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INTRODUCTION

The ability to metastasize is a hallmark ofmalignant tumors, and metastasis is the prin-

cipal cause of death among cancer patients.It is the single most challenging obstacle to

successful cancer management and may alsobe viewed as the last frontier of cancer re-

search. Metastasis is the process whereby can-cer cells spread throughout the body, estab-

lishing new colonies in organs at a distancefrom the one where the primary tumor orig-

inated. It is well established that metastasis isa complex, multistep process, which although

considered highly inefficient from the cellularpoint of view, virtually constitutes a death sen-

tence for the patient. Its prevention and man-agement are therefore among the key goals in

clinical and basic cancer research.

There are three major routes for tu-mor dissemination: lymphatic vessels, blood

vessels, and serosal surfaces. Tumor metas-tases occurring via these three routes are re-

ferred to as lymphatic, hematogenous, andtranscoelomic, respectively. Epithelial malig-

nancies, or carcinomas, typically begin theirdissemination by the lymphatic route, with

hematogenous metastases occurring at a latertime. In contrast, bone and soft tissue tu-

mors, or sarcomas, preferentially metasta-

size by the hematogenous route, whereastranscoelomic metastasis is the property ofa relatively small group of tumors that in-

cludes mesotheliomas and ovarian carcino-mas. Because of their prevalence, lymphatic

and hematogenous metastases will provide themain focus of the present review.

A tumor cell that initiates a metastaticcolony must (a) detach from the primary

mass, (b) invade the local host tissue stroma,(c) penetrate local lymphatic and blood ves-

sels, (d) survive within the circulation, (e) be-come arrested in capillaries or venules of

other organs, (f) penetrate the correspondingparenchyma, (g) adapt to the newly colonized

milieu or subvert the local microenvironmentto suit its own needs, and (h) divide to form

the new tumor (Figure 1). Although much

has been learned regarding the properties that

such a cell requires, several key questions arestill unresolved. For example, do cells display-

ing metastatic proclivity emerge late in tu-mor progression as a result of multiple mu-

tations and selection, or are they part of thecell population that constitutes early malig-

nant growth? What role does the host mi-croenvironment play in tumor cell dissemina-

tion, and which components of tumor-stromacross talk might provide potential therapeutic

targets?

At least five functions are required for atumor cell to successfully complete the se-

quence of events outlined above. They in-clude interaction with the local microenvi-

ronment, migration, invasion, resistance toapoptosis, and the ability to induce angio-

genesis. All five functions are regulated byadhesion and proteolysis, which together pro-

vide the most fundamental molecular effectormechanisms upon which a metastatic cell re-

lies (Table 1). Adhesion and proteolysis de-termine tumor cell interaction with other cells

and with the extracellular matrix (ECM), helpcreate a path for migration, promote angio-

genesis, and both directly and indirectly trig-ger survival signals.

Transformation results in major pheno-typic changes that affect cell surface receptor

expression, cytoskeletal function, growthfactor and cytokine secretion, proteolytic

enzyme production, and the glycosyltrans-ferase and glycosidase repertoire (Figure 2)

These combined changes alter the way inwhich the transformed cell communicates

with its microenvironment and, by the sametoken, the way in which it is perceived by

normal surrounding cells. They not onlyprovide transformed cells with the ability to

disrupt the barriers that keep their normalcounterparts confined to a defined tissue

compartment, but also with the means tosubject the host tissue microenvironment to

their rules and use its resources for their own

survival, growth, and dissemination. It is in-creasingly clear that tumor cells depend upon

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Basement membranedegradation

Basement membrane

Migration

Growing metastases

Proliferation, angiogenesis,

microenvironment activation

Dormantmetastases

Collagen fibers

Detachment

Intravasation

Extravasation Circulation

Normal epithelia

In situ carcinoma

Invasive carcinoma

No proliferation

Invasion

Figure 1

Principal steps in metastasis. Transformation of normal epithelial cells leads to carcinoma in situ, which,as a result of loss of adherens junctions, evolves toward the invasive carcinoma stage. Following basementmembrane degradation, tumor cells invade the surrounding stroma, migrate and intravasate into blood orlymph vessels, and become transported until they arrest in the capillaries of a distant organ.

their microenvironment to metastasize and

that they even rely on host tissue stromal cellsto provide functions, such as a diversity ofproteolytic activity, that they themselves may

lack. Understanding tumor-host interactionsmay therefore provide a key to understanding

metastasis. A more recently emerging view,based on gene expression profile analysis of

diverse primary and metastatic tumors, is

that metastatic cells may constitute part ofthe early makeup of a malignant tumor. Thisreview highlights our current understanding

of tumor cell properties and host tissueresponses whose combination culminates in

cancer metastasis and discusses the origin ofmetastatic cells in light of recent observations.

www.annualreviews.org Metastatic Cancer Cell 223


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Table 1 Summary of adhesion molecules and proteolytic enzymes discussed in the text that are implicated in tumor

metastasis on the basis of experimental evidence

Mechanism Candidate functions Reference(s)

Adhesion

Cadherins

E-cadherin Promotes cell-cell adhesion; prevents cell detachment; cleaved by MMP-3, MMP-7,

and ADAMs; tumor suppressor

(1, 2, 4, 5)

N-cadherin Promotes migration and invasion; regulates activation of FGFRs (19, 20)Integrins

21, 31 Enhance metastasis in selected experimental models (28, 29)

v3 Promotes migration and invasion; mediates tumor cell-platelet interactions (30, 123)

64 Promotes migration, invasion, and proliferation; cooperates with RTKs (31, 32)

41 Mediates tumor cell adhesion to endothelial cells (126)

Immunoglobulin superfamily

VCAM-1 Mediates tumor cellendothelial cell adhesion (126)

L1 Promotes tumorigenicity and motility (55)

NrCAM Promotes tumorigencity and migration (56)

NCAM Its downregulation is associated with enhanced lymph node metastasis in some tumor

models

(57)

Selectins Mediate tumor cellendothelial interactions and tumor cellplatelet/leukocyte adhesion (119122)

Cell surface proteoglycans

CD44 Mediates interaction with hyaluronan; interacts with RTKs; specific isoforms provide a

scaffold for the assembly of molecular complexes that can promote metastasis

(5860, 62, 63)

Proteolysis

Matrix metalloproteinases

MMP-1 Degrades collagen; promotes invasion (79, 85)

MMP-2 Activates growth factors, including TGF-; promotes invasion; interacts with v3

integrin on the cell surface

(79, 85)

MMP-3 Promotes tuumorigenesis; activates growth factors, including HB-EGF (79, 85, 93)

MMP-7 Promotes cell survival; activates HB-EGF; interacts with CD44 on the cell surface (63, 79, 85)MMP-9 Promotes invasion; enhances angiogenesis; promotes intravasation; activates TGF-;

interacts with CD44 on the cell surface

(62, 79, 85)

MMP-14, -15, -16 Degrade native basement membrane; promote invasion (7779, 85)

Cathepsins Promote tumor growth and invasion; may be expressed by tumor cells or exclusively by

stromal cells

(74)

FUNDAMENTAL MOLECULAREFFECTOR MECHANISMSOF METASTASIS

Adhesion

Detachment, cadherins, and epithelial-

to-mesenchymal transition. Interepithelial

cell interactions are regulated by complex ad-hesion mechanisms, including tight and ad-

herens junctions and desmosomes (1). Trans-

formed and malignant cells that become de-tached from the epithelium display loss of ad-

herens junctions, which, in epithelial cells

are constituted primarily by E-cadherin (12). Like other members of the cadherin fam-ily, E-cadherin displays homophilic binding

specificity. Its adhesive functions are stabi-lized by-catenin, which binds to its cyto-

plasmic domain, providing a link to-cateninand the actin cytoskeleton (3). Experiments



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performedinvitroandinvivohaveshownthat

genetic and antibody-mediated inhibition ofE-cadherin function can alter the phenotype

of epithelial cells from noninvasive to invasive

(4). Conversely, introduction of E-cadherininto E-cadherin-deficient invasive carcinoma

cells abrogated their invasiveness (3). Down-regulation of E-cadherin activity in vivo us-

ing a dominant negative E-cadherin constructin the transgenic Rip-Tag mouse model of

pancreatic islet cell carcinoma resulted in thetransition of a well-differentiated -cell ade-

noma to an invasive carcinoma (5). Crossingthe Rip-Tag mice with mice that maintain

E-cadherin expression in transformed -cellsarrested tumor progression in the adenoma

stage (5). Together, these observations arguethat E-cadherin functions as a tumor suppres-

sor.Although loss of function mutations occur

in E-cadherin, they are not commonly ob-served in malignant tumors (3). Mechanisms

that decrease or abrogate E-cadherin func-tion in carcinoma cells include transcriptional

repression, followed by promoter methyla-tion (6, 7), disruption of cytoskeletal connec-

tions, increased intracellular degradation, andproteolytic cleavage of the extracellular do-

main by matrix metalloproteinases (MMPs)(3). Several of the transcriptional repressors

that control E-cadherin expression in devel-opment, including Snail, Slug, SIP1, Twist,

dEF1, and E12/E47 (812), are implicatedin E-cadherin downregulation in malignant

cells. An alternative mechanism that can in-activate E-cadherin function in tumor cells

is disruption of the link between E-cadherin

and the cytoskeleton. One example is pro-vided by mutations in -catenin that abro-

gate its binding to -catenin and result in anonadhesive phenotype (3). Coordinate re-

ceptor tyrosine kinase (RTK)-integrin sig-naling can also interfere with E-cadherin

function. Activated RTKs and Src family ki-nases induce tyrosine phosphorylation of the

E-cadherin-catenin complex, which is thenrecognized by the Cbl-like E3 ubiquitin lig-

ase and downregulated by endocytosis (13). In

Selectin ligands

CD44v3

MMP-7

N-cadherin

pro-HB-EGF

Glycosaminoglycanchains

ErbB4

MMP-14

FGFR

c-Met

c-Met

MMP-15

CD44inactive

E-cadherin

5 1 3

v

6

1

3 4

BM

Extracellular matrix

ErbB2

Extracellular matrix

Figure 2

Changes of adhesive properties in transformed cells. Normal epithelial cellcommunication with its microenvironment is regulated byE-cadherin-mediated cell-cell interaction and 1-integrin-mediatedadhesion to the basement membrane (BM). Transformation results in thecadherin switch that leads to E-cadherin loss and replacement byN-cadherin, which plays an important role in invasion by regulatingfibroblast growth factor receptor (FGFR) function. Carcinomas frequentlyexpress v3 and 64 integrins, which promote invasion andproliferation, in part through their interactions with receptor tyrosinekinases, including ErbB2 and Met. Transformation also results in changes

in glycosylation of cell surface proteoglycans such as CD44, whichregulates invasion by coordinating MMP-7-mediated heparin-bindingepidermal growth factor (HB-EGF) activation and ErbB4 signaling.Changes in the glycosyltransferase repertoire can also result in thedecoration of cell surface receptors by oligosaccharides that constituteselectin ligands. Several transmembrane MMPs are expressed by tumorcells and mediate BM degradation.



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v-src-transformed cells, this process is de-

pendent upon integrin signaling and fo-cal adhesion kinase (FAK) phosphorylation

(14, 15). Integrin signaling operates through

Snail/Slug to suppress E-cadherin expres-sion and disrupt adherens junctions. In some

epithelial cells, this process may be medi-ated by integrin-linked kinase (16, 17). Fi-

nally, proteolytic cleavage of the extracellu-lar domain of E-cadherin by MMP-3 and

MMP-7 disrupts its ability to promote cellularinteractions (18). Abrogation of E-cadherin-

mediated cell-cell adhesion results in detach-ment of tumor cells from the epithelial cell

layer and affects signaling pathways impli-cated in cell migration and growth, includ-

ing Rho GTPase-mediated modulation of theactin cytoskeleton and the canonical Wnt sig-

naling pathway (3).Loss of E-cadherin in malignant cells

may be replaced by other cadherins, mostcommonly, N-cadherin (Figure 2). This

process, known as the cadherin switch, isassociated with a phenotypic change observed

in vitro known as epithelial-to-mesenchymaltransition (EMT). EMT, defined as the

conversion of epithelial cells to motile,fibroblast-like cells that express mesenchymal

rather than epithelial cell markers, is acommon event during normal embryonic

development and is observed as epithelial cellprogress through the stages of carcinogenesis

in vitro. It is proposed to reflect invasive andmetastatic properties of transformed epithe-

lial cells, but remains somewhat controversialbecause full EMT is difficult to prove in

vivo. Nevertheless, expression of N-cadherin

may make a critical contribution to invasionthrough both its adhesive and signaling

functions. N-cadherin can mediate cell-cellinteractions with N-cadherin-expressing

stromal cells, which may play an importantrole in the ability of tumor cells to direct host

responses. N-cadherin binds to and regulatesthe activation of fibroblast growth factor

receptors (FGFRs), thereby helping assemblethe FGFR-signaling complex, which triggers

downstream signaling pathways, including

phospholipase C-, phosphatidylinosito

3-kinase, and mitogen-activated protein ki-nase (MAPK). The combined action of these

signaling pathways promotes cell survivalmigration, and invasion (19, 20). Similar to

E-cadherin, the extracellular domain of N-cadherin is susceptible to proteolytic cleavage

by MMPs. The N-cadherin cleavage productcan block N-cadherin-mediated cell-cell ad-

hesion, but can also stimulate FGFR signalingon adjacent cells in paracrine fashion (19

20). Cleavage of N-cadherin at a site withinthe transmembrane or cytoplasmic domain

by a -secretase-type protease results in thetranslocation of its carboxy-terminal segment

to the nucleus, where it represses transcrip-tion mediated by the CREB-binding protein

(21).

Interaction with the extracellular matrix

integrins. Tumor cell interactions with theECM are mediated primarily by integrins and

play a key role in tumor invasion and spreadIntegrins form a large family of adhesion re-

ceptors, each member consisting of an anda transmembrane chain (22). In mammals

18 and8 chainsassociateinvariouscombi-nations to give rise to 24 integrins that recog-

nize distinct ECM ligands, with, neverthelesssome degree of overlap (22). When integrins

bind to ECM, they aggregate in the planeof the cell membrane and associate with a

molecular complex composed of adaptor, sig-naling, and cytoskeletal proteins that orches-

trate the organization of actin filaments. Or-ganization of actin filaments into stress fibers

in turn, promotes further integrin aggrega-tion that results in increased ECM binding in

a positive feedback loop. The outcome is anintegrin-mediated assembly of ECM and cy-

toskeletal protein clusters on each side of thecell membrane known as focal adhesions and

ECM contacts (23).Integrins trigger both mechanical and

chemical signals that organize and remodelthe cytoskeleton of the cell, regulate adhesive

versus migratory interactions with the ECM

impart polarity, and control proliferation



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and survival. To exert their effects, integrins

cooperatewith RTKs, thereby jointly control-ling survival and mitogenic pathways. A re-

ductionist view might be that integrins me-

diate cell adhesion and impose positionalcontrol on RTK activity, which together de-

termine whether cells migrate and proliferatein response to cytokines and growth factors.

Although seemingly straightforward, integrinimplication in cellular functions is compli-

cated by their diverse adhesive and signalingproperties that provide them the ability to

affect cell function in a variety of ways thatare often context dependent. Thus, 21 and31 integrins mediate epithelial cell adhe-sion to basal lamina and maintain them in

a quiescent state. These integrins are oftendownregulated in carcinomas and their re-

expression in carcinoma cells can decrease oreven revert the malignant phenotype (2427).

However, both of these integrins can enhancemetastasis in selected experimental models

(28, 29), underscoring the cell context and tu-mor stage dependence of the effects of1 in-

tegrins. By contrast, v3 (30) and 64 (31)integrins are frequently upregulated in car-

cinomas, where they may promote migration,invasion, and proliferation. In addition to par-

ticipating in hemidesmosome organization,the 64 integrin cooperates with epider-

mal growth factor receptor (EGFR), ErbB2,and Met, and is likely to promote the growth

of carcinomas in which activating mutationsof the corresponding growth factor receptor

genes represent the oncogenic driving force(3133). In support of this view, introduction

of the 4 chain into 4-negative breast car-

cinoma cells activates the phosphatidylinosi-tol 3-kinase pathway, which results in activa-

tion of Rac and increased invasiveness of thesecells in vitro (31). The cytoplasmic domain

of 4 also acts as an adaptor and amplifierof proinvasive signals induced by the hepato-

cyte growth factor receptor Met in cells un-dergoing Met-mediated transformation (33).

Both EGF and Met induce phosphorylationof4 and enhance SHC signaling, which dis-

rupts hemidesmosomes and increases epithe-

lial cell migration and carcinoma cell invasion

(32). RTKs may therefore augment the sig-naling functions of 64 at the expense of

its ability to mediate stable adhesion. Simi-larly, cooperation betweenv3 and platelet-

derived growth factor receptor (PDGFR) mayenhance growth and migration of tumor cells

overexpressing PDGF (34). By altering theirintegrin repertoire, neoplastic cells can hone

part of the molecular machinery that under-lies adhesion, migration, survival, and growth

to optimally serve their needs (Figure 2).Upon clustering in focal adhesions, inte-

grins activate several protein tyrosine kinases,central among which is FAK (35, 36). The

adaptor proteins paxillin and talin mediate in-tegrin interaction with FAK, which coordi-

nates many of the key events that constitute

or are related to integrin signaling (36). Acti-vation of FAK is initiated by autophosphory-

lation at Tyr397, which results in a structuralmotif recognized by SH2-domain-containing

proteins, including Src. Binding to FAK acti-vates Src, resulting in phosphorylation of ad-

ditional FAK residues and recruitment of sev-eral signaling adaptor and effector proteins,

guanine nucleotide exchange factors, GTPaseactivating proteins, cytoskeletal adaptors, and

proteolytic enzymes (23, 36, 37). The N-terminal domain of FAK provides a signal-

ing linkage between integrins and RTKs,especially EGFR and PDGFR (38). FAK

thus constitutes a platform for the coordi-nated growth-factor-receptorintegrin signal

exchange, regulation of Rho GTPase activ-ity, and focal complex turnover. Binding of

the GRB2 adaptor protein to FAK generatesan important link to the activation of Ras

and the MAPK/extracellular-signal-regulatedkinase-2 (ERK2) cascade. ERK2 phospho-

rylation can modulate focal contact dynam-ics in motile cells in addition to promoting

both proliferation and survival. Recruitmentof guanine nucleotide exchange factors, in-

cluding p190RhoGEF, may provide a directlink to RhoA activation (39) and provide reg-

ulation of promigratory versus adhesive inter-actions with substrate. Cytoskeletal adaptors,



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including ezrin, and proteolytic enzymes,

including calpain, regulate intracellular link-age of focal contacts to the actin cytoskeleton

and focal contact turnover (40).

Cell migration induced by EGF or PDGFrequires FAK association with both RTKs

and integrin-containing focalcomplexes,con-sistent with the notion that FAK can inte-

grate promigratory signals from integrins andRTKs. These signals culminate in the activa-

tion of Rho GTPases and downstream effec-tors of the MAPK pathway, including ERK

and Jun-amino-terminal kinase ( JNK) (23).ERK and JNK participate in regulating cell

migration by phosphorylating and activatingthe myosin light chain kinase, which induces

contraction of actomyosin fibers (41). JNKin-duces phosphorylation of paxillin, which may

participate in cell migration by facilitating fo-cal adhesion turnover (42). Rho GTPases ac-

tivated by FAK include Rho, Rac, and Cdc42(43, 44). Cdc42 and Rac have both been

implicated in carcinoma invasion, as theypromote actin polymerization at the leading

edge and, consequently, formation of filopo-dia and lamellipodia, respectively (43). Both

GTPases activate the ARP2/3 complex andinduce actinfilament assembly coordinated by

the Wiskott-Aldrich syndrome protein (45).They also activate p21-activatedkinase, which

enhances actin polymerization by activatingLIM kinase. Whereas Cdc42 and Rac pro-

mote actin polymerization at the leading edge,Rho orchestrates the assembly and contrac-

tion of actomyosin fibers, which pulls thetrailing edge forward during migration. At

least two Rho effector molecules, Rho kinase

(ROCK) and mammalian diaphanous, func-tion jointly to induce the assembly of acto-

myosin fibers (46). By inhibiting myosin lightchain phosphatase, ROCK promotes myosin

light chain phosphorylation and actomyosinfiber contraction (47). Rho-ROCK signaling

appears to regulate several aspects of carci-noma dissemination and is required for cancer

cells to invade three-dimensional matrices byamoeboid movement (48). Gene expression

profile comparison between melanoma cells

with low and high colony-forming abilities

in the lung in experimental metastasis assaysidentified RhoC as one of the most robustly

upregulated genes in the highly metastaticvariants (49).

Although FAK is commonly associatedwith coordinating migration signals, there

is abundant evidence to suggest that it canalso promote invasion in both normal and

neoplastic cells (50, 51). Malignant cells fre-quently display elevated FAK levels and activ-

ity(50),which is associated with shape changepodosome formation, and induction of in-

vadopodia (52). In experimental models, theinvasive tumor phenotype was associated with

the accumulation of FAK-Src signaling com-plexes within invadopodia, specialized cel

protrusions enriched in integrins and MMPs

Consistent with these observations, overex-pression of FAK in some tumor cell types was

reported to induce invasion (53, 54). Inter-estingly, the invasion-promoting property of

FAK appears to be distinct from its ability toinduce migration (52).

Immunoglobulin superfamily adhesion

receptors. Adhesion receptors belonging tothe immunoglobulin superfamily have been

implicated in the progression of some carci-nomas. Theadhesion receptorL1 is highlyex-

pressed at the invasive front of colorectal can-cers. L1 is a direct Wnt/-catenin signaling

pathway target in colorectal cancer cells, andin experimental model systems has been ob-

served to enhance motility and tumorigenicity(55).

Similar to L1, the neuronal cell adhe-sion receptor NrCAM, which is also a tar-

get of Wnt signaling, promotes tumorigenic-ity and migration in various tumor cell types

(56). By contrast, downregulation of neuralcell adhesion molecule is associated with en-

hanced lymph node metastasis in the Rip-Tagtransgenic mouse model of pancreatic islet

cell carcinogenesis (57). Like N-cadherinL1 and neural cell adhesion molecule bind

to and activate FGFRs in neurons and tu-

mor cells, thereby participating in modulating



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integrin-mediated cell adhesion to and migra-

tion on the ECM (20).

Cell surface proteoglycan function

changes. Transformation may also result inthe activation and differential glycosylation

of cell surface proteoglycans. One suchproteoglycan relevant to tumor metastasis

is CD44, the principal cell surface receptorfor hyaluronan. In addition to mediating cell

attachment to hyaluronan-coated surfacesand participating in hyaluronan metabolism,

CD44, by virtue of its structural polymor-phism and facultative decoration with a

variety of glycosaminoglycans, can serveas a multipurpose cell surface scaffold that

orchestrates the assembly of complexescomprising various classes of molecules (58).

Thus, CD44 associates with and promotesoligomerization of the ErbB family of

growth factor receptors (59) and Met (60).It also localizes various MMPs and some of

their substrates to the cell surface (6163).Its cytoplasmic domain interacts with the

ezrin-radixin-moesin family of cytoskeletalinteractors (64), including merlin, which is

implicated in tumor metastasis (65). Themechanisms whereby CD44 promotes tumor

invasion and dissemination include enhance-ment of cell migration (66), coordination of

proteolytic activity on the cell surface (62,

63), and enhancement of survival (62, 63, 67).Although it is expressed in most carcinomas,

CD44 promotes invasion and metastasis ina selective manner. By analogy to integrins,

CD44 may be expressed in an inactive confor-mation, and its activation requires, at the very

least, partial desialylation of the extracellulardomain and a critical cell surface expression

density (6871). CD44 appears to be consti-tutively active in fibroblasts and mesenchymal

progenitor cells, and to regulate migrationand adhesion of both cell types. Consistent

with this observation, as well as observationsin mouse model systems, sarcomas may

retain CD44 function and display at leastpartial dependence upon its expression for

migration, invasion, and metastasis (72).

Proteolysis, Invasion,and Tumor-Host Interactions

Following several rounds of division within

the epithelial compartment, generating acarcinoma in situ, malignant cells disrupt the

basement membrane (BM), allowing themto come into direct contact with structural

and cellular components of the stroma. Thisphase can be subdivided into several events

key for subsequent tumor dissemination,including (a) expression by the tumor cells

of the proteolytic arsenal required to de-grade the BM; (b) interaction with stromal

fibroblasts and modification of their functionto better serve the requirements of tumor

cell growth, migration, and survival; (c)

recruitment of leukocytes that may amplifythe stromal reaction and further facilitate

tumor cell dissemination; (d) angiogenesis;and finally (e) intravasation (Figure 3). The

stromal reaction to invading tumor cells isvariable, depending in part upon tumor cell

properties and in part upon the local stromalcomposition. Tumor cells typically produce

mediators that can initiate local stromalcell activation, leading to ECM remodeling

and recruitment of additional stromal cellpopulations, which provides permissive

conditions for tumor growth. The combined

proteolytic machinery of the tumor and acti-vated stromal cells degrades ECM proteins,uncovering cryptic sites that may display pro-

migratory properties and releasing se-questered growth and survival factors,

including insulin-like growth factor-1, trans-forming growth factor- (TGF-), PDGF,

vascular endothelial growth factor (VEGF),fibroblast growth factor, and hepatocyte

growth factor, thereby augmenting theirbioavailability (73).

Recent assessment of the expression pro-file of stroma associated with invasive can-

cer in a transgenic model of multistage car-cinogenesis of the prostate revealed a gene

signature reminiscent of that associated withwound healing (74). Importantly, genes that

were upregulated in the reactive stroma were



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BM

Normal epithelia

VEGFR3

Fibroblast

Monocyte/TAM

Granulocyte

Blood vessel

TGF-

Proliferation/differentiation

EMT/invasion

Recruitment,

proliferation,

and activation

HPCs and EPCs

Release ofsequestered

growth factors

IGF-1, TGF-, PDGF,VEGF, bFGF, HGF/SF

Tumor cell

CAF

Activatedfibroblast

ECM remodeling

MMP-9, uPA,cathepsins

VEGF-A

VEGF-C, VEGF-D

Lymphatic vessel

Lymphangiogenesis

Angiogenesis

EPC recruitmentSDF-1

VEGFR1

Collagen

fibers

Figure 3

Tumor-host interactions. Basement membrane (BM) degradation allows tumor cells to enter into contactwith stromal fibroblasts and alter their phenotype toward that of myofibroblasts (CAFs). Invading tumor

cells secrete numerous growth factors that stimulate angiogenesis, including VEGF-A, which binds toVEGFR1 on endothelial hematopietic precursor cells (HPCs) and endothelial precursor cells (EPCs), aswell as on monocytes/tumor-associated macrophages (TAMs), resulting in their recruitment andactivation. Tumor cells also secrete VEGF-C and -D, which bind and activate VEGFR3 on lymphaticendothelial cells and stimulate lymphangiogenesis. Activated CAFs, TAMs, and tumor cells secretenumerous extracellular matrix (ECM)-degrading enzymes, including matrix metalloproteinases (MMPs),cathepsins, and uPA, whose combined activity releases numerous ECM-sequestered growth factors thatfurther stimulate stromal fibroblast proliferation and tumor cell invasion.

found to have predictive value for both overall

and metastasis-free survival of prostate andbreast carcinoma patients. Several of these

genes were found to be upregulated in tumor-associatedstromal remodelingin studies using

different approaches to address tumor-hostinteractions (75, 76). Together these obser-

vations strongly suggest that the stromal re-sponse to primary carcinoma growth may

hold the key to subsequent development anddissemination.

Disruption of the basement membrane

The first barrier to invasion by carcinoma

cells is constituted by the BM. Conventional

wisdom would have it that a broad range ofMMPs can degrade the various components

of the BM. However, most of the evidencesupporting this view was derived from studies

of tumor invasion of matrigel, which iscomposed of denatured BM but does not

recapitulate its native structure. Recent workhas provided direct evidence that proteolytic



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degradation of the BM is mediated by trans-

membrane MMPs (MT-MMPs), includingMT1-, MT2-, and MT3-MMP (MMP-14,

-15, and -16, respectively), all three of which

are commonly expressed by cancer cells(77). Importantly, the ability to degrade

native BM has been shown to be restrictedto these three MMPs. Neither soluble nor

artificially membrane-tethered MMP-2 andMMP-9, which are commonly associated

with tumor invasion, were able to promotecell penetration of the BM (77). The same

held true for several other secreted MMPs,providing the first clear evidence that native

BM degradation by tumor cells is dependentupon MT-MMP-mediated proteolysis (77).

MT-MMPs display collagenase activityand are capable of degrading collagen IV,

which is a major constitutent of the BM.Their ability to degrade other collagens

and numerous other ECM componentssuggests that MT-MMPs may be implicated

in events that occur beyond BM invasion.Consistent with this view, experiments using

three-dimensional collagen gels showed thatMT1-MMP expression provides tumor cells

with a growth advantage in vitro and in vivo(78). The replicative advantage conferred by

MT1-MMP requires pericellular ECM pro-teolysis, as proliferation is abrogated in tumor

cells suspended in protease-resistant collagengels. In the absence of proteolysis, tumor

cells embedded in physiologically relevantECM matrices adopt a spherical config-

uration and fail to display shape changesand cytoskeletal reorganization required for

three-dimensional growth. These obser-

vations suggest that MT1-MMP regulatesproliferation by controlling cell geometry

within the the three-dimensional ECM (78).In addition to these essential roles in

the early steps of tumor metastasis, MT1-MMP activity provides the principal source

of MMP-2 activation (79) and promotes an-giogenesis (80) by degrading the fibrin ma-

trix that surrounds newly formed blood ves-sels, facilitating endothelial cell penetration of

tumor tissue (81). Consistent with their role

in invasion, MT-MMPs have been observed

to colocalize with integrins to invadopodia(82). MMP-14 has also been shown to bind

and cleave the extracellular domain of CD44,which helps detach tumor cells from the ECM

and promotes migration (83, 84).

Proteolytic events within the extracellular

matrix. The evidence that MT-MMPs me-diate tumor cell disruption of the BM as well

as subsequent ECM invasion raises the ques-

tion as to what role secreted MMPs play intumor metastasis, particularly because the ma-

jority of them appear to be supplied by stro-malcells(79). Several linesof evidencesuggest

that at least some of the secreted MMPs mayhave a robust tumor-initiating effect (79, 85).

However, secreted MMPs may also partici-pate in tumor dissemination (79, 85). Secreted

MMPs can interact with cell surface adhesionreceptors and proteoglycans, leading to co-

operation between adhesive and proteolyticmechanisms (79, 85), by analogy to the well-

established functional relationship betweenintegrins and the urokinase receptor (86).

Hyaluronan-dependent association betweenCD44 and MMP-9, which is cell-context de-

pendent, has been shown to promote pro-teolytic activation of latent TGF- relevant

to both inflammation (87) and cancer (62).In a mouse model of mammary carcinoma

dissemination, the functional CD44/MMP-9/TGF- complex promoted angiogenesis

and survival of metastatic tumor cells (62, 67).Similarly, the cell surface complex comprising

CD44HSPG, proheparin-binding epider-mal growth factor (HB-EGF), MMP-7, and

ErbB4 promotes both normal and malignantcell survival by facilitating MMP-7-mediated

HB-EGF activation and its engagement ofErbB4 (Figure 2) (63). The sum of these ob-

servations suggests that secreted MMPs teth-ered to the surface of stromal or tumor cells

may indirectly participate in metastasis by ac-tivating relevant growth factors, promoting

angiogenesis, and probably further disrupting

structural ECM barriers to invading cells.



12/29

Interaction with fibroblasts and soluble

regulators of tumor-host cross talk. Hav-ing crossed the disrupted BM, tumor cells for

the first time find themselves in direct contact

with stromal fibroblasts. Tumor-derived anddegraded ECM-released growth factors,

including PDGF and TGF-, alter thefibroblast phenotype to one reminiscent

of myofibroblasts (Figure 3) (88). Thesetumor-conditioned stromal fibroblasts are

referred to as carcinoma-associated fibrob-lasts (CAFs), and their contribution to tumor

initiation and growth is now well established(8991). However, the mechanisms whereby

CAFs promote tumor progression are onlybeginning to emerge. CAFs are an abundant

source of proteolytic enzymes, includingMMPs and cathepsins (74, 92, 93), which

may stimulate tumor cell growth and inva-sion at both primary and secondary sites.

Increased deposition of collagen I and IIIas well as de novo expression of tenascin C

may provide additional signals that facilitatetumor invasion and metastasis; by secreting

chemokines such as monocyte chemotacticprotein-1 and cytokines such as interleukin-1

(IL-1), CAFs participate in regulating theinflammatory response to tumor invasion. In

addition, CAF-derived stromal-cell-derivedfactor-1 (SDF-1) has been shown to mediate

bone marrowderived endothelial cell pre-cursor recruitment and to directly increase

tumor cell proliferation (94).Among the soluble factors implicated in

coordinating tumor-host cross talk, TGF-plays a leading role. Although TGF- in-

hibits proliferation of normal epithelial cells

and carcinoma cells at early stages of progres-sion, it stimulates fibroblast growth and ECM

secretion and promotes late-stage carcinomainvasiveness (88). Both transgenic models

(95) and experimental metastasis assays haveshown that TGF- can enhance dissemina-

tion of at least some carcinomas (67). Accord-ingly, soluble TGF-receptor fusion proteins

were observed to reduce metastatic growthof tumor cells injected into immunocompro-

mised mice (96). TGF- can induce EMT

in tumor cells resistant to its cytostatic ef-

fects and, as already discussed, may be a majorplayer in the activation of normal fibroblasts

to display tumor-promoting functions (88).

Migration. ECM remodeling and the pres-ence of activated stroma fibroblasts create

conditions favorable for tumor cell migra-tion. Although the molecular mechanisms

that underlie migration are reasonably wellunderstood (see above), the question as to

how tumor cells actually migrate in a three-

dimensional structure has only recently beenaddressed. Real-time imaging of invading tu-

mor cells in three-dimensional collagen gelshasgiven rise to some surprising observations

Single tumor cells that had detached fromthe original tumor mass were shown to dis-

play two possible migration patterns. Mes-enchymal cells, or malignant epithelial cells

that had undergone EMT, migrate along aclassical scheme that includes protrusion of

the leading edge, formation of focal contactswith the ECM, recruitment of surface pro-

teases to ECM contacts resulting in localizedproteolysis, Rho-mediated contraction of ac-

tomyosin leading to cell contraction, and fi-nally detachment of the trailing edge. How-

ever, other malignant cells display amoeboidmovement through collagen gels. This type

of movement relies on cell deformability andrelatively weak interactions with the ECM

Movement is generated by cortical filamen-tousactin, whereasfocalcontacts,stress fibers

and localized proteolysis at cell-ECM con-tacts are lacking (97). Cells displaying amoe-

boid movement typically circumscribe but donot degrade collagen fibers (97). Although

thistype of movementcharacterizes lymphoidcells, carcinoma cells can adopt it as well

(97).Tumor cells can also migrate in groups

or aggregates (Figure 2). Here again twotypes of movement have been described. One

consists of chain migration, where cells followeach other in single file and form a chain-like

image. It is displayed by neural crest cells

(98) and normal myoblasts (99), but can also



13/29

be observed in melanomas. Lobular invasive

breast carcinoma as well as ovarian carcinomacells often display a chain-type arrangement.

The second type of group movement of

malignant cells is referred to as collectivemigration and invasion and mimics a well-

described phenomenon that occurs duringdevelopment. Following neural tube closure,

cells in the blastoderm or ectoderm migrate insheets(100), andsimilar migration is observed

during branching morphogenesis of mam-mary glands and ducts (101). Malignant cells

also have the ability to aggregate and migrateas a functional unit (97, 102). In contrast to

individual migrating cells, cell-cell adhesionthat occurs in cell aggregates leads to a specific

form of cortical actin filament assembly alongcell junctions that allows the formation of a

larger-sized, multicellular contractile body(97, 102). The cells at the front of the body are

designated path-finding cells and are the onesthat generate traction via pseudopod activity

and expression of clusters of integrins andMMPs within the corresponding invadopodia

(102, 103). Cells in the inner and trailingregions are passively pulled along. Recent

work has shown that the type-1 mucin-likecell surface receptor podoplanin is upregu-

lated at the outer edge of growing tumorsand may promote collective tumor cell mi-

gration (104). Collective movement has beenobserved in several types of carcinoma (97).

Inflammation. Recruitment of leukocytesmay have different effects in different tu-

mor types. Thus, accumulation of myeloidcells, including neutrophils monocytes and

macrophages, is associated with indolent evo-lution in some cancers, but bears a much more

somber prognosis in others (105). Infiltrat-ing tumor-associated macrophages (TAMs)

present antigen and secrete cytokines thatsupport an adaptive antitumor immune re-

sponse. On the other hand, if tumor cells re-sist the immune reponse, which most solid

tumors appear to do succesfully, the TAM-derived chemokine/cytokine repertoire may

promote tumor progression (106). In addition

to CAFs, tumor cells themselves may recruit

hematopoietic precursors and leukocytes.Myeloid precursors as well as monocytes

express receptors for several growth fac-tors/cytokinessecreted by tumorcells, includ-

ing VEGFR1, which binds tumor-derivedVEGF-A and placental growth factor PIGF,

facilitating their recruitment to the tumor mi-croenvironment where they can differentiate

into TAMs and promote tumor growth anddissemination. In a model of skin carcinogen-

esis in the mouse, TAM-derived MMP-9 was

shown to play a key role in promoting tu-mor angiogenesis (107). Macrophages were

also found to play an essential role in tumorintravasation (108), whereas macrophage de-

pletion hasbeen observed to repress late-stagetumor progression and metastasis but not pri-

mary tumor growth (109, 110). Together withCAFs, TAMs and possibly other leukocytes

may supply tumor cells with proinvasive fac-tors that facilitate metastasis (111).

Angiogenesis. One of the prerequisites for

metastatic tumor growth is the inductionof angiogenesis (112, 113). Angiogenesis is

frequently induced by transforming eventsthat promote tumor progression and aug-

ment expression of angiogenic factors. Thus,VEGF-A expression is induced by the MAPK

signaling pathway and hypoxia that accom-panies rapid primary tumor growth. Hypoxia

induces and stabilizes expression of hypoxia-inducible-factor-1, which drives VEGF-A

transcription. VEGF-A is among growthfactors deposited in the ECM and whose

bioavailability, in addition to that of otherproangiogenic factors including basic fibrob-

last growth factor and TGF-, is increased asa result of tissue remodeling (114).

Studies in cancer patients as well as in

mouse models provide evidence that lym-phangiogenesis, defined as the outgrowth

of new lymphatic vessels from preexistingones, promotes metastasis to regional drain-

ing lymph nodes of a tumor (115, 116). Lym-phangiogenesis is induced by tumor-derived

VEGF-C and -D members of the VEGF



14/29

family, which bind to VEGFR3 on the sur-

face of lymphatic endothelial cells. VEGF-Cexpression is regulated, at least in part, by in-

flammatory responses triggered by IL-1 and

tumor necrosis factor signaling. VEGF-Dis the product of an immediate early, Fos-

regulated gene, and its expression may there-fore be regulatedby oncogenicsignaling path-

ways (115, 116).

Intravasation. It would seem logical thatan increased lymphatic and vascular network

may facilitate penetration of the vascular lu-men by invading tumor cells. Carcinomas ini-

tiallyform metastasis in local lymph nodes andonly at a later stage in other organs. There

is an ongoing and as yet unresolved debateas to whether distal hematogenous metastases

in carcinomas develop as a result of vascularinvasion and penetration at the primary tu-

mor site or whether they are derived fromcells that have colonized local lymph nodes.

In the first case, tumor cells would need todegrade vascular BM and irrupt into the cir-

culation. An argument favoring this possibil-ity is that vascular invasion and penetration

by tumor cells is observed by microscopic ex-amination of tissue sections. This is further

supported by experimental approaches using

an in vivo assay (117), where intravasation de-pended upon MMP-9 activity and constituteda rate-limiting step in metastasis. Tumor cell

dissemination from lymph nodes could occurby migration of the cells to efferent lymph

vessels and transport to the vena cava fromwhere hematogenous spread would be possi-

ble. Currently, it would seem plausible thatboth mechanisms might be operational and

that the relative ease of lymph vessel inva-sionand penetration might explain that lymph

nodes are usually the first metastatic site incarcinomas. It is also possible, however, that

colonization of both lymphoid and nonlym-

phoid organs occurs within a comparable timeframe, but owing to local conditions tumor

growth proceeds more rapidly in lymphoidtissues.

Tumor Dissemination

Survival in lymph and blood and interac-

tions with endothelium. Once tumor cells

have penetrated the blood circulation, theyare exposed to shear stress and to interac-

tions with leukocytes that may lead to theirdestruction. It wouldappear, however, thattu-

mor cells are capable of resisting shear stresspossibly aided by platelets and leukocytes, and

that they may rely on some of the mechanismsused by leukocytes to adhere to endothelium

Transformed cells often express an alteredglycosyltranferase repertoire with respect to

normal counterparts. Glycosyl- and sialyl-transferases expressed in many carcinoma

types decorate cell surface receptors witholigosaccharide structures that correspond to

ligands of selectins, a C-type lectin class of

cell surface adhesion molecules that regulateleukocyte endothelial interactions and leuko-

cyte trafficking (118). P-selectin/CD62P isexpressed on the surface of activated platelets

and endothelial cells, and E-selectin/CD62Eis predominantly induced in activated en-

dothelium. L-selectin/CD62L is expressed onthe surface of a broad range of leukocyte

subpopulations.A variety of potentially metastatic tu-

mor cells, particularly those that are mucin

richoften derived from colonexpress se-lectin ligands (119121). Circulating tumorcells that express selectin ligands can become

coated with platelets and leukocytes, creat-ing a microembolus that may obstruct cap-

illaries of various organs (119); they may alsoadhere to activated endothelial cells. In an ex-

perimental metastasis model, B16 melanomacells engineered to synthesize oligosaccha-

rides that constitute selectin ligands displayedan altered pattern of organ colonization

compared with their parental counterparts(122).

Platelets and leukocytes can also inter-act with tumor cells via v3-dependent

adhesion (123). Experimental models sug-gest that tumor-cellplatelet/leukocyte inter-

actions may favor tumor metastasis (120, 121



15/29

124, 125), but whether such a mechanism is

important in human cancer metastasis is sub-ject to debate.

Experimental evidence suggests that inte-

grins and immunoglobulin superfamily adhe-sion molecules are also implicated in tumor

cell adhesion to endothelium. The 41 in-tegrin, associatedprimarily with lymphocytes,

is expressed on a variety of tumor cell types,and its ligand VCAM-1 was shown to support

melanoma cell adhesion to endothelial cells(126).

Leukocyte adhesion to activated en-dothelium typically relies on three sets of

events: selectin-mediated low-affinity inter-actions responsible for leukocyte rolling on

the endothelium; endothelial cell-derivedchemokine-mediated leukocyte activation

that changes leukocyte 2 integrin confor-mation from low to high affinity; and high-

affinity interaction between leukocyte 2integrins and endothelial ICAM-1, which is

required for theleukocyte arrest that precedesand is necessary for diapedesis/extravasation

(118). Despite being larger and having dif-ferent morphological properties than circu-

lating leukocytes, carcinoma cells can makeuse of at least some of the adhesive mecha-

nisms that govern leukocyte trafficking to in-teract with vascular endothelium. However,

it remains to be demonstrated whether theseadhesive events are relevant to human cancer

metastasis.

Random arrest or programmed organ-

specific homing. Intravital microscopy im-ages argue that tumor cells tend to obstruct

capillaries, particularly if they are aggregatedor bound to leukocytes and platelets. Follow-

ing arrest in the capillary bed, they proliferatelocally and disrupt the capillary wall, whereby

they penetrate the local parenchyma (127).It would therefore seem that tumor cell ar-

rest in capillaries and subsequent extravasa-tion are primarily mechanical processes that

could occur in all organs. Organ-specific tu-mor cell homing would then require spe-

cific mechanims. Three nonmutually exclu-

sive candidate mechanisms proposed thus

far are chemokine-receptor-mediated chemo-taxis, the establishment of a metastatic niche,

and a tumor cell genetic program that facili-tates adaptation to a particular microenviron-

ment.

Chemokine-receptor-mediated chemo-taxis. Chemokines are believed to cooperate

with adhesion receptors in determining wheretumor cells arrest and extravasate. Tumor

cells can express a variety of chemokinereceptors, including CXCR4, that serves as

a receptor for CXCL12/SDF. Secretion ofSDF by host tissue stromal fibroblasts is

suggested to promote chemotaxis of tumorcells expressing CXCR4 and to determine, at

least in part, the localization of metastases of

certain tumor types (128).

Preparation of the metastatic microenvi-

ronment. Recent studies have suggested thatby virtue of their cytokine and chemokine

repertoire, tumorsmayhave theability to pre-pare the microenvironment of distant organs

to receive disseminating cells and allow theirproliferation. Tumor and associated stromal

cellderived chemokines can recruit endothe-lial and hematopoietic progenitor cells (EPCs

and HPCs, respectively) to the relevant or-gans prior to tumor cell arrival, which, to-

gether with tumor cellderived deposition offibronectin, appear to precondition the local

microenvironment (129, 130). Targeted inhi-bition of these cells using anti-VEGFR1 neu-

tralizing antibodiessuggeststhattheproposedpreconditioning is necessary for metastatic

spread. The mechanisms that govern HPCrecruitment to potential metastatic sites are

still unclear, as is the manner in which HPCs

render anygiven site permissive for metastatictumor growth. It is possible that recruitment

is initiated by the very first tumor cells thatarrive at a distant site and that local HPC

activity then alters the local microenviron-ment, amplifying tumor cell recruitment and

allowing subsequent division. However, the



16/29

existence of metastatic niches remains to be

confirmed.

Gene expression patterns that determine

organ-specific homing of tumor cells. Pi-oneeringworkusingan experimentalmetasta-

sis approach has shown that repeated cycles ofin vivo passage of tumor cells that develop few

lung tumors following tail vein injection inmice result in selection of cells that preferen-

tially develop lung metastasis (131). As men-tioned above, thegeneexpression profileanal-

ysis of B16 melanoma cells selected for lungcolonization revealed upregulation of numer-

ous genes including RhoC (49), and forcedexpression if RhoC in parental B16 cells re-

sulted in the preferential homing to the lung(49).

To address this issue in human tumors,one study focused on the human breast

carcinoma MDA-MB231 cell line derivedfrom the pleural effusion of a patient with

widespread metastasis. Repeated rounds of invivo passage and selection allowed isolation of

different sublines of MDA-MB231 cells thatpredictably formed tumors in given organs

following intravenous injection (132). Geneexpression profiling of these sublines shows

that they express a specific set of genes thatcorrelates with general metastatic proclivity.

However, the selected cell lines expressed

additional gene signatures that correlatedwith the organ to which they metastasized

(132). Thus, a set of 54 genes was identi-fied that distinguished cell lines displaying

lung tropism (132). Although none of theidentified genes alone could recapitulate the

metastatic phenotype of selected cell lines,combinations of the genes could induce the

poorly metastatic parental MDA-MB231cells to colonize the organ from which the

metastatic cell variants were retrieved.Prior to this study, the same cell line had

been used to identify genes that may be in-volved in promoting bone metastasis (133).

Most of the highly overexpressed genes incell lines derived from and selected for their

ability to induce bone metastases encoded cell

membrane or secreted molecules that were in

some way relevant to the bone microenviron-ment. Expression of any one of these genes in

parental MDA-MB231 cellsfailedto augmenttheir intrinsic metastatic potential to bone

However, the combination of three to four ofthe genes augmented the metastatic activity

of parental cells to levels comparable to thosedisplayed by the most aggressive cell lines ex-

pressing the entire bone metastasis gene set(133). These observations strongly suggest

that cooperation among several genes that en-

code proteins with complementary functionsunderlies the metastatic phenotype. Cells ex-

pressing the required genes were identified inthe initiating tumor cell line, suggesting that

breast cancer cells that display a gene expres-sion signature associated with bone or lung

metastatic proclivity exist in the parental tu-mor cell population.

Establishment of new colonies. Extrava-sation was long thought to be a rate-

limiting step in metastasis. However, RAS-transformed NIH3T3 cells and parenta

counterparts were found to extravasate intothe liver at a comparable rate following in-

jection into the portal vein, whereas only theRAS-transformed cells could form metastatic

growth (134). These observations provideconvincing evidence that the rate-limiting

step is not extravasation, but rather the abilityof the cells to establish themselves in the new

host tissue microenvironment.Once tumor cells have penetrated the

parenchyma of an organ other than the onewhere they originated, they must create a mi-

croenvironment conducive to their survivaland proliferation. The situation is analogous

to that of invading cells of the primary tu-

mor penetrating its BM and entering into di-rect physical contact for the first time with the

stroma. However, the stromal composition ofthe secondary site may differ from that sur-

rounding theprimary tumor, andtheability ofthetumor cells to subvert the local microenvi-

ronment will most likely determine their fate



17/29

Dormancy. Metastases from some human

cancers occur as many as 20 years follow-ing removal of the primary tumor. These

metastatic lesions are believed to be dormant

for an extended period of time and to becomeproliferative once localconditionsbecome ap-

propriately permissive.Dormancy is a concealed state, and as such

does not readily lend itself to direct study. Ex-perimental work, however, has provided ev-

idence for the existence of micrometastasesthat fail to induce angiogenesis and in which

cell proliferation is balanced by cell death be-cause of inadequate blood supply (135). These

small metastatic lesions may therefore consti-tute one source of tumor dormancy. Another

possible source of tumor dormancy are iso-lated tumor cells that arrive at secondary sites

where they may persist for long periods oftime without being able to divide (127). Inter-

estingly, recovery of isolated dormant mam-mary carcinoma cells from liver tissue showed

that they retain tumorigenicity when injectedinto immunocompromised mice (127). These

observations suggest that the host tissue mayprovide permissive or restrictive cues that de-

termine whether metastatic cells may prolif-erate and generate secondary tumors. Recent

evidence suggests that changes in the ECMthat lead to a shift in the equilibrium between

natural stimulators and inhibitors of angio-genesis, many of which are ECM degradation

products, may allow dormant metastatic le-sions to induce an angiogenic switch and de-

velop into full-blown secondary tumors (136).

EARLY EMERGENCE ORLATE-STAGE SELECTIONOF METASTATIC CELLS?

Until recently, the prevailing reasoning was

that metastasis arises from rare tumor cellsthat emerge relatively late in tumor progres-

sion. The genetic makeup of these cells wasbelieved to be the consequence of stochastic

accumulation of mutations that provide themwith all of the properties required for dissem-

ination. This view is supported by the well-

documented correlation between primary tu-

mor size and risk of metastasis. However, theassociation of clinical features such as tumor

grade with metastatic proclivity and the oc-currence of bone micrometastases early in the

evolution of cancer are inconsistent with astrictly stochastic model.

The use of DNA microarray studies toidentify transcriptional signatures that may

distinguish metastatic from primary tumorgrowth has further challenged this view, sug-

gesting that relevant signatures may be de-tected in early-stage primary tumors that are

destined to metastasize. This second viewis supported by observations from several

independent and distinctly designed studies(Figure 4). The first of these studies showed

that the clinical outcome of breast cancer pa-

tients can be predicted by a poor progno-sis gene expression signature present in the

majority of early-stage primary tumors (137,138). This gave rise to the notion that certain

tumors may have the properties required formetastasis from the very beginning. The size

of the primary tumor would then be irrele-vant in terms of the risk of developing metas-

tases, and even small tumors may be expectedto contain cells with metastatic potential.

In a second study, comparison of the geneexpression profile of metastatic adenocarci-

noma lesions to unmatched primary carcino-mas revealed a 17-gene expression signature

thatdistinguishedprimary frommetastatic tu-mors (139). Subsequent analysis revealed that

numerous early-stage primary solid tumors ofdiverse histotypes harbored the same gene ex-

pression signature, suggesting that these tu-mors were most likely associated with metas-

tases and poor clinical outcome.Because the bone marrow is a major hom-

ing site for breast cancer, a third study un-dertook the task of analyzing genomic differ-

ences between single bone marrowderivedmicrometastatic cells and the primary tumor

by comparative genomic hybridization (140).Single viable disseminated breast cancer cells

hadan abundance of chromosomal copy num-ber changes in their genome, with significant



18/29

Metastasis

is an

early event

in tumor

progression

Primary tumors vs. metastatic nodules

17-genesignature

Breast ProstateLung

Good

Poor

The metastatic potential is encoded in the

bulk of primary tumors

(Ramaswamy et al. 2003)

Early-stage primary

invasive breast tumors

Tumor microenvironment

70-genesignature

BreastGood

Poor

Prostate Breast

Good

Poor

Wound-

response

genes

No mets

Mets

Metastatic proclivity is present in early tumors

(vant Veer et al. 2002)

Wound response signature in primary tumors

predicts increased risk of metastasis

and poor outcome

(Bacac et al. 2006, Chang et al. 2005)

In situ Invasive

LCM stroma

Bone metastases vs. primary tumor

Dissemination occurs early during tumorigenesis

(Gangnus et al. 2004)

Short-termculture

CGH

11-genesignature

Metastases vs.primarytumor

Breast ProstateLung

Good

Poor

Stem cellness and cancer

Tumors with stem celllike signatures are

likely to have poor prognosis

(Glinsky et al. 2005)

Bmi/

Bmi+/+Mouse-human

translational

genomics



19/29

intercellular heterogeneitybut more impor-

tantly, they displayed numerous differences incomparison to the matching primary tumors.

These observations led to the conclusion that

metastasis is an early event in malignant tu-mors and that disseminated cells evolve inde-

pendently of the primary tumor.A fourth study addressed the possibility

that cancers with poor prognosis and highmetastatic proclivity may display some prop-

erties that characterize normal stem cells.Comparison of primary and metastatic mouse

prostate cancers with normal stem cells thathad retained or lost their self-renewing po-

tential identifieda common 11-gene signaturethat was then used to probe human cancers

(141143). Expression of this gene signaturein 11 distinct types of primary cancers was

consistently a powerful predictor of a shortinterval to disease recurrence, distant metas-

tasis, and death following therapy. These ob-servations are consistent with the possibility

that cancer cells with high metastatic procliv-ity display features that are at least in part

reminiscent of those of normal stem cells. In-terestingly, a fraction of each of the gene ex-

pression signatures identified in these stud-ies comprised stromal cell transcripts, some

of which were part of the recently describedpredictive stromal gene set (74). This further

underscores the notion that the stroma mayactively participate in determining the ability

of cancer to metastasize (74, 75, 144, 145).The emerging, and still controversial, con-

cept of cancer stem cells suggests that amongthe heterogeneous populations of cells that

compose primary tumors is a small popula-

tion of stem cellresembling malignant cellsthat constitute the driving force of the tumor

and are essential for progression and metasta-sis. In support of this view, eight out of nine

tumor samples that served as a source for theidentification of breast cancerinitiating cells

were derived from metastatic lesions (146).These cells may share attributes of normal

stem cells that are relevant to their natural be-havior and resistance to chemotherapy. Nor-

mal stem cells express multidrug-resistancegenes, which, coupled to their slow prolifer-

ative rate, may play a key role in their abilityto withstand cytotoxic drugs and repopulate

tissues that had been depleted of their nor-mal cells by chemotherapy. Metastatic cancer

lesions are notorious for resistance to con-ventional chemotherapy, at least in part be-

cause of multidrug-resistance gene expres-sion. Whether or not the stem cell connection

turns out to be correct, the lack of respon-siveness of metastatic lesions to conventional

therapy and the increasingly clear evidencethat the stroma is implicated in tumor metas-

tasis warrant a closer look at the molecularmechanisms that govern tumor-host interac-

tions at both primary and metastatic sites.

SUMMARY AND FUTURE

DIRECTIONSRecent studies strongly support the view that

the capacity of a tumor to disseminate is ac-quired at early steps during the multistep pro-

cess of tumorigenesis. They also suggest thatspecific genes uniquely responsible for cancer

cell dissemination and metastatic growth are

Figure 4

Overview of recent microarray and CGH studies that led to the notion that metastasis is an early event intumorigenesis. Validation of signatures identified by Ramaswamy et al. (139) on patient cohorts withearly-stage tumors showed that metastatic proclivity is present early in tumor evolution. Studiesperformed by Gangnus et al. (140) on bone micrometastasis confirmed this notion. More recently,Glinsky et al. (141) proposed that tumors with a stem cell expression signature are likely to have a poorprognosis. Most of these signatures contained stroma-associated genes, consistent with the notion thatthe tumor microenvironment plays an important role in dissemination. This was also suggested by recentstudies focusing on stromal reactions to tumor invasion and injury that showed that a wound-responsesignature in primary tumors predicts increased risk of metastasis.



20/29

unlikely to be discovered. Rather, the com-

bined effect of oncogene signaling and tu-mor suppressor gene loss in the appropriate

cellular environment is likely to determine

whether a cancer cell has the potential to col-onize distant organs. Success of secondary tu-

mor growth is then determined by the natureof the host response and the tumor cells abil-

ity or inability to subvert it.The realization that early cancer har-

bors metastatic potential should warrant pre-ventive treatment of metastatic disease. Our

extensive understanding of the mechanismswhereby cancer cells spread should allow the

development of strategies that can effectivelyblock tumor dissemination. Approaches likely

to meet with success are those that simul-

taneously target several mechanisms uponwhich tumor cell dissemination depends, in-

cluding angiogenesis, proteolysis, and growthfactor signaling. Much more challenging

is the prospect of reversing already estab-lished metastatic growth. The formidable

ability of metastatic lesions to evade cyto-toxic drug effects, at least in part due to

multidrug resistance family gene expressionindicates that we need to look elsewhere

for therapeutic solutions. As metastatic tu-mors require local stroma support for growth

identifying targetable tumor-host interac-tion mechanisms appears to be an appealing

pursuit.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.

ACKNOWLEDGMENTS

This work was supported by the Fonds de la Recherche Scientifique grant number 3100A0-

105833 and by the National Center of Competence in Research Molecular Oncology.

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