Review2008 - Metastasisb Cancer Cell

Metastatic Cancer Cell
Marina Bacac and Ivan Stamenkovic

Annu. Rev. Pathol. Mech. Dis. 2008.3:221-247. Downloaded from arjournals.annualreviews.org by SEOUL NATIONAL UNIVERSITY on 05/21/09. For personal use only.
Experimental Pathology Unit, Department of Pathology, University of Lausanne, Switzerland; email: Ivan.Stamenkovic@chuv.ch, Marina.Bacac@chuv.ch
Annu. Rev. Pathol. Mech. Dis. 2008. 3:22147 First published online as a Review in Advance on September 17, 2007 The Annual Review of Pathology: Mechanisms of Disease 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 microenvironment at a distance from the primary tumor. Metastatic cancer cells require properties that allow them not only to adapt to a foreign microenvironment but to subvert it in a way that is conducive to their continued proliferation and survival. Recent conceptual and technological advances have contributed to our understanding of the role of the host tissue stroma in promoting tumor cell growth and 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 of malignant tumors, and metastasis is the principal cause of death among cancer patients. It is the single most challenging obstacle to successful cancer management and may also be viewed as the last frontier of cancer research. Metastasis is the process whereby cancer cells spread throughout the body, establishing new colonies in organs at a distance from the one where the primary tumor originated. It is well established that metastasis is a complex, multistep process, which although considered highly inefcient from the cellular point of view, virtually constitutes a death sentence for the patient. Its prevention and management are therefore among the key goals in clinical and basic cancer research. There are three major routes for tumor dissemination: lymphatic vessels, blood vessels, and serosal surfaces. Tumor metastases occurring via these three routes are referred to as lymphatic, hematogenous, and transcoelomic, respectively. Epithelial malignancies, or carcinomas, typically begin their dissemination by the lymphatic route, with hematogenous metastases occurring at a later time. In contrast, bone and soft tissue tumors, or sarcomas, preferentially metastasize by the hematogenous route, whereas transcoelomic metastasis is the property of a relatively small group of tumors that includes mesotheliomas and ovarian carcinomas. Because of their prevalence, lymphatic and hematogenous metastases will provide the main focus of the present review. A tumor cell that initiates a metastatic colony must (a) detach from the primary mass, (b) invade the local host tissue stroma, (c) penetrate local lymphatic and blood vessels, (d ) survive within the circulation, (e) become arrested in capillaries or venules of other organs, ( f ) penetrate the corresponding parenchyma, ( g) adapt to the newly colonized milieu or subvert the local microenvironment to 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 are still unresolved. For example, do cells displaying metastatic proclivity emerge late in tumor progression as a result of multiple mutations and selection, or are they part of the cell population that constitutes early malignant growth? What role does the host microenvironment play in tumor cell dissemination, and which components of tumor-stroma cross talk might provide potential therapeutic targets? At least ve functions are required for a tumor cell to successfully complete the sequence of events outlined above. They include interaction with the local microenvironment, migration, invasion, resistance to apoptosis, and the ability to induce angiogenesis. All ve functions are regulated by adhesion and proteolysis, which together provide the most fundamental molecular effector mechanisms upon which a metastatic cell relies (Table 1). Adhesion and proteolysis determine tumor cell interaction with other cells and with the extracellular matrix (ECM), help create a path for migration, promote angiogenesis, and both directly and indirectly trigger survival signals. Transformation results in major phenotypic changes that affect cell surface receptor expression, cytoskeletal function, growth factor and cytokine secretion, proteolytic enzyme production, and the glycosyltransferase and glycosidase repertoire (Figure 2). These combined changes alter the way in which the transformed cell communicates with its microenvironment and, by the same token, the way in which it is perceived by normal surrounding cells. They not only provide transformed cells with the ability to disrupt the barriers that keep their normal counterparts conned to a dened tissue compartment, but also with the means to subject the host tissue microenvironment to their rules and use its resources for their own survival, growth, and dissemination. It is increasingly clear that tumor cells depend upon
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Invasive carcinoma
Detachment
In situ carcinoma
Normal epithelia
Basement membrane
Basement membrane degradation Invasion Collagen fibers Migration
Dormant metastases
No proliferation Intravasation
Extravasation
Circulation
Proliferation, angiogenesis, microenvironment activation
Growing metastases
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 basement membrane degradation, tumor cells invade the surrounding stroma, migrate and intravasate into blood or lymph 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 cells to provide functions, such as a diversity of proteolytic activity, that they themselves may lack. Understanding tumor-host interactions may therefore provide a key to understanding metastasis. A more recently emerging view, based on gene expression prole analysis of
diverse primary and metastatic tumors, is that metastatic cells may constitute part of the early makeup of a malignant tumor. This review highlights our current understanding of tumor cell properties and host tissue responses whose combination culminates in cancer metastasis and discusses the origin of metastatic cells in light of recent observations.
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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 Adhesion Cadherins E-cadherin N-cadherin Integrins 21, 31 v3 Enhance metastasis in selected experimental models Promotes migration and invasion; mediates tumor cell-platelet interactions Promotes migration, invasion, and proliferation; cooperates with RTKs Mediates tumor cell adhesion to endothelial cells Mediates tumor cellendothelial cell adhesion Promotes tumorigenicity and motility Promotes tumorigencity and migration Its downregulation is associated with enhanced lymph node metastasis in some tumor models Mediate tumor cellendothelial interactions and tumor cellplatelet/leukocyte adhesion Mediates interaction with hyaluronan; interacts with RTKs; specic isoforms provide a scaffold for the assembly of molecular complexes that can promote metastasis (28, 29) (30, 123) (31, 32) (126) (126) (55) (56) (57) (119122) (5860, 62, 63) Promotes cell-cell adhesion; prevents cell detachment; cleaved by MMP-3, MMP-7, and ADAMs; tumor suppressor Promotes migration and invasion; regulates activation of FGFRs (1, 2, 4, 5) (19, 20) Candidate functions Reference(s)
64 41 VCAM-1 L1 NrCAM NCAM Selectins CD44 Proteolysis
Immunoglobulin superfamily
Cell surface proteoglycans
Matrix metalloproteinases MMP-1 MMP-2 MMP-3 MMP-7 MMP-9 MMP-14, -15, -16 Cathepsins Degrades collagen; promotes invasion Activates growth factors, including TGF-; promotes invasion; interacts with v3 integrin on the cell surface Promotes tuumorigenesis; activates growth factors, including HB-EGF Promotes cell survival; activates HB-EGF; interacts with CD44 on the cell surface Promotes invasion; enhances angiogenesis; promotes intravasation; activates TGF-; interacts with CD44 on the cell surface Degrade native basement membrane; promote invasion Promote tumor growth and invasion; may be expressed by tumor cells or exclusively by stromal cells (79, 85) (79, 85) (79, 85, 93) (63, 79, 85) (62, 79, 85) (7779, 85) (74)
FUNDAMENTAL MOLECULAR EFFECTOR MECHANISMS OF METASTASIS Adhesion

Detachment, cadherins, and epithelialto-mesenchymal transition. Interepithelial cell interactions are regulated by complex adhesion mechanisms, including tight and adherens junctions and desmosomes (1). Trans224 Bacac
formed and malignant cells that become detached from the epithelium display loss of adherens junctions, which, in epithelial cells, are constituted primarily by E-cadherin (1, 2). Like other members of the cadherin family, E-cadherin displays homophilic binding specicity. Its adhesive functions are stabilized by -catenin, which binds to its cytoplasmic domain, providing a link to -catenin and the actin cytoskeleton (3). Experiments
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performed in vitro and in vivo have shown that genetic and antibody-mediated inhibition of E-cadherin function can alter the phenotype of epithelial cells from noninvasive to invasive (4). Conversely, introduction of E-cadherin into E-cadherin-decient invasive carcinoma cells abrogated their invasiveness (3). Downregulation of E-cadherin activity in vivo using a dominant negative E-cadherin construct in the transgenic Rip-Tag mouse model of pancreatic islet cell carcinoma resulted in the transition of a well-differentiated -cell adenoma to an invasive carcinoma (5). Crossing the Rip-Tag mice with mice that maintain E-cadherin expression in transformed -cells arrested tumor progression in the adenoma stage (5). Together, these observations argue that E-cadherin functions as a tumor suppressor. Although loss of function mutations occur in E-cadherin, they are not commonly observed in malignant tumors (3). Mechanisms that decrease or abrogate E-cadherin function in carcinoma cells include transcriptional repression, followed by promoter methylation (6, 7), disruption of cytoskeletal connections, increased intracellular degradation, and proteolytic cleavage of the extracellular domain by matrix metalloproteinases (MMPs) (3). Several of the transcriptional repressors that control E-cadherin expression in development, including Snail, Slug, SIP1, Twist, dEF1, and E12/E47 (812), are implicated in E-cadherin downregulation in malignant cells. An alternative mechanism that can inactivate E-cadherin function in tumor cells is disruption of the link between E-cadherin and the cytoskeleton. One example is provided by mutations in -catenin that abrogate its binding to -catenin and result in a nonadhesive phenotype (3). Coordinate receptor tyrosine kinase (RTK)-integrin signaling can also interfere with E-cadherin function. Activated RTKs and Src family kinases induce tyrosine phosphorylation of the E-cadherin-catenin complex, which is then recognized by the Cbl-like E3 ubiquitin ligase and downregulated by endocytosis (13). In
CD44 inactive
E-cadherin 5 1 3 1
BM Extracellular matrix
Selectin ligands N-cadherin c-Met CD44v3 Glycosaminoglycan chains pro-HB-EGF FGFR MMP-7 ErbB4 3 MMP-14 MMP-15 ErbB2 6 4 c-Met
Extracellular matrix
Figure 2 Changes of adhesive properties in transformed cells. Normal epithelial cell communication with its microenvironment is regulated by E-cadherin-mediated cell-cell interaction and 1-integrin-mediated adhesion to the basement membrane (BM). Transformation results in the cadherin switch that leads to E-cadherin loss and replacement by N-cadherin, which plays an important role in invasion by regulating broblast growth factor receptor (FGFR) function. Carcinomas frequently express v3 and 64 integrins, which promote invasion and proliferation, in part through their interactions with receptor tyrosine kinases, including ErbB2 and Met. Transformation also results in changes in glycosylation of cell surface proteoglycans such as CD44, which regulates invasion by coordinating MMP-7-mediated heparin-binding epidermal growth factor (HB-EGF) activation and ErbB4 signaling. Changes in the glycosyltransferase repertoire can also result in the decoration of cell surface receptors by oligosaccharides that constitute selectin ligands. Several transmembrane MMPs are expressed by tumor cells and mediate BM degradation.
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v-src-transformed cells, this process is dependent upon integrin signaling and focal adhesion kinase (FAK) phosphorylation (14, 15). Integrin signaling operates through Snail/Slug to suppress E-cadherin expression and disrupt adherens junctions. In some epithelial cells, this process may be mediated by integrin-linked kinase (16, 17). Finally, proteolytic cleavage of the extracellular domain of E-cadherin by MMP-3 and MMP-7 disrupts its ability to promote cellular interactions (18). Abrogation of E-cadherinmediated cell-cell adhesion results in detachment of tumor cells from the epithelial cell layer and affects signaling pathways implicated in cell migration and growth, including Rho GTPase-mediated modulation of the actin cytoskeleton and the canonical Wnt signaling pathway (3). Loss of E-cadherin in malignant cells may be replaced by other cadherins, most commonly, N-cadherin (Figure 2). This process, known as the cadherin switch, is associated with a phenotypic change observed in vitro known as epithelial-to-mesenchymal transition (EMT). EMT, dened as the conversion of epithelial cells to motile, broblast-like cells that express mesenchymal rather than epithelial cell markers, is a common event during normal embryonic development and is observed as epithelial cell progress through the stages of carcinogenesis in vitro. It is proposed to reect invasive and metastatic properties of transformed epithelial cells, but remains somewhat controversial because full EMT is difcult to prove in vivo. Nevertheless, expression of N-cadherin may make a critical contribution to invasion through both its adhesive and signaling functions. N-cadherin can mediate cell-cell interactions with N-cadherin-expressing stromal cells, which may play an important role in the ability of tumor cells to direct host responses. N-cadherin binds to and regulates the activation of broblast growth factor receptors (FGFRs), thereby helping assemble the FGFR-signaling complex, which triggers downstream signaling pathways, including
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phospholipase C-, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase (MAPK). The combined action of these signaling pathways promotes cell survival, migration, and invasion (19, 20). Similar to E-cadherin, the extracellular domain of Ncadherin is susceptible to proteolytic cleavage by MMPs. The N-cadherin cleavage product can block N-cadherin-mediated cell-cell adhesion, but can also stimulate FGFR signaling on adjacent cells in paracrine fashion (19, 20). Cleavage of N-cadherin at a site within the transmembrane or cytoplasmic domain by a -secretase-type protease results in the translocation of its carboxy-terminal segment to the nucleus, where it represses transcription mediated by the CREB-binding protein (21). Interaction with the extracellular matrix integrins. Tumor cell interactions with the ECM are mediated primarily by integrins and play a key role in tumor invasion and spread. Integrins form a large family of adhesion receptors, each member consisting of an and a transmembrane chain (22). In mammals, 18 and 8 chains associate in various combinations to give rise to 24 integrins that recognize distinct ECM ligands, with, nevertheless, some degree of overlap (22). When integrins bind to ECM, they aggregate in the plane of the cell membrane and associate with a molecular complex composed of adaptor, signaling, and cytoskeletal proteins that orchestrate the organization of actin laments. Organization of actin laments into stress bers, in turn, promotes further integrin aggregation that results in increased ECM binding in a positive feedback loop. The outcome is an integrin-mediated assembly of ECM and cytoskeletal protein clusters on each side of the cell membrane known as focal adhesions and ECM contacts (23). Integrins trigger both mechanical and chemical signals that organize and remodel the 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 cooperate with RTKs, thereby jointly controlling survival and mitogenic pathways. A reductionist view might be that integrins mediate cell adhesion and impose positional control on RTK activity, which together determine whether cells migrate and proliferate in response to cytokines and growth factors. Although seemingly straightforward, integrin implication in cellular functions is complicated by their diverse adhesive and signaling properties that provide them the ability to affect cell function in a variety of ways that are often context dependent. Thus, 21 and 31 integrins mediate epithelial cell adhesion to basal lamina and maintain them in a quiescent state. These integrins are often downregulated in carcinomas and their reexpression in carcinoma cells can decrease or even revert the malignant phenotype (2427). However, both of these integrins can enhance metastasis in selected experimental models (28, 29), underscoring the cell context and tumor stage dependence of the effects of 1 integrins. By contrast, v3 (30) and 64 (31) integrins are frequently upregulated in carcinomas, where they may promote migration, invasion, and proliferation. In addition to participating in hemidesmosome organization, the 64 integrin cooperates with epidermal growth factor receptor (EGFR), ErbB2, and Met, and is likely to promote the growth of carcinomas in which activating mutations of 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 carcinoma cells activates the phosphatidylinositol 3-kinase pathway, which results in activation of Rac and increased invasiveness of these cells in vitro (31). The cytoplasmic domain of 4 also acts as an adaptor and amplier of proinvasive signals induced by the hepatocyte growth factor receptor Met in cells undergoing Met-mediated transformation (33). Both EGF and Met induce phosphorylation of 4 and enhance SHC signaling, which disrupts hemidesmosomes and increases epithe-
lial cell migration and carcinoma cell invasion (32). RTKs may therefore augment the signaling functions of 64 at the expense of its ability to mediate stable adhesion. Similarly, cooperation between v3 and plateletderived growth factor receptor (PDGFR) may enhance growth and migration of tumor cells overexpressing PDGF (34). By altering their integrin repertoire, neoplastic cells can hone part of the molecular machinery that underlies adhesion, migration, survival, and growth to optimally serve their needs (Figure 2). Upon clustering in focal adhesions, integrins activate several protein tyrosine kinases, central among which is FAK (35, 36). The adaptor proteins paxillin and talin mediate integrin interaction with FAK, which coordinates many of the key events that constitute or are related to integrin signaling (36). Activation of FAK is initiated by autophosphorylation at Tyr397, which results in a structural motif recognized by SH2-domain-containing proteins, including Src. Binding to FAK activates Src, resulting in phosphorylation of additional FAK residues and recruitment of several signaling adaptor and effector proteins, guanine nucleotide exchange factors, GTPase activating proteins, cytoskeletal adaptors, and proteolytic enzymes (23, 36, 37). The Nterminal domain of FAK provides a signaling linkage between integrins and RTKs, especially EGFR and PDGFR (38). FAK thus constitutes a platform for the coordinated growth-factor-receptorintegrin signal exchange, regulation of Rho GTPase activity, and focal complex turnover. Binding of the GRB2 adaptor protein to FAK generates an important link to the activation of Ras and the MAPK/extracellular-signal-regulated kinase-2 (ERK2) cascade. ERK2 phosphorylation can modulate focal contact dynamics in motile cells in addition to promoting both proliferation and survival. Recruitment of guanine nucleotide exchange factors, including p190RhoGEF, may provide a direct link to RhoA activation (39) and provide regulation of promigratory versus adhesive interactions with substrate. Cytoskeletal adaptors,
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including ezrin, and proteolytic enzymes, including calpain, regulate intracellular linkage of focal contacts to the actin cytoskeleton and focal contact turnover (40). Cell migration induced by EGF or PDGF requires FAK association with both RTKs and integrin-containing focal complexes, consistent with the notion that FAK can integrate promigratory signals from integrins and RTKs. These signals culminate in the activation of Rho GTPases and downstream effectors of the MAPK pathway, including ERK and Jun-amino-terminal kinase ( JNK) (23). ERK and JNK participate in regulating cell migration by phosphorylating and activating the myosin light chain kinase, which induces contraction of actomyosin bers (41). JNK induces phosphorylation of paxillin, which may participate in cell migration by facilitating focal adhesion turnover (42). Rho GTPases activated by FAK include Rho, Rac, and Cdc42 (43, 44). Cdc42 and Rac have both been implicated in carcinoma invasion, as they promote actin polymerization at the leading edge and, consequently, formation of lopodia and lamellipodia, respectively (43). Both GTPases activate the ARP2/3 complex and induce actin lament assembly coordinated by the Wiskott-Aldrich syndrome protein (45). They also activate p21-activated kinase, which enhances actin polymerization by activating LIM kinase. Whereas Cdc42 and Rac promote actin polymerization at the leading edge, Rho orchestrates the assembly and contraction of actomyosin bers, which pulls the trailing edge forward during migration. At least two Rho effector molecules, Rho kinase (ROCK) and mammalian diaphanous, function jointly to induce the assembly of actomyosin bers (46). By inhibiting myosin light chain phosphatase, ROCK promotes myosin light chain phosphorylation and actomyosin ber contraction (47). Rho-ROCK signaling appears to regulate several aspects of carcinoma dissemination and is required for cancer cells to invade three-dimensional matrices by amoeboid movement (48). Gene expression prole comparison between melanoma cells
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with low and high colony-forming abilities in the lung in experimental metastasis assays identied RhoC as one of the most robustly upregulated genes in the highly metastatic variants (49). Although FAK is commonly associated with coordinating migration signals, there is abundant evidence to suggest that it can also promote invasion in both normal and neoplastic cells (50, 51). Malignant cells frequently display elevated FAK levels and activity (50), which is associated with shape change, podosome formation, and induction of invadopodia (52). In experimental models, the invasive tumor phenotype was associated with the accumulation of FAK-Src signaling complexes within invadopodia, specialized cell protrusions enriched in integrins and MMPs. Consistent with these observations, overexpression of FAK in some tumor cell types was reported to induce invasion (53, 54). Interestingly, the invasion-promoting property of FAK appears to be distinct from its ability to induce migration (52). Immunoglobulin superfamily adhesion receptors. Adhesion receptors belonging to the immunoglobulin superfamily have been implicated in the progression of some carcinomas. The adhesion receptor L1 is highly expressed at the invasive front of colorectal cancers. L1 is a direct Wnt/-catenin signaling pathway target in colorectal cancer cells, and in experimental model systems has been observed to enhance motility and tumorigenicity (55). Similar to L1, the neuronal cell adhesion receptor NrCAM, which is also a target of Wnt signaling, promotes tumorigenicity and migration in various tumor cell types (56). By contrast, downregulation of neural cell adhesion molecule is associated with enhanced lymph node metastasis in the Rip-Tag transgenic mouse model of pancreatic islet cell carcinogenesis (57). Like N-cadherin, L1 and neural cell adhesion molecule bind to and activate FGFRs in neurons and tumor cells, thereby participating in modulating
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integrin-mediated cell adhesion to and migration on the ECM (20). Cell surface proteoglycan function changes. Transformation may also result in the activation and differential glycosylation of cell surface proteoglycans. One such proteoglycan relevant to tumor metastasis is CD44, the principal cell surface receptor for hyaluronan. In addition to mediating cell attachment to hyaluronan-coated surfaces and participating in hyaluronan metabolism, CD44, by virtue of its structural polymorphism and facultative decoration with a variety of glycosaminoglycans, can serve as a multipurpose cell surface scaffold that orchestrates the assembly of complexes comprising various classes of molecules (58). Thus, CD44 associates with and promotes oligomerization 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 cytoskeletal interactors (64), including merlin, which is implicated in tumor metastasis (65). The mechanisms whereby CD44 promotes tumor invasion and dissemination include enhancement 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 in a selective manner. By analogy to integrins, CD44 may be expressed in an inactive conformation, and its activation requires, at the very least, partial desialylation of the extracellular domain and a critical cell surface expression density (6871). CD44 appears to be constitutively active in broblasts and mesenchymal progenitor cells, and to regulate migration and adhesion of both cell types. Consistent with this observation, as well as observations in mouse model systems, sarcomas may retain CD44 function and display at least partial 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 a carcinoma in situ, malignant cells disrupt the basement membrane (BM), allowing them to come into direct contact with structural and cellular components of the stroma. This phase can be subdivided into several events key for subsequent tumor dissemination, including (a) expression by the tumor cells of the proteolytic arsenal required to degrade the BM; (b) interaction with stromal broblasts and modication of their function to better serve the requirements of tumor cell growth, migration, and survival; (c) recruitment of leukocytes that may amplify the stromal reaction and further facilitate tumor cell dissemination; (d ) angiogenesis; and nally (e) intravasation (Figure 3). The stromal reaction to invading tumor cells is variable, depending in part upon tumor cell properties and in part upon the local stromal composition. Tumor cells typically produce mediators that can initiate local stromal cell activation, leading to ECM remodeling and recruitment of additional stromal cell populations, which provides permissive conditions for tumor growth. The combined proteolytic machinery of the tumor and activated stromal cells degrades ECM proteins, uncovering cryptic sites that may display promigratory properties and releasing sequestered growth and survival factors, including insulin-like growth factor-1, transforming growth factor- (TGF-), PDGF, vascular endothelial growth factor (VEGF), broblast growth factor, and hepatocyte growth factor, thereby augmenting their bioavailability (73). Recent assessment of the expression prole of stroma associated with invasive cancer in a transgenic model of multistage carcinogenesis of the prostate revealed a gene signature reminiscent of that associated with wound healing (74). Importantly, genes that were upregulated in the reactive stroma were
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Recruitment, proliferation, and activation HPCs and EPCs
Angiogenesis VEGF-A
VEGFR1 Tumor cell Blood vessel VEGFR3 Lymphangiogenesis VEGF-C, VEGF-D
Lymphatic vessel Proliferation/ differentiation TGF-

BM
Normal epithelia
EMT / invasion ECM remodeling MMP-9, uPA, cathepsins CAF Granulocyte Monocyte/TAM Release of sequestered growth factors IGF-1, TGF-, PDGF, VEGF, bFGF, HGF/SF SDF-1 EPC recruitment
Fibroblast Collagen fibers
Activated fibroblast
Figure 3 Tumor-host interactions. Basement membrane (BM) degradation allows tumor cells to enter into contact with stromal broblasts and alter their phenotype toward that of myobroblasts (CAFs). Invading tumor cells secrete numerous growth factors that stimulate angiogenesis, including VEGF-A, which binds to VEGFR1 on endothelial hematopietic precursor cells (HPCs) and endothelial precursor cells (EPCs), as well as on monocytes/tumor-associated macrophages (TAMs), resulting in their recruitment and activation. Tumor cells also secrete VEGF-C and -D, which bind and activate VEGFR3 on lymphatic endothelial cells and stimulate lymphangiogenesis. Activated CAFs, TAMs, and tumor cells secrete numerous extracellular matrix (ECM)-degrading enzymes, including matrix metalloproteinases (MMPs), cathepsins, and uPA, whose combined activity releases numerous ECM-sequestered growth factors that further stimulate stromal broblast proliferation and tumor cell invasion.
found to have predictive value for both overall and metastasis-free survival of prostate and breast carcinoma patients. Several of these genes were found to be upregulated in tumorassociated stromal remodeling in studies using different approaches to address tumor-host interactions (75, 76). Together these observations strongly suggest that the stromal response to primary carcinoma growth may hold the key to subsequent development and dissemination.
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Disruption of the basement membrane. The rst barrier to invasion by carcinoma cells is constituted by the BM. Conventional wisdom would have it that a broad range of MMPs can degrade the various components of the BM. However, most of the evidence supporting this view was derived from studies of tumor invasion of matrigel, which is composed of denatured BM but does not recapitulate its native structure. Recent work has provided direct evidence that proteolytic
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degradation of the BM is mediated by transmembrane MMPs (MT-MMPs), including MT1-, 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 restricted to these three MMPs. Neither soluble nor articially membrane-tethered MMP-2 and MMP-9, which are commonly associated with tumor invasion, were able to promote cell penetration of the BM (77). The same held true for several other secreted MMPs, providing the rst clear evidence that native BM degradation by tumor cells is dependent upon MT-MMP-mediated proteolysis (77). MT-MMPs display collagenase activity and are capable of degrading collagen IV, which is a major constitutent of the BM. Their ability to degrade other collagens and numerous other ECM components suggests that MT-MMPs may be implicated in events that occur beyond BM invasion. Consistent with this view, experiments using three-dimensional collagen gels showed that MT1-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 proteolysis, as proliferation is abrogated in tumor cells suspended in protease-resistant collagen gels. In the absence of proteolysis, tumor cells embedded in physiologically relevant ECM matrices adopt a spherical conguration and fail to display shape changes and cytoskeletal reorganization required for three-dimensional growth. These observations suggest that MT1-MMP regulates proliferation by controlling cell geometry within the the three-dimensional ECM (78). In addition to these essential roles in the early steps of tumor metastasis, MT1MMP activity provides the principal source of MMP-2 activation (79) and promotes angiogenesis (80) by degrading the brin matrix that surrounds newly formed blood vessels, 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 mediate tumor cell disruption of the BM as well as subsequent ECM invasion raises the question as to what role secreted MMPs play in tumor metastasis, particularly because the majority of them appear to be supplied by stromal cells (79). Several lines of evidence suggest that at least some of the secreted MMPs may have a robust tumor-initiating effect (79, 85). However, secreted MMPs may also participate in tumor dissemination (79, 85). Secreted MMPs can interact with cell surface adhesion receptors and proteoglycans, leading to cooperation between adhesive and proteolytic mechanisms (79, 85), by analogy to the wellestablished functional relationship between integrins and the urokinase receptor (86). Hyaluronan-dependent association between CD44 and MMP-9, which is cell-context dependent, has been shown to promote proteolytic activation of latent TGF- relevant to both inammation (87) and cancer (62). In a mouse model of mammary carcinoma dissemination, the functional CD44/MMP9/TGF- complex promoted angiogenesis and survival of metastatic tumor cells (62, 67). Similarly, the cell surface complex comprising CD44HSPG, proheparin-binding epidermal growth factor (HB-EGF), MMP-7, and ErbB4 promotes both normal and malignant cell survival by facilitating MMP-7-mediated HB-EGF activation and its engagement of ErbB4 (Figure 2) (63). The sum of these observations suggests that secreted MMPs tethered to the surface of stromal or tumor cells may indirectly participate in metastasis by activating relevant growth factors, promoting angiogenesis, and probably further disrupting structural ECM barriers to invading cells.
Interaction with broblasts and soluble regulators of tumor-host cross talk. Having crossed the disrupted BM, tumor cells for the rst time nd themselves in direct contact with stromal broblasts. Tumor-derived and degraded ECM-released growth factors, including PDGF and TGF-, alter the broblast phenotype to one reminiscent of myobroblasts (Figure 3) (88). These tumor-conditioned stromal broblasts are referred to as carcinoma-associated broblasts (CAFs), and their contribution to tumor initiation and growth is now well established (8991). However, the mechanisms whereby CAFs promote tumor progression are only beginning to emerge. CAFs are an abundant source of proteolytic enzymes, including MMPs and cathepsins (74, 92, 93), which may stimulate tumor cell growth and invasion at both primary and secondary sites. Increased deposition of collagen I and III as well as de novo expression of tenascin C may provide additional signals that facilitate tumor invasion and metastasis; by secreting chemokines such as monocyte chemotactic protein-1 and cytokines such as interleukin-1 (IL-1), CAFs participate in regulating the inammatory response to tumor invasion. In addition, CAF-derived stromal-cell-derived factor-1 (SDF-1) has been shown to mediate bone marrowderived endothelial cell precursor 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- inhibits proliferation of normal epithelial cells and carcinoma cells at early stages of progression, it stimulates broblast growth and ECM secretion and promotes late-stage carcinoma invasiveness (88). Both transgenic models (95) and experimental metastasis assays have shown that TGF- can enhance dissemination of at least some carcinomas (67). Accordingly, soluble TGF-receptor fusion proteins were observed to reduce metastatic growth of tumor cells injected into immunocompromised mice (96). TGF- can induce EMT
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in tumor cells resistant to its cytostatic effects and, as already discussed, may be a major player in the activation of normal broblasts to display tumor-promoting functions (88). Migration. ECM remodeling and the presence of activated stroma broblasts create conditions favorable for tumor cell migration. Although the molecular mechanisms that underlie migration are reasonably well understood (see above), the question as to how tumor cells actually migrate in a threedimensional structure has only recently been addressed. Real-time imaging of invading tumor cells in three-dimensional collagen gels has given rise to some surprising observations. Single tumor cells that had detached from the original tumor mass were shown to display two possible migration patterns. Mesenchymal cells, or malignant epithelial cells that had undergone EMT, migrate along a classical scheme that includes protrusion of the leading edge, formation of focal contacts with the ECM, recruitment of surface proteases to ECM contacts resulting in localized proteolysis, Rho-mediated contraction of actomyosin leading to cell contraction, and nally detachment of the trailing edge. However, other malignant cells display amoeboid movement through collagen gels. This type of movement relies on cell deformability and relatively weak interactions with the ECM. Movement is generated by cortical lamentous actin, whereas focal contacts, stress bers, and localized proteolysis at cell-ECM contacts are lacking (97). Cells displaying amoeboid movement typically circumscribe but do not degrade collagen bers (97). Although this type of movement characterizes lymphoid cells, carcinoma cells can adopt it as well (97). Tumor cells can also migrate in groups or aggregates (Figure 2). Here again two types of movement have been described. One consists of chain migration, where cells follow each other in single le and form a chain-like image. It is displayed by neural crest cells (98) and normal myoblasts (99), but can also
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be observed in melanomas. Lobular invasive breast carcinoma as well as ovarian carcinoma cells often display a chain-type arrangement. The second type of group movement of malignant cells is referred to as collective migration and invasion and mimics a welldescribed phenomenon that occurs during development. Following neural tube closure, cells in the blastoderm or ectoderm migrate in sheets (100), and similar migration is observed during branching morphogenesis of mammary glands and ducts (101). Malignant cells also have the ability to aggregate and migrate as a functional unit (97, 102). In contrast to individual migrating cells, cell-cell adhesion that occurs in cell aggregates leads to a specic form of cortical actin lament assembly along cell 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-nding cells and are the ones that generate traction via pseudopod activity and expression of clusters of integrins and MMPs within the corresponding invadopodia (102, 103). Cells in the inner and trailing regions are passively pulled along. Recent work has shown that the type-1 mucin-like cell surface receptor podoplanin is upregulated at the outer edge of growing tumors and may promote collective tumor cell migration (104). Collective movement has been observed in several types of carcinoma (97). Inammation. Recruitment of leukocytes may have different effects in different tumor types. Thus, accumulation of myeloid cells, including neutrophils monocytes and macrophages, is associated with indolent evolution in some cancers, but bears a much more somber prognosis in others (105). Inltrating tumor-associated macrophages (TAMs) present antigen and secrete cytokines that support an adaptive antitumor immune response. On the other hand, if tumor cells resist the immune reponse, which most solid tumors appear to do succesfully, the TAMderived 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 factors/cytokines secreted by tumor cells, including VEGFR1, which binds tumor-derived VEGF-A and placental growth factor PIGF, facilitating their recruitment to the tumor microenvironment where they can differentiate into TAMs and promote tumor growth and dissemination. In a model of skin carcinogenesis in the mouse, TAM-derived MMP-9 was shown to play a key role in promoting tumor angiogenesis (107). Macrophages were also found to play an essential role in tumor intravasation (108), whereas macrophage depletion has been observed to repress late-stage tumor progression and metastasis but not primary tumor growth (109, 110). Together with CAFs, TAMs and possibly other leukocytes may supply tumor cells with proinvasive factors that facilitate metastasis (111). Angiogenesis. One of the prerequisites for metastatic tumor growth is the induction of angiogenesis (112, 113). Angiogenesis is frequently induced by transforming events that promote tumor progression and augment expression of angiogenic factors. Thus, VEGF-A expression is induced by the MAPK signaling pathway and hypoxia that accompanies rapid primary tumor growth. Hypoxia induces and stabilizes expression of hypoxiainducible-factor-1, which drives VEGF-A transcription. VEGF-A is among growth factors deposited in the ECM and whose bioavailability, in addition to that of other proangiogenic factors including basic broblast growth factor and TGF-, is increased as a result of tissue remodeling (114). Studies in cancer patients as well as in mouse models provide evidence that lymphangiogenesis, dened as the outgrowth of new lymphatic vessels from preexisting ones, promotes metastasis to regional draining lymph nodes of a tumor (115, 116). Lymphangiogenesis is induced by tumor-derived VEGF-C and -D members of the VEGF
family, which bind to VEGFR3 on the surface of lymphatic endothelial cells. VEGF-C expression is regulated, at least in part, by inammatory responses triggered by IL-1 and tumor necrosis factor signaling. VEGF-D is the product of an immediate early, Fosregulated gene, and its expression may therefore be regulated by oncogenic signaling pathways (115, 116). Intravasation. It would seem logical that an increased lymphatic and vascular network may facilitate penetration of the vascular lumen by invading tumor cells. Carcinomas initially form metastasis in local lymph nodes and only at a later stage in other organs. There is an ongoing and as yet unresolved debate as to whether distal hematogenous metastases in carcinomas develop as a result of vascular invasion and penetration at the primary tumor site or whether they are derived from cells that have colonized local lymph nodes. In the rst case, tumor cells would need to degrade vascular BM and irrupt into the circulation. An argument favoring this possibility is that vascular invasion and penetration by tumor cells is observed by microscopic examination of tissue sections. This is further supported by experimental approaches using an in vivo assay (117), where intravasation depended upon MMP-9 activity and constituted a rate-limiting step in metastasis. Tumor cell dissemination from lymph nodes could occur by migration of the cells to efferent lymph vessels and transport to the vena cava from where hematogenous spread would be possible. Currently, it would seem plausible that both mechanisms might be operational and that the relative ease of lymph vessel invasion and penetration might explain that lymph nodes are usually the rst metastatic site in carcinomas. It is also possible, however, that colonization of both lymphoid and nonlymphoid organs occurs within a comparable time frame, but owing to local conditions tumor growth proceeds more rapidly in lymphoid tissues.
Tumor Dissemination
Survival in lymph and blood and interactions with endothelium. Once tumor cells have penetrated the blood circulation, they are exposed to shear stress and to interactions with leukocytes that may lead to their destruction. It would appear, however, that tumor cells are capable of resisting shear stress, possibly aided by platelets and leukocytes, and that they may rely on some of the mechanisms used by leukocytes to adhere to endothelium. Transformed cells often express an altered glycosyltranferase repertoire with respect to normal counterparts. Glycosyl- and sialyltransferases expressed in many carcinoma types decorate cell surface receptors with oligosaccharide structures that correspond to ligands of selectins, a C-type lectin class of cell surface adhesion molecules that regulate leukocyte endothelial interactions and leukocyte trafcking (118). P-selectin/CD62P is expressed on the surface of activated platelets and endothelial cells, and E-selectin/CD62E is predominantly induced in activated endothelium. L-selectin/CD62L is expressed on the surface of a broad range of leukocyte subpopulations. A variety of potentially metastatic tumor cells, particularly those that are mucin richoften derived from colonexpress selectin ligands (119121). Circulating tumor cells that express selectin ligands can become coated with platelets and leukocytes, creating a microembolus that may obstruct capillaries of various organs (119); they may also adhere to activated endothelial cells. In an experimental metastasis model, B16 melanoma cells engineered to synthesize oligosaccharides that constitute selectin ligands displayed an altered pattern of organ colonization compared with their parental counterparts (122). Platelets and leukocytes can also interact with tumor cells via v3-dependent adhesion (123). Experimental models suggest that tumor-cellplatelet/leukocyte interactions may favor tumor metastasis (120, 121,
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124, 125), but whether such a mechanism is important in human cancer metastasis is subject to debate. Experimental evidence suggests that integrins and immunoglobulin superfamily adhesion molecules are also implicated in tumor cell adhesion to endothelium. The 41 integrin, associated primarily 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 endothelium typically relies on three sets of events: selectin-mediated low-afnity interactions responsible for leukocyte rolling on the endothelium; endothelial cell-derived chemokine-mediated leukocyte activation that changes leukocyte 2 integrin conformation from low to high afnity; and highafnity interaction between leukocyte 2 integrins and endothelial ICAM-1, which is required for the leukocyte arrest that precedes and is necessary for diapedesis/extravasation (118). Despite being larger and having different morphological properties than circulating leukocytes, carcinoma cells can make use of at least some of the adhesive mechanisms that govern leukocyte trafcking to interact with vascular endothelium. However, it remains to be demonstrated whether these adhesive events are relevant to human cancer metastasis. Random arrest or programmed organspecic homing. Intravital microscopy images argue that tumor cells tend to obstruct capillaries, particularly if they are aggregated or bound to leukocytes and platelets. Following arrest in the capillary bed, they proliferate locally and disrupt the capillary wall, whereby they penetrate the local parenchyma (127). It would therefore seem that tumor cell arrest in capillaries and subsequent extravasation are primarily mechanical processes that could occur in all organs. Organ-specic tumor cell homing would then require specic mechanims. Three nonmutually exclu-
sive candidate mechanisms proposed thus far are chemokine-receptor-mediated chemotaxis, the establishment of a metastatic niche, and a tumor cell genetic program that facilitates adaptation to a particular microenvironment. Chemokine-receptor-mediated chemotaxis. Chemokines are believed to cooperate with adhesion receptors in determining where tumor cells arrest and extravasate. Tumor cells can express a variety of chemokine receptors, including CXCR4, that serves as a receptor for CXCL12/SDF. Secretion of SDF by host tissue stromal broblasts is suggested to promote chemotaxis of tumor cells expressing CXCR4 and to determine, at least in part, the localization of metastases of certain tumor types (128). Preparation of the metastatic microenvironment. Recent studies have suggested that by virtue of their cytokine and chemokine repertoire, tumors may have the ability to prepare the microenvironment of distant organs to receive disseminating cells and allow their proliferation. Tumor and associated stromal cellderived chemokines can recruit endothelial and hematopoietic progenitor cells (EPCs and HPCs, respectively) to the relevant organs prior to tumor cell arrival, which, together with tumor cellderived deposition of bronectin, appear to precondition the local microenvironment (129, 130). Targeted inhibition of these cells using anti-VEGFR1 neutralizing antibodies suggests that the proposed preconditioning is necessary for metastatic spread. The mechanisms that govern HPC recruitment to potential metastatic sites are still unclear, as is the manner in which HPCs render any given site permissive for metastatic tumor growth. It is possible that recruitment is initiated by the very rst tumor cells that arrive at a distant site and that local HPC activity then alters the local microenvironment, amplifying tumor cell recruitment and allowing subsequent division. However, the
existence of metastatic niches remains to be conrmed. Gene expression patterns that determine organ-specic homing of tumor cells. Pioneering work using an experimental metastasis approach has shown that repeated cycles of in vivo passage of tumor cells that develop few lung tumors following tail vein injection in mice result in selection of cells that preferentially develop lung metastasis (131). As mentioned above, the gene expression prole analysis of B16 melanoma cells selected for lung colonization revealed upregulation of numerous genes including RhoC (49), and forced expression if RhoC in parental B16 cells resulted 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 derived from the pleural effusion of a patient with widespread metastasis. Repeated rounds of in vivo passage and selection allowed isolation of different sublines of MDA-MB231 cells that predictably formed tumors in given organs following intravenous injection (132). Gene expression proling of these sublines shows that they express a specic set of genes that correlates with general metastatic proclivity. However, the selected cell lines expressed additional gene signatures that correlated with the organ to which they metastasized (132). Thus, a set of 54 genes was identied that distinguished cell lines displaying lung tropism (132). Although none of the identied genes alone could recapitulate the metastatic phenotype of selected cell lines, combinations of the genes could induce the poorly metastatic parental MDA-MB231 cells 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 involved in promoting bone metastasis (133). Most of the highly overexpressed genes in cell lines derived from and selected for their ability to induce bone metastases encoded cell
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membrane or secreted molecules that were in some way relevant to the bone microenvironment. Expression of any one of these genes in parental MDA-MB231 cells failed to augment their intrinsic metastatic potential to bone. However, the combination of three to four of the genes augmented the metastatic activity of parental cells to levels comparable to those displayed by the most aggressive cell lines expressing the entire bone metastasis gene set (133). These observations strongly suggest that cooperation among several genes that encode proteins with complementary functions underlies the metastatic phenotype. Cells expressing the required genes were identied in the initiating tumor cell line, suggesting that breast cancer cells that display a gene expression signature associated with bone or lung metastatic proclivity exist in the parental tumor cell population.
Establishment of new colonies. Extravasation was long thought to be a ratelimiting step in metastasis. However, RAStransformed NIH3T3 cells and parental counterparts were found to extravasate into the liver at a comparable rate following injection into the portal vein, whereas only the RAS-transformed cells could form metastatic growth (134). These observations provide convincing evidence that the rate-limiting step is not extravasation, but rather the ability of the cells to establish themselves in the new host tissue microenvironment. Once tumor cells have penetrated the parenchyma of an organ other than the one where they originated, they must create a microenvironment conducive to their survival and proliferation. The situation is analogous to that of invading cells of the primary tumor penetrating its BM and entering into direct physical contact for the rst time with the stroma. However, the stromal composition of the secondary site may differ from that surrounding the primary tumor, and the ability of the tumor cells to subvert the local microenvironment will most likely determine their fate.
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Dormancy. Metastases from some human cancers occur as many as 20 years following removal of the primary tumor. These metastatic lesions are believed to be dormant for an extended period of time and to become proliferative once local conditions become appropriately permissive. Dormancy is a concealed state, and as such does not readily lend itself to direct study. Experimental work, however, has provided evidence for the existence of micrometastases that fail to induce angiogenesis and in which cell proliferation is balanced by cell death because of inadequate blood supply (135). These small metastatic lesions may therefore constitute one source of tumor dormancy. Another possible source of tumor dormancy are isolated tumor cells that arrive at secondary sites where they may persist for long periods of time without being able to divide (127). Interestingly, recovery of isolated dormant mammary carcinoma cells from liver tissue showed that they retain tumorigenicity when injected into immunocompromised mice (127). These observations suggest that the host tissue may provide permissive or restrictive cues that determine whether metastatic cells may proliferate and generate secondary tumors. Recent evidence suggests that changes in the ECM that lead to a shift in the equilibrium between natural stimulators and inhibitors of angiogenesis, many of which are ECM degradation products, may allow dormant metastatic lesions to induce an angiogenic switch and develop into full-blown secondary tumors (136).
EARLY EMERGENCE OR LATE-STAGE SELECTION OF METASTATIC CELLS?

Until recently, the prevailing reasoning was that metastasis arises from rare tumor cells that emerge relatively late in tumor progression. The genetic makeup of these cells was believed to be the consequence of stochastic accumulation of mutations that provide them with all of the properties required for dissemination. This view is supported by the well-
documented correlation between primary tumor size and risk of metastasis. However, the association of clinical features such as tumor grade with metastatic proclivity and the occurrence of bone micrometastases early in the evolution of cancer are inconsistent with a strictly stochastic model. The use of DNA microarray studies to identify transcriptional signatures that may distinguish metastatic from primary tumor growth has further challenged this view, suggesting that relevant signatures may be detected in early-stage primary tumors that are destined to metastasize. This second view is supported by observations from several independent and distinctly designed studies (Figure 4). The rst of these studies showed that the clinical outcome of breast cancer patients can be predicted by a poor prognosis 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 for metastasis from the very beginning. The size of the primary tumor would then be irrelevant in terms of the risk of developing metastases, and even small tumors may be expected to contain cells with metastatic potential. In a second study, comparison of the gene expression prole of metastatic adenocarcinoma lesions to unmatched primary carcinomas revealed a 17-gene expression signature that distinguished primary from metastatic tumors (139). Subsequent analysis revealed that numerous early-stage primary solid tumors of diverse histotypes harbored the same gene expression signature, suggesting that these tumors were most likely associated with metastases and poor clinical outcome. Because the bone marrow is a major homing site for breast cancer, a third study undertook the task of analyzing genomic differences between single bone marrowderived micrometastatic cells and the primary tumor by comparative genomic hybridization (140). Single viable disseminated breast cancer cells had an abundance of chromosomal copy number changes in their genome, with signicant
Primary tumors vs. metastatic nodules

17-gene signature
Early-stage primary invasive breast tumors

70-gene signature
No mets
Mets
Good Poor
Good Breast Poor
Lung
Breast
Prostate
The metastatic potential is encoded in the bulk of primary tumors

(Ramaswamy et al. 2003)
Metastatic proclivity is present in early tumors

(vant Veer et al. 2002)
Bone metastases vs. primary tumor Tumor microenvironment

Metastasis is an early event in tumor progression
CGH
Short -term culture
Dissemination occurs early during tumorigenesis In situ Invasive

(Gangnus et al. 2004)
Stem cellness and cancer

LCM stroma Mouse-human translational genomics Bmi +/+ Bmi / Metastases vs. primary tumor 11-gene signature
Woundresponse genes Prostate Breast
Good Poor
Good Poor Lung Breast Prostate
Wound response signature in primary tumors predicts increased risk of metastasis and poor outcome
(Bacac et al. 2006, Chang et al. 2005)
Tumors with stem cell like signatures are likely to have poor prognosis
(Glinsky et al. 2005)
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intercellular heterogeneitybut more importantly, they displayed numerous differences in comparison to the matching primary tumors. These observations led to the conclusion that metastasis is an early event in malignant tumors and that disseminated cells evolve independently of the primary tumor. A fourth study addressed the possibility that cancers with poor prognosis and high metastatic proclivity may display some properties that characterize normal stem cells. Comparison of primary and metastatic mouse prostate cancers with normal stem cells that had retained or lost their self-renewing potential identied a common 11-gene signature that was then used to probe human cancers (141143). Expression of this gene signature in 11 distinct types of primary cancers was consistently a powerful predictor of a short interval to disease recurrence, distant metastasis, and death following therapy. These observations are consistent with the possibility that cancer cells with high metastatic proclivity display features that are at least in part reminiscent of those of normal stem cells. Interestingly, a fraction of each of the gene expression signatures identied in these studies comprised stromal cell transcripts, some of which were part of the recently described predictive stromal gene set (74). This further underscores the notion that the stroma may actively participate in determining the ability of cancer to metastasize (74, 75, 144, 145). The emerging, and still controversial, concept of cancer stem cells suggests that among the heterogeneous populations of cells that compose primary tumors is a small popula-
tion of stem cellresembling malignant cells that constitute the driving force of the tumor and are essential for progression and metastasis. In support of this view, eight out of nine tumor samples that served as a source for the identication 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 behavior and resistance to chemotherapy. Normal stem cells express multidrug-resistance genes, which, coupled to their slow proliferative rate, may play a key role in their ability to withstand cytotoxic drugs and repopulate tissues that had been depleted of their normal cells by chemotherapy. Metastatic cancer lesions are notorious for resistance to conventional chemotherapy, at least in part because of multidrug-resistance gene expression. Whether or not the stem cell connection turns out to be correct, the lack of responsiveness of metastatic lesions to conventional therapy and the increasingly clear evidence that the stroma is implicated in tumor metastasis warrant a closer look at the molecular mechanisms that govern tumor-host interactions at both primary and metastatic sites.
SUMMARY AND FUTURE DIRECTIONS

Recent studies strongly support the view that the capacity of a tumor to disseminate is acquired at early steps during the multistep process of tumorigenesis. They also suggest that specic 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 in tumorigenesis. Validation of signatures identied by Ramaswamy et al. (139) on patient cohorts with early-stage tumors showed that metastatic proclivity is present early in tumor evolution. Studies performed by Gangnus et al. (140) on bone micrometastasis conrmed this notion. More recently, Glinsky et al. (141) proposed that tumors with a stem cell expression signature are likely to have a poor prognosis. Most of these signatures contained stroma-associated genes, consistent with the notion that the tumor microenvironment plays an important role in dissemination. This was also suggested by recent studies focusing on stromal reactions to tumor invasion and injury that showed that a wound-response signature in primary tumors predicts increased risk of metastasis.
unlikely to be discovered. Rather, the combined effect of oncogene signaling and tumor suppressor gene loss in the appropriate cellular environment is likely to determine whether a cancer cell has the potential to colonize distant organs. Success of secondary tumor growth is then determined by the nature of the host response and the tumor cells ability or inability to subvert it. The realization that early cancer harbors metastatic potential should warrant preventive treatment of metastatic disease. Our extensive understanding of the mechanisms whereby cancer cells spread should allow the development of strategies that can effectively block tumor dissemination. Approaches likely
to meet with success are those that simultaneously target several mechanisms upon which tumor cell dissemination depends, including angiogenesis, proteolysis, and growth factor signaling. Much more challenging is the prospect of reversing already established metastatic growth. The formidable ability of metastatic lesions to evade cytotoxic drug effects, at least in part due to multidrug resistance family gene expression, indicates that we need to look elsewhere for therapeutic solutions. As metastatic tumors require local stroma support for growth, identifying targetable tumor-host interaction 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 of this review.
ACKNOWLEDGMENTS
This work was supported by the Fonds de la Recherche Scientique grant number 3100A0105833 and by the National Center of Competence in Research Molecular Oncology.
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Annual Review of Pathology: Mechanisms of Disease
Contents
Volume 3, 2008
The Relevance of Research on Red Cell Membranes to the Understanding of Complex Human Disease: A Personal Perspective Vincent T. Marchesi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Molecular Mechanisms of Prion Pathogenesis Adriano Aguzzi, Christina Sigurdson, and Mathias Heikenwalder p p p p p p p p p p p p p p p p p p p p 11 The Aging Brain Bruce A. Yankner, Tao Lu, and Patrick Loerch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 41 Gene Expression Proling of Breast Cancer Maggie C.U. Cheang, Matt van de Rijn, and Torsten O. Nielsen p p p p p p p p p p p p p p p p p p p p p p 67 The Inammatory Response to Cell Death Kenneth L. Rock and Hajime Kono p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 99 Molecular Biology and Pathogenesis of Viral Myocarditis Mitra Esfandiarei and Bruce M. McManus p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p127 Pancreatic Cancer Anirban Maitra and Ralph H. Hruban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p157 Kidney Transplantation: Mechanisms of Rejection and Acceptance Lynn D. Cornell, R. Neal Smith, and Robert B. Colvin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p189 Metastatic Cancer Cell Marina Bacac and Ivan Stamenkovic p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p221 Pathogenesis of Thrombotic Microangiopathies X. Long Zheng and J. Evan Sadler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p249 Anti-Inammatory and Proresolving Lipid Mediators Charles N. Serhan, Stephanie Yacoubian, and Rong Yang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p279 Modeling Morphogenesis and Oncogenesis in Three-Dimensional Breast Epithelial Cultures Christy Hebner, Valerie M. Weaver, and Jayanta Debnath p p p p p p p p p p p p p p p p p p p p p p p p p p p p313
The Origins of Medulloblastoma Subtypes Richard J. Gilbertson and David W. Ellison p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p341 Molecular Biology and Pathology of Lymphangiogenesis Terhi Karpanen and Kari Alitalo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p367 Endoplasmic Reticulum Stress in Disease Pathogenesis Jonathan H. Lin, Peter Walter, and T.S. Benedict Yen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p399 Autophagy: Basic Principles and Relevance to Disease Mondira Kundu and Craig B. Thompson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p427
The Osteoclast: Friend or Foe? Deborah V. Novack and Steven L. Teitelbaum p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p457 Applications of Proteomics to Lab Diagnosis Raghothama Chaerkady and Akhilesh Pandey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p485 The Pathology of Inuenza Virus Infections Jeffrey K. Taubenberger and David M. Morens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p499 Airway Smooth Muscle in Asthma Marc B. Hershenson, Melanie Brown, Blanca Camoretti-Mercado, and Julian Solway p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p523 Molecular Pathobiology of Gastrointestinal Stromal Sarcomas Christopher L. Corless and Michael C. Heinrich p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p557 Notch Signaling in Leukemia Jon C. Aster, Warren S. Pear, and Stephen C. Blacklow p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p587 The Role of Hypoxia in Vascular Injury and Repair Tony E. Walshe and Patricia A. DAmore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p615 Indexes Cumulative Index of Contributing Authors, Volumes 13 p p p p p p p p p p p p p p p p p p p p p p p p p p p645 Cumulative Index of Chapter Titles, Volumes 13 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p647 Errata An online log of corrections to Annual Review of Pathology: Mechanisms of Disease articles may be found at http://pathol.annualreviews.org
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Review2008 - Metastasisb Cancer Cell

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Metastatic Cancer Cell

Marina Bacac and Ivan Stamenkovic

Basement membrane degradation Invasion Collagen fibers Migration

Proliferation, angiogenesis, microenvironment activation

www.annualreviews.org Metastatic Cancer Cell

64 41 VCAM-1 L1 NrCAM NCAM Selectins CD44 Proteolysis

Cell surface proteoglycans

FUNDAMENTAL MOLECULAR EFFECTOR MECHANISMS OF METASTASIS Adhesion

www.annualreviews.org Metastatic Cancer Cell

Proteolysis, Invasion, and Tumor-Host Interactions

www.annualreviews.org Metastatic Cancer Cell

Recruitment, proliferation, and activation HPCs and EPCs

VEGFR1 Tumor cell Blood vessel VEGFR3 Lymphangiogenesis VEGF-C, VEGF-D

Lymphatic vessel Proliferation/ differentiation TGF-

Fibroblast Collagen fibers

EARLY EMERGENCE OR LATE-STAGE SELECTION OF METASTATIC CELLS?

Primary tumors vs. metastatic nodules

Early-stage primary invasive breast tumors

Good Breast Poor

The metastatic potential is encoded in the bulk of primary tumors

Metastatic proclivity is present in early tumors

Bone metastases vs. primary tumor Tumor microenvironment

Dissemination occurs early during tumorigenesis In situ Invasive

Stem cellness and cancer

Woundresponse genes Prostate Breast

Good Poor Lung Breast Prostate

SUMMARY AND FUTURE DIRECTIONS

Annual Review of Pathology: Mechanisms of Disease

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