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CANCER METASTASIS

Metastases to regional lymph nodes are detected atdiagnosis and surgery in approximately one-third of breast, colorectal, uterine cervix, and oral cavity and pharynx cancer patients, and one-quarter of esophageal, lung  pancreas,  gastric  and  bladder  cancer  patients. The high mortality rates associated with cancer are caused by the metastatic spread of tumor cells from the site of their origin. In fact, metastases are the cause of 90% of cancer deaths. The prognosis for a patient who is diagnosed with advanced invasive or metastatic disease remains little better than it was decades ago. Tumor cells invade either the blood or lymphatic vessels to access the general circulation and then establish themselves in other (visceral) tissues. Ultimately, they become surgically unresectable, with pharmacological or radiological long-term control being uncommon. In this review, we summarize recent advances in understanding the molecular mechanisms that govern metastatic progression along an axis from the primary tumor to regional lymph nodes to distant organ sites.

 Normal tissue homeostasis and cancer development

Although the genetic basis of tumorigenesis may vary greatly between different cancer types, the cellular and molecular steps required for metastasis are generally similar for all solid tumor cells. Not surprisingly, the molecular mechanisms that propel invasive growth and metastasis are also found in embryonic development, and, however to a less perpetual/chronic/aggressive/quantitatively different extent, in adult tissue maintenance (e.g. involving stem cell differentiation) and repair processes (‘tumors are wounds that do not heal’). We now view cancer as a complex tissue resulting from disrupted organ homeostasis, rather than focusing on the cancer cell, and the genes within it, alone. Normal tissue homeostasis is maintained between epithelial cells and their microenvironment, such as fibroblasts, endothelial and immunocompetent cells, and the extracellular matrix (ECM). Similarly, during malignant transformation and progression, there are (however deregulated) reciprocal and conspirational interactions between the neoplastic cells and the adjacent stromal cells. A series of recent investigations have shown that aberrations in the stroma can both precede and stimulate the development of  cancers.

Metastatic cascade: the Ying and Yang of tumor cell–host interaction

 The process of metastasis involves an intricate interplay between altered cell adhesion, survival, proteolysis, migration, lymph-/angiogenesis, immune escape mechanisms, and homing on target organs (Table 1). However, there is still very little knowledge of how these events are coordinated by the cancer cell, with conspirational help by the stromal component (microenvironment) of the host. This process is usually said to be ‘uncontrolled’. As we shall see, however, it is by no means purely stochastic, but rather a finely tuned molecular machinery with active tumor cell–host collaboration. Thus, all explanations of ‘success’ of the metastatic axis contain a strong element of determinism. Whereas the early steps in the metastastic campaign are completed very efficiently, metastasis is an inefficient process in its later steps, especially the regulation of cancer cell growth at the secondary sites. Given that spread of the tumor to distant organs is usually lethal, more intense studies of these molecular mechanisms assume general importance to develop more effective anticancer strategies. In the following discussion of specific molecular mechanisms, we have often chosen to draw mainly from examples that pertain to melanoma progression, although similar processes are most likely also operating during oncogenesis of a wide range of cancers.

 Redefining the metastatic cascade/axis: dominant plasticity of cancer cells

 The classical metastatic cascade encompasses intravasa-tion by tumor cells, their circulation in lymph and blood vascular systems, arrest in distant organs, extravasation, and growth into metastatic foci. Ann Chambers et al. (2001, 2002) have demonstrated in murine models that the limiting factor for metastasis formation is growth after extravasation (Figure 1a). Recently, this extravasation model has been challenged by Ruth Muschel and co-workers, who showed that tumor cells can readily proliferate after arrest in blood vessels, suggesting that extravasation is not a prerequisite for metastatic growth (Figure 1b). In a separate series of experiments, Mary Hendrix and co-workers described that tumor cells can even have endothelial cell-like functions and form channels that allow fluid flow (Figure 1c). The group has identified some of the players, such as EphA2 and VE-cadherin, on aggressive melanoma cells that are shared with endothelial cells and that are likely involved in ‘vasculogenic mimicry’. Vasculogenic mimicry is the ability of aggressive cancer cells to form de novo vessellike networks in vitro in the absence of endothelial cells or fibroblasts, concomitant with their expression of vascular-associated cellular marker. Tumor cell plasticity is demonstrated by the ability of tumor cells to adopt a variety of phenotypes, including an endothelial phenotype. These exciting new findings underscore the plasticity of malignant cells from advanced tumor progression stages, and they require from tumor biologists a more dynamic view of the metastatic cascade. If the biological phenotype of metastasis must be portrayed flexibly, we then need a new ‘yardstick,’ a normal cell, to better characterize and understand the many faces of metastasis. We need to understand how the malignant cells exert cooperation from the normal cells. Our central hypothesis is that both normal and malignant cells utilize the same molecules for invasion, but that differences in downstream signaling events allow the tumor cells to dominate over normal cells in the microenvironment. This ‘dominant plasticity’ model of cancer metastasis takes into account the flexible response of malignant cells to microenvironmental pressures while maintaining dominance over the normal parenchymal and stromal cells.

 Table 1. The long and winding roads to metastatic colonization

Biological capability

Molecular examples/pathway entities

Survival

IGF survival factors

Adhesion and deadhesion

CAMs, cadherins, integrins

Migration

Met-SF/HGF signaling, FAK

Proteolysis/ECM remodeling

MMPs, uPA, ADAMs, heparanase

Immune escape

Downregulation of intrinsic immunogenicity, MHC loss

Lymph-/angiogenesis

VEGF, PDGF, bFGF

Homing on target organs

Chemokines/chemokine receptors, CD44, osteopontin

Cell–cell interactions and adhesion molecules: linking proliferation, survival, and motility

At the core of the metastatic process lie the changing adhesive preferences of the cancer cells (i.e. from epithelial cells to fibroblasts and endothelial cells) that dictate their reciprocal interactions with the ECM and neighboring stromal cells. The majority of cell adhesion molecules (CAMs) fall into three gene families, the integrins, the immunoglobulin superfamily, and the cadherins, all of which have been implicated in metastasis.

Integrins: outside-in and inside-out signaling

 This large group of membrane proteins is formed from at least 18a and 8b subunits, which dimerize to yield at least 24 different integrin heterodimers, each with distinct ligand binding and signaling properties. Integ-rins are essential for progression because of their ability to mediate physical interactions with ECMs and their ability to regulate signaling pathways that control actin dynamics and cell movement. At the molecular level, adhesion and deadhesion as well as cytoskeletal remodeling are not only a prerequisite for cellular motility, but are also linked to proliferation and survival (antiapoptotic) pathways through integrins. Proinvasive and prosurvival messages converge at numerous molecular relay stations, diverging again into multiple effector molecules (Figure 1). Located at the communication interface between the cell and the ECM are the integrins. Integrin engagement activates a battery of downstream molecules crucial for motile function and survival. Focal adhesion kinase (FAK), whose phosphorylation is necessary for functional adhesion signaling and migration, was shown to be an early component of prosurvival pathways. FAK also links integrin-mediated signals to the Ras-Raf-MAPK-ERK pathway.

 

Figure 1. Models of metastasis. (a) According to Chambers and co-workers, only a very small population of injected cells (2%) form micrometastases, although over 87% are arrested in the liver. Furthermore, not all of the micrometastases persist, and the progressively growing metastases that kill the mice arise only from a small subset (0.02%) of the injected cells. (b) Muschel and co-workers recently proposed a new model for pulmonary metastasis in which endothelium-attached tumor cells that survived the initial apoptotic stimuli proliferate intravascularly. Thus, a principal tenet of this new model is that the extravasation of tumor cells is not a prerequisite for metastatic colony formation and that the initial proliferation takes place within the blood vessels. (c) The unique ability of aggressive tumor cells to generate patterned networks, similar to the patterned networks during embryonic vasculogenesis, and concomitantly to express vascular markers associated with endothelial cells, their precursors and other vascular cells has been termed ‘vasculogenic mimicry’ by Hendrix and co-workers

 Many other signaling molecules are involved in regulating cytoskeletal organization and cell motility, including phosphatidylinositol 3-OH kinase (PI(3)K), β-catenin, Ras, and the Rho GTPases (RhoA, RhoC, Rac1, and Cdc42). Conversely, RhoA, Rac1, and Cdc42 modulate cell–cell adhesion by regulating cadherin activity. Notably, a recent report highlighted the role of one member of the Rho family, RhoC, in conferring a metastatic phenotype to melanoma cells. Thus, there is remarkable overlap, bidirectional crosstalk and redundancy between these pathways, resulting in proliferation, survival, and motility (‘out-side-in’ signaling). Moreover, these downstream effector molecules in return have repercussions on the expression and function of cell surface molecules (‘inside-out’ signaling).

One of the best-studied integrins is avb3, the vitronectin receptor. In melanoma, for example, it is currently the best molecular marker correlating with the change from radial growth phase (RGP) to the metastatically competent vertical growth phase (VGP). Introduction of b3-integrin into RGP melanoma cell lines converted them into VGP-like melanoma cells. A series of studies have shown that this integrin is a good prognostic indicator of poor survival and short disease-free interval. A number of potent small-molecule antagonists of the avb3-integrin have now been identified and are progressing in the clinic.

 CAMs of the immunoglobulin superfamily: not just superglue

 CAMs of the immunoglobulin superfamily mediate cation-independent adhesion with themselves (homo-philic) or via a heterophilic ligand, but also act as receptors for integrins and ECM proteins. Hence, these molecules do not simply function as a molecular ‘superglue’ organizing cells into static structures. Instead, they support and direct the dynamic interchange of information between two cells by actively transducing signals into the cells through interaction of their cytoplasmatic regions with kinases as well as through interaction with growth factor receptors.

One of the best-characterized CAMs is MCAM (MUC18/Mel-CAM/CD146) in cutaneous melanoma. MCAM is overexpressed in advanced primary and metastatic melanomas when compared to benign nevus cells. More than 80% of melanoma metastases express this molecule, and among primary melanomas expression increases with vertical thickness (Breslow’s index). Moreover, overexpression of MCAM in melanoma cells correlates with the ability to grow and form metastases in nude mice. Conversely, inhibition of MCAM expression in metastatic melanoma cells using genetic suppressor elements leads to inhibition of adhesion between melanoma cells and downregulation of the tumorigenic phenotype. Melanoma–endothelial cell interactions during metastasis may occur through the binding of MCAM on endothelial cells to its ligand expressed by melanoma cells. MCAM is involved in outside-in signaling and contributes to focal adhesion assembly, reorganization of the cytoskeleton, intercellular interaction, maintenance of cell shape, and control of cell migration and proliferation. In contrast, a wide variety of other carcinomas, sarcomas, and neuroendocrine tumors do not show MCAM expression. Interestingly, MCAM appears to act differently in the progression of breast carcinoma. MCAM expression is frequently lost in breast carcinomas and overexpression of MCAM in breast carcinoma cells results in more cohesive cell growth and the formation of smaller metastases in nude mice.

In various cancers, N-CAM expression shifts from the adult 120 kDa isoform to the embryonic 140 and 180 kDa isoforms.   The   biological significance of this isoform switch and its role in tumor initiation and/or progression are not understood. Besides this isoform switch, a correlation between reduced N-CAM expression and poor prognosis has been reported for a number of cancer types.

 Cadherin expression and function. Cadherin switch during cancer progression

 In functional adherens junctions, the extracellular domain of E-cadherin interacts in a homophilic manner with E-cadherin molecules on the surface of neighboring cells. Loss or reduction of E-cadherin expression correlates with enhanced aggressiveness and dedifferentiation in most carcinomas and serves as a prognostic indicator. The cytoplasmic domain of E-cadherin is the site of interaction with the catenin molecules, which mediate its binding to the actin cytoskeleton. Alterations of proteins involved in the E-cadherin–catenin complex are early incidents in cancer development. These include reduction or loss of E-cadherin expression, induced by genetic and epigenetic events (i.e. mutation or reduced transcription of the genes), redistribution of E-cadherin to different sites within the cell, shedding of E-cadherin, and competition for binding sites from other proteins. Indeed, reconstitution of a functional E-cadherin adhesion complex suppresses the invasive phenotype of many different tumor cell types.

Recent studies highlight the role of other members of the cadherin family in tumor cell invasion and metastasis. N-cadherin, in particular, enhances cell motility of various tumor cell types, in some cases even overcoming E-cadherin-dependent cell–cell adhesion. In addition, de novo expression of N-cadherin in tumor cells, which have lost functional E-cadherin, has been repeatedly reported. These data raise the intriguing possibility that a ‘cadherin switch’ from proadhesive, epithelial cadherins (e.g. E-cadherin) to mesenchymal, promigratory cadherins (e.g. N-cadherin) promotes tumor invasion and metastasis. It is thus conceivable that a change in cadherin repertoire results in a change of cellular partners, to support the interaction of tumor cells with stromal cells such as fibroblasts and endothelial cells (Figure 1).

 β-catenin: a dual role in cell–cell adhesion and Wnt-mediated signal transduction

 β-catenin, one of the components of the adhesion complex, also plays a significant role in cell signal transduction, gene activation, apoptosis inhibition, and increased cellular proliferation and migration. β-catenin exerts a dual role: Besides being important   for   cadherin-mediated   cell–cell   adhesion, it plays a key role in Wnt-mediated signal transduction. β-catenin is usually sequestered in the E-cadherin adherens junction or in tight-junction complexes. Nonsequestered, free β-catenin is rapidly phosphory-lated by glycogen synthetase kinase 3b (GSK-3b) in the adenomatous polyposis coli (APC)/GSK-3b/axin complex and subsequently degraded by the ubiquitin-proteasome pathway. If the tumor suppressor APC is nonfunctional, as for example is the case in many colon cancer cells, or GSK-3b activity is blocked by activated Wnt-signaling, β-catenin accumulates at high levels in the cytoplasm. Subsequently, it translocates to the nucleus, where it binds to a member of the TCF/LEF-1 family of transcription factors and possibly activates the expression of TCF/LEF-1-target genes. Target genes of TCF/β-catenin that could be relevant for tumor progression include the proto-oncogenes c-Myc and cyclin D1.

 Ephrins/Eph receptors: control of cell behavior by bidirectional communication

 Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell–cell communication regulating cell shape, attachment, and mobility. Eph signaling is crucial for the development of many tissues and organs including the nervous and cardiovascular systems. Eph receptors and ephrins are unique in that they mediate bidirectional signaling. Both Ephs and ephrins are membrane bound and their interactions at sites of cell–cell contact occur, where information is transduced in both the receptor-and the ligand-expressing cells. Both Eph receptors and ephrins are divided into two subclasses, based on sequence conservation and their binding affinities. In general, the 8different EphA RTKs (EphA1–A8) promiscuously interact with five A-ephrins (ephrinA1– A5). The EphB subclass receptors (EphB1–B6) interact with three different Β-ephrins (ephrinB1–B3).

Increasing evidence also implicates Eph family proteins in cancer. Eph–ephrin interactions regulate critical steps in tumor angiogenesis and possibly metastasis. Eph receptors and ephrins are frequently overexpressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. Ephrin-A1 is upregulated during melanoma progression and may correlate with invasion by endothelial cells. Ephrin-B2 is also overexpressed in melanoma and correlates with tumor-igenicity and metastatic potential. EphA2 is overexpressed in many cancers, including 40% of breast cancers. EphA2 can also transform breast epithelial cells in vitro to display properties commonly associated with the development of metastasis. However, their role in oncogenesis is complex and remains ill-defined. More specifically, oncogenecity may depend on the capacity of unactivated (unphosphorylated) EphA2 to interact with a variety of signaling molecules, while stimulation of EphA2 by its ligand (ephrin-A1) results in EphA2 autophosphorylation, the stimulation reverses the oncogenic transformation.

 Manipulation of the microenvironment and ECM proteolysis: Vanguard of the metastatic campaign?

 The ECM provides a mechanical support for migration and prevents the induction of apoptosis (anoikis). Remodeling (‘landscaping’) of the tumor microenvironment and ECM through lysis of matrix proteins is a necessary step in local invasion and metastasis (Chang and Werb, 2001). The principal classes of enzymes that degrade the ECM- and cell-associated proteins are: (1) the matrix metalloproteinases (MMPs), a family of membrane-anchored and -secreted proteinases; (2) tissue serine proteinases, including urokinase plasminogen activator, thrombin, and plasmin; (3) the adamalysin-related membrane proteases (ADAM family); (4) the bone morphogenetic protein (BMP)-1-type metalloproteinases; (5) heparanase; and (6) cathepsins.

 MMPS/matrixins

 Degradation and remodeling of the ECM, including the basement membrane, by proteolytic enzymes are essential steps in the process of cancer invasion, intra- and extravasation, and colonization at distant sites. For nomenclature, classification, domain structure, and biological functions of MMPs, we refer to excellent recent reviews. These enzyme cascades are tightly regulated by a series of activation steps and specific inhibitors. As a striking example of tumor–host conspiration, most of these matrix-degrading enzymes and inhibitors are contributed by ‘conscripted’ host stromal cells, not by the invading tumor cells. Their expression is regulated by tumor cells through chemokines, cytokines, and ECM metallopro-tease inducers (EMMPRIN). EMMPRIN is enriched on the surface of tumor cells and stimulates the production of MMPs by adjacent stromal cells, which in turn activates tumor cell invasion. Conversely, MMPs may exert their effects in cancer progression by releasing growth factors tethered to the ECM. Moreover, they may also activate latent forms of growth factors and proenzymes through proteolytic cleavage.

Metalloproteases are thus important in many aspects of invasion and metastasis, ranging from cell proliferation and remodeling of the ECM to angiogenesis and cell migration. Most of these processes require a delicate balance between the functions of MMPs (or ADAMs) and tissue inhibitors of metalloproteases (TIMPs). TIMPs are a family of secreted proteins that selectively, but reversibly, inhibit metalloproteases in a 1 : 1 stoichiometric manner. During the invasive events, TIMPs are expressed primarily by the cancer cells and are thought to serve as a regulatory mechanism for fine tuning the activity of stromal MMPs, so that the cancer cells can have an active role in determining where and when they invade.

 ADAMs: multifunctional cell surface proteins with adhesion and protease activity

The ADAMs are a multifunctional gene family, some of which play a role in diverse physiological processes, which have been the subject of several recent reviews. In contrast to MMPs, ADAM function is more focused, regulating growth signaling and tumor cell adhesion. ADAMs are transmembrane proteins that contain disintegrin and metallopro-tease domains, indicative of cell adhesion and protease activities. The extracellular domain of 440 plasma membrane-anchored cytokines, growth factors, receptors, adhesion molecules, and enzymes can be cleaved, and thereby released (shed) from the plasma membrane by various proteases (called sheddases or secretases). These sheddases are themselves transmembrane proteins and, in several cases, are ADAMs. One of the best-studied cases of shedding is the release of the tumor-necrosis factor a (TNF-a), a cytokine that is involved in the inflammatory response, by TNF-a converting enzyme (TACE or ADAM 17). Another example is metalloproteinase disintegrin cysteine-rich 9 (MDC9 or ADAM 9), which has been reported to shed the heparin-binding EGF-like receptor. Kuzbanian (ADAM 19) is also a sheddase that releases a soluble form of delta, the Notch1 ligand. Notch1 is a surface receptor that regulates cell fate determination in development and angiogenesis. At present, however, no studies have yet comprehensively examined the expression or regulation of ADAMs in solid tumors.

 Urokinase-type plasminogen activator system

 Accumulated clinical and experimental evidence indicates that the urokinase-type plasminogen activator (uPA) and its regulators are causatively involved in the metastatic phenotype of many types of cancers. uPA, a serine protease, can bind to its receptor, uPAR, a cell membrane-anchored protein. Binding enables activation of uPA proteolytic activity and at the same time, uPAR ensures that uPA is localized to particular regions of the cell membrane. The main function of uPA appears to be the proteolytic cleavage of the inactive serine protease plasminogen, thereby converting it into the active plasmin. Plasmin, in return, has broad substrate specifity as it can degrade ECM proteins including fibronectin, vitronectin, laminin, and fibrin. Plasmin also activates protocollagenases. Further, uPA and plasmin stimulate tumor growth by proteolytically activating latent forms of several growth factors, such as SF/HGF, bFGF and TGF-b. uPA activity itself is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. Enhanced expression of components of the uPA system has been found in a series of cancers, including carcinomas of the lung, colon, stomach, breast, and prostate, as well as melanomas and gliomas. In many breast and colon carcinomas, uPA is expressed specifically in myofibroblasts at the invasion front. Increased uPA activity has been correlated with tumor invasiveness and may be a prognostic indicator of disease recurrence and metastasis in multiple types of cancer. In experimental tumor models, invasion and metastasis could be inhibited by treatment of tumor cells with antibodies or pharmacologic agents that inhibit uPA activity.

 Heparanase and heparan sulfate proteoglycans

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules, which play a key role in the self-assembly, insolubility, and barrier properties of basement membranes and extracellular matrices, as well as in the sequestration and stabilization of bioactive molecules. Heparan sulfate (HS) chains bind a plethora of proteins (growth factors, chemokines, CAMs, and enzymes) and ensure that these molecules cling to the cell surface and ECM. HSPGs are also prominent components of blood vessels. They have key physiological roles in embryogenesis, morphogenesis, angiogenesis, and epithelial–mesenchymal interactions. Cleavage of HS affects the integrity and functional state of tissues and thus the cellular response to changes in the extracellular microenvironment as well as cell migration. Heparanase is preferentially expressed in metastatic cell lines and primary tumor tissues, where it correlates with a metastatic phenotype. The latter include prostate, bladder, pancreas, colon, breast, ovarian, hepatocellular, gastric, and oral squamous cell carcinomas, as well as melanoma. Over-expression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential, and treatment with heparanase inhibitors (PI-88) or antisense constructs markedly reduced the incidence of metastasis in experimental animals. Furthermore, elevated levels of heparanase were detected in the serum of animals bearing metastatic tumors and cancer patients.

The heparanase enzyme also releases ECM-resident angiogenic factors (e.g. bFGF) in vitro and its over-expression induces an angiogenic response in vivo. Taken collectively, heparanase may facilitate tumor cell invasion and neovascularization, both critical steps in cancer metastasis. It is the first functional mammalian HS-degrading enzyme that has been characterized, which may lead to the identification and cloning of other glycosaminoglycans-degrading enzymes.

 Migration/motility

 From proteolytic mesenchymal toward nonproteolytic ‘amoeboid’ movement

 Collective cell movement represents an efficient dissemination strategy in epithelial and mesenchymal cancers. Recently, Friedl and colleagues have shown that interference with β1-integrin function induces complex changes in cell polarity and cohesion, including development of two or several opposing leading edges, cluster disruption, and the detachment of individual cells followed by β1-integrin-independent ‘amoeboid’ crawling and dissemination. Hence, the conversion from β1-integrin-dependent collective movement to β1-integrin-independent single-cell motility suggests an efficient cellular and molecular plasticity in tumor cell migration strategies. The transition from proteolytic mesenchymal toward nonproteolytic ‘amoeboid’ movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

 Met-SF/HGF-signaling: a specific mediator of invasive growth

Local attractants include scatter factor/hepatocyte growth factor (SF/HGF), also termed plasminogen-related growth factor-1 (PRGF-1), which binds to the Met receptor (c-Met), a tyrosine kinase receptor. SF/HGF-binding to the Met receptor stimulates tyrosine phosphorylation of FAK and its association with the signal-transducing adaptor Grb2, thereby connecting c-Met to the Ras pathway, which promotes growth. Moreover, the autophosphorylation of c-Met is followed by activation of the PI3K/AKT pathway, thereby impinging on survival (suppressed apoptosis) and motility. Generally speaking, SF/HGF stimulates the facets of invasive growth in virtually every tissue of the body.

 ECM proteins

Another group of motility factors corresponds to ECM proteins surrounding the tumor cells (microenvironment). As they are cleaved by proteolytic enzymes secreted by the tumors, the soluble matrix proteins can stimulate the tumor cells to migrate. Thus, the motility stimulation by matrix proteins may be an important component of tumor metastasis, coupling matrix protein degradation to motility. A recent study, based on a whole-genomic analysis of metastasis, reveals that enhanced expression of several genes normally involved in ECM assembly correlates with progression to a metastatic phenotype.

Matrix proteins that are known to induce motility are vitronectin, fibronectin, laminin, type I collagen, type IV collagen, and thrombospondin. These proteins stimulate chemotaxis (motility toward a chemical gradient) and haptotaxis (motility stimulation toward a bound substrate). Several of these matrix proteins stimulate motility through integrin receptors. Type I collagen, which comprises 90% of the bone matrix, has been shown to stimulate the motility of tumor cells. In addition, many cytokines, such as members of the EGF family, TGF-b1, platelet-derived growth factor (PDGF), β-FGF, and IFN-g bind to various components of the ECM.

 Growth factors and cytokines: heterotypic signaling

 Another group of motility factors are host-secreted growth factors. Many of these paracrine motility factors also act as mitogens for the tumor cells in which they cause increased motility, once again supporting the ‘conspirational’ hypothesis in tumor cell–host interaction during cancer progression. Two potent growth factors expressed in the stroma of carcinomas are the insulin-like growth factors (IGF-I and IGF-II). IGFs probably exert both autocrine and paracrine effects on tumor growth. In breast cancers, IGF-II in particular correlates with tumor progression. However, while both IGF-I and IGF-II are typically expressed in fibroblasts of carcinomas, cell lines derived from metastases may acquire the ability to express IGF-II, which may assist in their ability to proliferate in a stroma-independent fashion.

Several studies demonstrate that the biological effects of tumor-derived growth factors and cytokines are biphasic, depending on the level of secretion and the tumor progression stage. For example, we could recently show that intermediate levels of monocyte chemoat-tractant protein-1 (MCP-1) elicit an angiogenic effect mediated through monocyte activation that results in melanoma growth, whereas high levels of MCP-1 lead to massive monocyte/macrophage accumulation and tumor destruction. Similarly, there is a differential response of primary and metastatic melanomas to neutrophils attracted by IL-8. Nontumorigenic primary melanomas depend on IL-8stimulation in vivo for growth. Tumor growth depends on the level of neutrophil infiltration, as at high IL-8transduction levels tumor growth was impaired due to massive neutrophil infiltration. In contrast, highly tumorigenic and metastatic melanoma cells proliferate in vivo independent of infiltrating neutrophils and show marked increases in tumor growth and number of metastatic foci in the lungs, depending on the expression levels of IL-8.

 Angiogenesis and lymphangiogenesis

 Tumor metastasis to regional lymph nodes is a crucial step in the progression of cancer. Clinicopathological data suggest that the lymphatics are an initial route for the spread of solid tumors. Detection of sentinel lymph nodes by biopsy provides significant information for staging, prognostic information, and designing therapeutic regimens. However, the molecular mechanisms that control the spread of cancer to the lymph nodes were unknown until recently. The proliferation of new lymphatic vessels (lymphangiogenesis) is controlled, in part, by members of the vascular endothelial growth factor (VEGF) family – namely, VEGF C, and VEGF D – and their cognate receptor on the lymphatic endothelium, VEGFR3.

 Metastasis suppressor genes – do metastasis-specific genes exist?

 Metastasis suppressor genes are a biologically diverse novel class of eight genes that were recently identified by their reduced expression in several highly metastatic tumor cells (breast, melanoma), when compared with tumorigenic, but poor or nonmetastatic lines. Following the re-expression of a metastatis suppressor gene in tumor cell lines, metastasis is inhibited in vivo, without a significant effect on tumorigenicity. They affect various cellular functions, including pathways involved in invasion, growth factor receptor signaling, MAP kinase pathway, cell–cell communication, and transcription (Table 2), MacDonald et al. (2001) identified a set of genes that could differentiate between medulloblastomas with and without metastases. The metastasis-associated ‘portrait’ or ‘signature’ strongly implicated the PDGF receptor (PDGFR) and the RAS/MAPK pathway.

More recently, however, doubts have been raised regarding the existence of metastasis-specific genes. It has been argued that the tendency of a  tumor to eventually metastasize is largely determined/preordained by the spectrum of mutant genes that are acquired relatively early during multistep carcinogenesis, rather than the emergence of rare cells with the metastatic phenotype. Thus, genes and genetic changes specifically and exclusively involved in orchestrating the metastatic process do not exist. Consequently, even relatively small tumors may already have the ability to metastasize. Data for this reasoning stem from cDNA-microarray analyses revealing that the gene-expression pattern of metastatic tumor cells is often strikingly similar to the cells of the primary tumor from which they were derived. Further more, primary breast tumors and metastases from the same individual are more similar to each other than either is to tumors from other individuals. These findings support the emerging view that the clinical outcome of patients with cancer can be predicted using gene-expression profiles of primary tumors at diagnosis.

 Organ preference for metastatic colonization. Homing

 The locations of distant secondary tumors are nonran-dom. Even though most metastases develop in the first capillary bed encountered after discharge from the primary tumor, it is now accepted that their distributions cannot be explained by anatomical or mechanical hypotheses based on the simple lodgement or trapping of tumor cell emboli alone. The high proportion of bone metastases in certain common cancers such as prostate carcinomas is an example of selective homing of cancer cells to a specific organ.

Three major types of homing mechanisms have been proposed. The first is selective growth. Under this mechanism, tumor cells extravasate ubiquitously but selectively grow only in the organs that have the appropriate growth factors or extracellular matrix environment. The second mechanism is selective adhesion to sites on the endothelial lumenal surface only at the site of organ homing. The third major mechanism is selective chemotaxis of circulating tumor cells to the organ producing soluble attraction factors. The precise regulation of metastatic colonization at distant sites remains a matter of discussion. All of these mechanisms have been found to play a role in experimental metastasis.

Table 2. Metastasis suppressor genes

Gene

Function

NM23

Histidine kinase; phosphorylates KSR, reduced ERK1/2 activation in response to signaling

MKK4

MAPKK; phosphorylates and activates p38and JNK kinase

КАП (CD82)

Tetraspanin; integrin interaction, EGFR desensitization

BRMS1

Gap-junctional communication

KiSSl

G-protein-coupled-receptor ligand (metastin or kisspeptin)

RHOGDI2

Regulates Rho and Rac function

CRSP3

Transcriptional coactivator

VDUP1 (TXNIP)

Thioredoxin (TRX) inhibitor

EGFR, epidermal growth factor receptor; ERK, extracellular signalregulated kinase; JNK, JUN-activated kinase; KSR, kinase suppressor of RAS; MAPKK, mitogen-activated protein kinase-kinase; BRMS1, breast cancer metastasis suppressor 1; VDUP1, vitamin D3 upregu-lated protein 1

Chemokines/chemokine receptors

It has been recognized for over a century that the organ preference for metastatic colonization is influenced by interactions between the circulating tumor cell (the ‘seed’) and the target host tissue (the ‘soil’). Circulating immune and stem cells are known to use chemokine-mediated signaling to home on specific organs. Chemokines are growth-factor-like molecules that bind to G-coupled receptors. They induce leukocytes to adhere tightly to endothelial cells and to migrate towards a gradient. Tumor cells co-opt the same mechanisms to direct metastatic organ preference. For example, the receptor/ligand pairs CXCR4/CXCL12 and CCR7/CCL21 fit the profile expected for breast cancer metastasis homing to bone, lung, and liver. Similarly, melanoma cells would find a CCL27/CCR10 chemokine-receptor ‘match’ in the skin.

However, it has been argued that this metastatic organ specificity is due to efficient organ-specific growth rather than preferential homing of cells. Accordingly, the initial delivery and arrest of cancer cells to specific metastatic organs seems to be primarily mechanical. Subsequently, molecular factors present in individual organs then influence whether or not a specific cancer cell will grow there. This model is based on in vivo videomicroscopy observations that most circulating cancer cells arrest by size restriction, and that both the lung and liver are very efficient at arresting the flow of cancer cells. Further, while leukocytes often arrest by adhering to the walls of vessels that are much larger than themselves, cancer cells usually do not arrest in this manner when injected into mice.

Metastasis to the bone

Bone is the third most common site of metastasis. Of the patients who die of breast cancer, 90% have bone metastases, and very often the bulk of the tumor burden at the time of death will be in the bone. Bone metastases cause severe pain and a dramatic reduction of the quality of life of affected patients. The mechanisms responsible for bone pain are poorly understood, but seem to be a consequence of osteolysis.

Cancer cells that metastasize to the bone are capable of having effects on osteoblasts that are stimulated to form new bone (osteoblastic or osteosclerotic metastasis) or on osteoclasts that cause bone resorption (osteolytic metastasis). In turn, the growth of bone metastases can be influenced by growth factors derived from the bone marrow, osteoblasts, or from the bone matrix products released by osteoclastic bone resorption, thereby causing a vicious circle. In most patients, however, bone metastases have both osteolytic and osteoblastic elements.

In breast cancer, bone metastases are predominantly osteolytic and the main mediator is parathyroid hormone-related peptide (PTHrP). PTHrP stimulates osteoclast activity by enhancing production of the cytokine receptor activator of nuclear factor-kB ligand (RANKL), which binds and activates its receptor RANK, which is expressed by osteoclasts. In prostate cancer, bone metastases are frequently osteo-blastic and known mediators include TGF-b, IGFs, FGFs, PDGF, endothelin-1, and uPA.

Tumor dormancy

It has been well documented that in melanoma or breast cancer, metastases occur decades after initial treatment. Mathematical modeling indicates that continuous slow growth is unlikely, favoring instead a model of discontinuous growth alternating with periods of quiescence. Folkman and co-workers hypothesize that tumor dormancy might be due to preangiogenic micometastases that subsequently acquire the ability to become vascularized, or solitary cells that persist for an extended period of time without division at a secondary site. Another potential cofactor is the persistence of solitary cells in secondary sites. More recently, a molecular mechanism for tumor dormancy has been identified, induced by down-regulation of urokinase receptor, which involves integrin and MAPK signaling.

Does hypoxia initiate the metastatic cascade?

The temporal and hierarchical organization of the metastatic campaign of the cancer cell remains elusive. What is the initiating event? Which are the molecular vanguards? Which ‘master’ genes mastermind the strategy and which molecules are merely ‘foot soldiers’? Clinical studies have shown that metastatic spread is associated with hypoxia in the primary tumor. The mechanism behind this association has not been identified and, in fact, it has not been established as to whether hypoxia induces metastasis or whether the most metastatic cell phenotypes develop the most hypoxic tumors. Several studies suggest that metastatic spread may be promoted by hypoxia in the primary tumor. Active   substances   released   by   hypoxia   and   tumor necrosis, which act to increase invasion and metastasis, include uPA and its receptor, interleukin-8, angiogenin, VEGF and hypoxia-inducible factor 1α.

Clinical applications

Many of the basic research findings are beginning to be translated into improved prognostic models as well as novel therapeutic strategies. A case in point is the manifold exploitation of the uPA system. For example, numerous studies have shown that patients with breast cancer and low levels of uPA and PAI-1 have a significantly better survival than patients with high levels of either factor. The particular combination of both factors, uPA/PAI-1 (both low vs either or both factors high), outperforms the single factors as well as other traditional prognostic factors with regard to risk group assessment, particularly in node-negative breast cancer. Another case in point is the tumor cell-selective cytotoxicity of MMP-activated anthrax toxin.

Inhibitors of the metastatic cascade in clinical trials

The requirement of growing tumors for a vascular supply has produced a diverse group of angiogenesis inhibitors that are currently in clinical testing. However, there is histomorpholo-gic evidence that some tumors may be vascularized without neoangiogenesis. A recent report suggests a nonangiogenic as well as an angiogenic pathway in breast cancer metastasis, which may further complicate antiangiogenic treatment strategies. On the other hand, if tumor cells mimic endothelial cells, it may be possible to find common inhibitors to combat both the angiogenic switch and the vasculogenic mimicry.

In a similar fashion, MMP inhibitors are also undergoing clinical evaluation, but the results with patients suffering from advanced stages of cancer have shown no clinical efficacy. However, recent data indicate that MMP inhibitors could be more successful when used in early-stage cancer or in combination with traditional treatment modalities.

Synthesis and perspectives

Metastasis development is an exceedingly complex series of parallel, yet intertwined, events involving the generation of new blood and lymph vessels, growth, invasion with breakdown of the host matrix, escape from immune surveillance, transport to other sites with adhesion, and subsequent invasion of the organ that hosts the metastasis. Successful invasion and metastasis not only depends upon the aforesaid capabilities, but also on other ‘hallmark capabilities’ of cancer. No single gene has been implicated as a metastasis-specific gene. Hence, the xenophilic tendency of cancer cells is propelled by the same mechanisms that, physiologically, account for tissue architecture maintenance and repair. In all these processes, the host microenvironment (the ‘stroma’) is not just an innocent bystander, but rather an active conspirator. It is only recently that the pathobiological basis for these events has been studied in more detail. Thus, the precise molecular mechanisms, regulatory circuits, and ‘master’ genes that govern these fatal changes remain elusive. Despite all similarities, they often seem to differ from one organ environment and one ‘cancer’ type to another. Another puzzling case in point are ‘dormant’ metastases, that is, metastases that appear only after several years. However, the understanding of the metastatic process is now increasingly leading to the development of rationally designed therapies that target one or more of the molecular components of this series of events. Evolving imaging techniques, such as time-lapse videomicro-scopy, and high-throughput analytic techniques, such as proteomics and SAGE libraries, should soon make it possible to construct comprehensive expression profiles of the crucial molecular players in individual cancers, which could then be targeted in return by novel technologies like RNA inhibition or molecular beacon photodynamic therapy. Yet, even more principal and conceptional questions remain. Do metastatic cells do fundamentally other things – or do they only do some things fundamentally different? That is, more of it and more perpetually, a question of ‘wrong time, wrong place’.

 

 

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Copyright (c) 2020 Konstantin Korchagin. All rights reserved.

k-korchagin@mail.ru