Chapter three - Metastasis Suppressor Genes: At the Interface Between the Environment and Tumor Cell Growth

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Abstract

The molecular mechanisms and genetic programs required for cancer metastasis are sometimes overlapping, but components are clearly distinct from those promoting growth of a primary tumor. Every sequential, rate-limiting step in the sequence of events leading to metastasis requires coordinated expression of multiple genes, necessary signaling events, and favorable environmental conditions or the ability to escape negative selection pressures. Metastasis suppressors are molecules that inhibit the process of metastasis without preventing growth of the primary tumor. The cellular processes regulated by metastasis suppressors are diverse and function at every step in the metastatic cascade. As we gain knowledge into the molecular mechanisms of metastasis suppressors and cofactors with which they interact, we learn more about the process, including appreciation that some are potential targets for therapy of metastasis, the most lethal aspect of cancer. Until now, metastasis suppressors have been described largely by their function. With greater appreciation of their biochemical mechanisms of action, the importance of context is increasingly recognized especially since tumor cells exist in myriad microenvironments. In this chapter, we assemble the evidence that selected molecules are indeed suppressors of metastasis, collate the data defining the biochemical mechanisms of action, and glean insights regarding how metastasis suppressors regulate tumor cell communication toā€“from microenvironments.

Introduction

Cancer metastasis is an arduous pathological process that is the major contributor to the morbidity and mortality of cancer patients (Eccles and Welch, 2007, Jemal et al., 2010). Upon diagnosis, a patient may feel that cancer has suddenly struck and their life has immediately changed. In reality, however, diagnosis follows a culmination of yearsā€”possibly decadesā€”of alterations occurring at the genetic, molecular, cellular, tissue, and organismal levels. Fortunately, the processes of tumor formation and, particularly, metastasis are extremely inefficient and only small fractions of cells from a tumor mass actually overcome the many hurdles to grow at a distant site (Eccles and Welch, 2007, Fidler, 1973a, Weiss, 1990). To metastasize, expression of particular genetic programs is required by a tumor cell to enable the appropriate interactions with changing microenvironments to promote continued survival and proliferation at secondary sites. Understanding these genetic programs and how they affect cellular interactions and signaling cascades is key to understanding the complex process of metastasis.

The existence of tumor suppressors and oncogenes is now accepted as dogma and is well supported by experimental and clinical data. Genes involved in the promotion of metastasis at distinct stages of the disease are also well accepted. However, the hypothesis for the existence of molecules that inhibit the process of metastasis without preventing primary tumor growth was initially met with much skepticism as demonstrated by the three-time rejection of the manuscript reporting the first metastasis suppressor gene NM23 (Steeg, 2004b). Since that time, multiple labs, using many different model systems, have demonstrated the existence of a multitude of protein coding and noncoding genes that significantly reduce metastasis without preventing primary tumor formation. It is now understood that metastasis, the ultimate step in tumor progression, involves many pathological processes, and, just as there are several hallmarks of primary tumor formation (Hanahan and Weinberg, 2000), there also exist hallmarks of metastatic cells (Fig. 3.1). Inhibition of a single step in the metastatic cascade leads to suppression of metastasis (Bruns et al., 2000, Eccles and Welch, 2007, Fidler and Radinsky, 1996). In this chapter, the process of metastasis and the functionality of metastasis suppressing molecules are discussed with the objective that this information can be utilized to identify potential antimetastatic therapeutic strategies. Before discussing metastasis suppressors, it is first necessary to establish the context in which they function.

The evolution of a normal cell into a neoplastic cell with progression to a potentially lethal macroscopic metastatic mass is referred to as neoplastic progression or, in the vernacular, tumor progression (Foulds, 1954, Welch and Tomasovic, 1985). There have been several distinct models to depict the cellular mechanisms for this progression including linear and parallel progression models, mutationā€“selection theory, cancer stem cells, and derivatives of each (Brabletz et al., 2005, Fidler, 2003, Fidler et al., 2007, Klein, 2009, Talmadge and Fidler, 2010, Welch, 1989, Welch and Tomasovic, 1985, Wellner et al., 2009). One of the primary difficulties in constructing generalized model systems for the study of cancer has been the fact that cancer is a heterogeneous disease. As the disease progresses, heterogeneity also increases (Heppner, 1984, Nowell, 1976, Nowell, 1986). In fact, metastatic cells are behaviorally distinct from cells remaining at the site of primary tumor origin (Steeg and Theodorescu, 2007). These behavioral differences arise at multiple levels including intrinsic cellular changes (genetic and epigenetic heterogeneity), from characteristics of the physical environment (positional heterogeneity; e.g., O2, pH, growth factors, cytokines, chemokines, etc.) and/or from transient events (temporal heterogeneity; e.g., stage of cell cycle, manipulation of the tumor; Nicolson, 1984, Rubin, 1990, Welch, 1989, Welch and Tomasovic, 1985). The intrinsic molecular mechanisms underlying phenotypic differences that characterize a metastatic cell are still being elucidated. However, appreciation for the interrelationships between the surrounding microenvironment and cancer cell-associated genes is increasing (Albini et al., 2007, Ben-Baruch, 2003, Bodenstine and Welch, 2008, Finger and Giaccia, 2010, Joyce and Pollard, 2009, Lin et al., 2009, Pietras and Ostman, 2010, Witz and Levy-Nissenbaum, 2006). Selective regulation of gene transcription also occurs through chemical modifications of DNA and chromatin. Epigenetic modifications are modulated, in part, by how cells interact with the microenvironment(s) in which they find themselves (Lin et al., 2009, Marusyk and Polyak, 2010).

Heterogeneity, for the most part, does not result from multicellular transformation. Data from isoenzyme patterns, karyotypes, and protein production all indicate that the vast majority of tumors are derived from a single cell (Frumkin et al., 2008, Heppner and Miller, 1998, Welch and Tomasovic, 1985). Likewise, analogous methods have been used to show that >Ā 90% of metastases are also the result of single-cell outgrowth (i.e., clonal origin) rather than emboli seeding various tissues (Jones et al., 2005, Jones et al., 2008, Talmadge et al., 1982, Wang et al., 2009a, Yamamoto et al., 2003).

Genetic instability may be the chief driver of heterogeneity during tumor progression by random (i.e., not sequentially acquired) generation of variants as described by the mutationā€“selection theory (Balmain, 2001, Boveri, 1914). However, there are others who advocate that metastatic ability may be a trait acquired early, or commensurate with, tumorigenesis (Bernards and Weinberg, 2002). Regardless, neoplastic cells are significantly more genetically unstable than normal counterparts as shown by fluctuation analyses for multiple genes and loci (Cifone and Fidler, 1981, Otto et al., 1989, Tlsty, 1990, Tlsty et al., 1989). As a result, progression is most often believed to occur as a result of mutation and coupled selection. Subpopulations of cells that have acquired the ability to migrate, invade, and colonize ectopic sites may have a selective advantage since these tumor cells ā€œacquiredā€ the ability to respond, adapt, and/or survive changing environments. Ultimately, with continued selection and variant generation, subpopulations of cells may acquire the ability to penetrate a basement membrane (i.e., invade). Invasion is the unequivocal hallmark that defines malignancy. It should be emphasized that tumor stage is typically measured in terms of the tumor mass and location, rather than individual cells within the mass. Grade is typically defined by the most malignant cells identified within a tumor. Even if the majority of individual cells within a neoplasm are indolent, the term malignant is applied even if a single cell has penetrated a basement membrane. Microdissection of tumor cells has identified chromosomal and genetic changes between subpopulations within a tumor mass (Frost et al., 2001, Steeg and Theodorescu, 2007). This information has been useful for the prediction of genetic underpinnings controlling tumorigenesis, invasiveness, and metastasis. However, it is important to note that adjacent, apparently normal cells also have evidence of genetic instability (Hida and Klagsbrun, 2005).

The complexity of tumor progression leading to a metastatic cell, as described above, showsā€”not surprisingly, given the numerous steps required to complete the process of metastasisā€”that a single genetic change is insufficient to accurately predict the likelihood of a lesion progressing to a metastatic phenotype. In fact, defined subsets of genes can be used as prognostic tools (Jorissen et al., 2009, Liu et al., 2007). While multiple genes are required for the progression from primary tumor formation to metastasis, expression of even a single gene that disrupts any of these events would have the ability to suppress metastasis. Although cofactors may be necessary for suppressor function, identification of metastasis suppressors is, overall, usually less technically challenging and easier to interpret than the identification of metastasis-promoting genes.

As alluded to, the cellular and molecular events along the progression of a tumor cell into a fully metastatic macroscopic lesion can be broken down into discrete steps. These steps are often discussed interchangeably, therefore, incorrectly. Thus, it is first critical to define metastasis. Doing so is necessary for two reasons. First, metastasis is both a verb and a noun. The process of metastasis (the verb) was defined above. And, the product of the process is a metastasis, the noun. Therefore, it is important to recognize the context in which the discussion of metastasis occurs. Second, the definitions provide the framework to understand the mechanisms involved and develop therapeutic strategies.

In recent years, five misconceptions regarding metastasis have crept into the scientific and medical literature (Welch, 2006, Welch, 2007). (1) Metastasis is an inherent property of cancer cells. (2) Metastasis and invasion are equivalent phenotypes. (3) Metastases arise only from cells disseminated via the blood or lymphatics. (4) Tumor cells at secondary sites are metastases. (5) Extravasated cells are metastases. By looking at the definition of metastasis and the mechanisms underlying the process of metastasis, we hope to dispel these misconceptions.

Usually, when a primary mass is apparent to the individual or the diagnosing physician, it often comprises at least 1010 cells based on the fact that a cubic centimeter of tissue contains ~Ā 109 cells (Tannock, 1983). Although histological analysis reveals these cells to be pleiomorphic and single-cell clones isolated from a tumor vary dramatically in terms of biological behavior, not all cells in a neoplasm are capable of completing the required steps for metastasis.

In their outstanding review, Hanahan and Weinberg described six hallmarks of cancer cells (Hanahan and Weinberg, 2000). Besides immortality (apparently limitless replicative potential), abnormal growth regulation (i.e., failure to respond to growth-inhibitory signals or hyperresponsiveness to progrowth signals), self-sufficient growth, evasion of apoptosis and sustained angiogenesis, invasion and metastasis were listed as distinguishing characteristics. Unfortunately, some have interpreted the list as meaning that all tumors are invasive and/or metastatic, which is certainly not true. Some tumors are highly aggressive and metastatic (e.g., small cell carcinoma of the lung, melanoma, pancreatic carcinoma), while others rarely metastasize despite being locally invasive (e.g., basal cell carcinomas of the skin, glioblastoma multiforme). Therefore, metastasis is not an inherent property of all neoplastic cells (Welch, 2007).

In fact, the process of metastasis begins before cells migrate from a primary tumor mass. Several groups have discovered that the presence of a tumor elicits mobilization of hematopoietic (Erier et al., 2009, Kaplan et al., 2005) and mesenchymal (Hurst and Welch, 2007, Karnoub et al., 2007, Kitamura et al., 2007, Ojalvo et al., 2010, Patsialou et al., 2009, Wyckoff et al., 2007, Yan et al., 2010) stem cells. Both cell types can facilitate tumor cell migration and invasion (Barkan et al., 2010, Ojalvo et al., 2010, Patsialou et al., 2009, Wyckoff et al., 2007) and reorganize tissues in order to manipulate a ā€œnicheā€ into which tumor cells migrate and/or proliferate (Psaila and Lyden, 2009).

In most textbooks, metastasis is described in terms of blood-borne (i.e., hematogenous) dissemination. However, secondary tumors can arise because tumor cells have migrated via lymphatics (i.e., lymph node metastases are extremely common in many carcinomas; Eccles et al., 2007, Nathanson, 2003); traversing body cavities (e.g., ovarian carcinoma cells most frequently establish secondary tumors by dissemination in the peritoneum while rarely forming metastases via hematogenous spread; Lengyel, 2010); along capillaries (i.e., many melanomas migrate along already-existing vessels; Lugassy et al., 2002, Lugassy et al., 2004, Lugassy et al., 2006, Shields et al., 2007); or along nerves (i.e., pancreatic and prostate carcinomas often exhibit perineural spread; Liebig et al., 2009). So, the route of dissemination is not inherent to a definition of metastasis (Eccles and Welch, 2007, Welch, 2006, Welch, 2007). Rather, development of a metastasis needs only incorporate spread of tumor cells to secondary sites.

Although proteolysis-dependent invasion is not an inherent requirement for all tumors to metastasize, it is required for the majority of cancers since physical barriers usually surround a tumor. Understanding the complexity of invasion is necessary to appreciate the mechanisms of many metastasis suppressor genes. Invasive cells have often acquired other traits necessary to metastasize; however, if an invasive cell cannot complete any other step in the metastatic cascade, it will not form a metastasis.

Invasion requires substantial changes of cell morphology and phenotype in addition to modifications of the surrounding environment. During invasion, three important processes are dynamically regulated, including adhesion, ECM reorganization, and motility (Liotta, 1992, Wolf and Friedl, 2006). Normally, epithelial cells form polarized sheets that are maintained by tight intercellular junctions and are anchored to basement membranes by hemidesmosomes, associated intermediate filaments, and integrins. Invading cells have altered cellā€“cell and cellā€“matrix adhesion that must be balanced. If a cell is too strongly adherent, it cannot move, and, like a person trying to walk or drive on ice, if to lose an adhesion, cells do not have the traction to move. The structural and functional proteins that regulate cell adhesion and migration are key downstream targets of oncogenes and tumor suppressor-controlled signaling pathways and provide insights into how oncogenic transformation results in progression to an invasive phenotype. Many of the proteins involved in tumor invasion also affect cell survival, growth, apoptosis and angiogenesis, and hallmarks of malignancy. This highlights the intricate network of interrelated pathways modulating cancer cell behavior.

Many dramatic changes in tumor cell morphology during invasion are reminiscent of a normal process that occurs during embryonic development (Hay, 2005, Thiery, 2002), known as epithelial-to-mesenchymal transition (EMT). The EMT describes conversion from an epithelial morphology to a nonpolarized, motile, spindle-shaped cell resembling a fibroblast (Polyak and Weinberg, 2009, Thiery, 2002, Thompson and Newgreen, 2005). EMT is associated with the loss of epithelial-specific E-cadherin from the adherens junctions, and a switch from the expression of keratins as the major intermediate filament to the mesenchymal intermediate filament, vimentin. EMT is influenced by the tumor microenvironment and has been observed primarily at the tumor stromal interface (Polyak and Weinberg, 2009, Thompson and Newgreen, 2005), but a role for EMT in cancer invasion is not universally observed (Cardiff, 2005, Cardiff, 2010, Tarin, 2005). A key regulator of EMT is transforming growth factor beta (TGF-Ī²) signaling (Bierie and Moses, 2006a, Bierie and Moses, 2006b, Creighton et al., 2010, Heldin et al., 2009, Huber et al., 2005, Oft et al., 1998, Pardali and Moustakas, 2007) but other mediators include hepatocyte growth factor/scatter factor (HGF/SF; Yang et al., 2009), PI3 kinase signaling pathway (Pon et al., 2008), MAP kinases (Bakin et al., 2002, Janda et al., 2002), Sprouty4 (Tennis et al., 2010), and the transcriptional factors ZEB1 (Wellner et al., 2009), Twist and Snail (Moreno-Bueno et al., 2008, Onder et al., 2008). Other signaling pathways implicated in stem cell maintenance that are linked to EMT are Wnt (Debies et al., 2008, ten Berge et al., 2008), Notch (Sahlgren et al., 2008), and Hedgehog (Bailey et al., 2007). Tumor cells may also reverse the process and undergo a mesenchymal-to-epithelial transition (MET) in the absence of EMT-inducing signals (Chaffer et al., 2006, Hugo et al., 2007). This transient nature of EMT helps explain why metastatic cells morphologically resemble primary tumor cells despite the fact that they by necessity accomplished all the steps of the metastatic cascade.

Cells induced to undergo EMT not only exhibit enhanced motility but are resistant to apoptosis (especially anoikis), another key requirement for successful metastasis. However, some cancer cells use EMT-independent modes of migration, including collective and amoeboid (Yilmaz and Christofori, 2010). For example in a clever study, Tsuji and colleagues isolated two populations from a single tumor (Tsuji et al., 2008). One population, herein designated Cell-I, exhibited properties of EMT and was able to enter the vasculature (i.e., intravasate). Cell-I was, however, unable to form metastases if injected directly into the vascular compartment. The second population, herein designated Cell-II, displayed an epithelial morphology and was not able to enter the blood stream or metastasize when injected orthotopically. However, Cell-II would colonize tissues when directly injected into the vasculature. If Cell-I and Cell-II were coinjected orthotopically, both were found in metastases. Critical to this review, however, cells undergoing EMT were not themselves successful for metastasis. The authors demonstrated that cellular cooperation existed within the primary tumor and was critical to form metastatic lesions. This would suggest that tumor heterogeneity not only exists but may also be essential for tumor progression.

The extracellular matrix (ECM) provides scaffolding for cells and spatial cues that dictate cellular behavior (Barkan et al., 2010). Matrices comprise proteins, primarily triple-helical collagens, glycoproteins such as laminin and fibronectin, and proteoglycans (Catchpole, 1982, Engbring and Kleinman, 2003, Iozzo et al., 2009, Liotta, 1986, Timpl, 1993, Timpl and Aumailley, 1989). Basement membranes are specialized ECM that form barriers separating polarized epithelial, endothelial, and muscle cells from the underlying tissue. Interstitial matrices provide structural characteristics to connective tissues (Erler and Weaver, 2009). The molecular composition of ECM varies between tissues and organs, and provides important contextual information to cellular constituents (Egeblad et al., 2010). In addition, the ECM interacts with many secreted molecules to serve as a repository for regulatory proteins and growth factors (Rozario and DeSimone, 2010). Thus, cell:matrix interactions dictate survival, growth, differentiation, and migration. Correspondingly, selective proteolysis of ECM components leads to release of fragments collectively known as matrikines (Arroyo and Iruela-Arispe, 2010, Duca et al., 2004, Tran et al., 2004), which further regulate protein function and may be involved in cell signaling.

Adhesion of cells to matrix occurs primarily through a family of transmembrane glycoproteins known as integrins, which are heterodimers assembled as specific combinations of 18 alpha and 8 beta subunits (Desgrosellier and Cheresh, 2010, Shattil et al., 2010). Each heterodimer binds distinct, but sometimes overlapping, ECM components. Integrinā€“ECM binding may be either tumor-promoting or -inhibitory. During tumor progression, cancer cells tend to downregulate the integrins that mediate adhesion and induce maintenance of a quiescent, differentiated state while simultaneously upregulating integrins that promote survival, migration, and proliferation. Although there is a cell-type dependency on integrin function, generally integrins Ī±2Ī²1 and Ī±3Ī²1 are viewed as suppressors of tumor progression, while Ī±vĪ²3, Ī±vĪ²6, and Ī±6Ī²4 promote cellular proliferation and migration (Desgrosellier and Cheresh, 2010).

Integrins bidirectionally mediate signals so that changes in intracellular signaling pathways can modulate cellular adhesion (i.e., inside-out signaling) and changes in cellular adhesion can alter cellular phenotype (i.e., outside-in signaling). Integrinā€“ECM interactions often modulate cell function by cooperative signaling with different growth factor receptors (Askari et al., 2010, Desgrosellier and Cheresh, 2010). Many cellular responses induced by activation of receptor tyrosine kinases are dependent upon proper cellular adhesion to ECM substrates in an integrin-dependent manner. Signaling in response to ECM interaction usually activates focal adhesion kinase (FAK) and nonreceptor tyrosine kinases of the Src-family.

The ECM can be remodeled by degradative enzymes that are produced by the tumor cells themselves and surrounding stromal cells (Bhowmick et al., 2004). These enzymes contribute to matrix degradation and facilitate tumor cell invasion. Proteolytic enzymes, representing virtually every class of proteases, have been implicated in tumor cell invasion (Boyd, 1996, Gabbert, 1985, Khokha and Denhardt, 1989, Liotta and Stetler-Stevenson, 1991, Nakajima and Chop, 1991, Nicolson, 1982b, Pauli et al., 1983, Roycik et al., 2009, Stracke et al., 1994). Tumor progression-associated proteases include, but are not limited to, serine proteinases (plasmin, plasminogen activator, seprase, hepsin, and several kallikreins), cysteine proteinases (e.g., cathepsin B), aspartyl proteinases (e.g., cathepsin D), and metal-dependent proteinases (e.g., matrix metalloproteinasesā€”MMP and a disintegrin and metalloproteinasesā€”ADAM families). Other matrix-degrading enzymes such as heparanase, an endoglycosidase that cleaves heparin sulfate proteoglycans, and hyaluronidase that cleaves hyaluronic acid, have also been causally associated with tumor progression and invasion (Nakajima et al., 1983, Sanderson et al., 2004, Vlodavsky et al., 1990, Vlodavsky et al., 2002).

Liotta and colleagues observed that metastatic potential correlates with the degradation of type IV collagen found predominantly in the basement membrane and focused attention on the metal-dependent type IV collagenases or gelatinases that are now recognized as MMP-2 and MMP-9 (Thorgeirsson et al., 1985, Turpeenniemi-Hujanen et al., 1985). Subsequently, many of the 23 members of the MMP family of matrix-degrading metalloproteinases have been associated with tumor progression (Nelson et al., 2000). Elevated MMP levels correlate with invasion, metastasis, and poor prognosis in many cancer types. Animal models provide evidence for a causal role for MMP activity in cancer progression (Coussens et al., 2001, McCawley and Matrisian, 2000, Sternlicht and Werb, 2001, Sternlicht et al., 1999). Additionally, the plasminogen activator/plasmin system has been causally implicated in cancer invasion, and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are validated prognostic and predictive markers for breast cancer (Andreasen et al., 1997, Carlsen et al., 1984, DeClerck et al., 1997, Hildenbrand et al., 2009).

Regulation of matrix proteolysis occurs at multiple levels. In addition to the expression of proteases themselves, many cells also produce endogenous inhibitors including the tissue inhibitors of metalloproteinases (TIMPs; Chirco et al., 2006), serine proteinase inhibitors (SERPINs Bailey et al., 2006), and cysteine protease inhibitors (CYSTATINs; Cox, 2009). Some inhibitors accumulate in high concentrations within the ECM and paradoxically exhibit tumor-promoting functions, including protease activation (Jiang et al., 2002). Conversion of pro-MMP-2 to active MMP-2 requires the activity of MT1-MMP (MMP-14), a transmembrane MMP that is activated intracellularly by the propeptidase family member furin, and TIMP-2 (Hernandez-Barrantes et al., 2000). The stoichiometry of each of these molecules is critical for proper function and regulation. Other proteolytic cascades are important for regulating protease activity during the degradation of ECM, including cathepsin(s)Ā ā†’Ā uPAĀ ā†’Ā plasminĀ ā†’Ā MMP (Affara et al., 2009). Each protease in this cascade can cleave ECM components; therefore, attribution of function requires detailed and systematic evaluation of each component in the cascade.

The original view that proteolytic enzymes function predominantly to remove physical ECM barriers has been expanded with the realization that proteolysis regulates multiple steps of tumor progression. For example, MMP substrates in the matrix or on the cell surface that modulate cellular growth, differentiation, apoptosis, angiogenesis, chemotaxis, and migration have been identified (Kessenbrock et al., 2010). The abundant evidence for a role of MMPs in tumor progression led to the design and testing of synthetic MMP inhibitors for cancer therapy. These inhibitors proved to be disappointingly ineffective in clinical trials (Coussens et al., 2002), results that have been explained by problems with inhibitor or clinical trial design, as well as a lack of understanding of the broad range of MMP activities resulting in both cancer-promoting and cancer-inhibitory effects (KrĆ¼ger et al., 2010, Lopez-Otin and Matrisian, 2007).

In addition to ECM remodeling, cell locomotion occurs via coordinated polymerization and depolymerization of the actin cytoskeleton to extend pseudopodia at the leading edge of the cell, known as invadopodia (Buccione et al., 2009, Weaver, 2006), followed by contraction associated with disassembly of cell:matrix adhesive contacts at the trailing edge (Wolf and Friedl, 2006). Adhesion molecules, including several Ī²1 integrins and CD44, and proteases, including MMP and ADAM, are an intricate part of the invadopodia. Inside the plasma membrane, invadopodia contain actin and actin assembly molecules as well as multiple signaling molecules, including FAK, Rac1, and synaptojanin 2; src associated proteins such as p130Cas and Tks5/FISH; and the small GTPases cdc42, Arf1 and Arf6 (Chuang et al., 2004, Guarino, 2010, Muralidharan-Chari et al., 2009, Seals et al., 2005, Tannock, 1983, Yamaguchi et al., 2005). Actin cytoskeletal reorganization involves the Arp2/3 complex and its regulators, WASP, cortactin, and the GTPase Rac (TenKlooster et al., 2006). Actin contractility is regulated by myosin light chain kinase and upstream small GTPases, in particular, Rho and its effector ROCK (Kosako et al., 2000, Olson and Sahai, 2009). Many of these molecules have been targeted since invadopodia are implicated as key cellular structures that coordinate and regulate the process of invasion (Buccione et al., 2009, Poincloux et al., 2009, Weaver, 2006).

As alluded to above in the discussion of EMT, single cells migrate either with a spindle-shaped morphology, referred to as mesenchymal migration, or with the less adhesive ellipsoid shape used by leukocytes and Dictyostelium termed amoeboid migration (Wyckoff et al., 2006). Collective migration can occur when the cells retain cell:cell junctions and clusters of cells move in single file through a tissue (Sahai, 2005, Yilmaz and Christofori, 2010). It is noted, however, that the ability of cells to utilize amoeboid migration has been called into question since methods used for reconstitution of matrix resulted in inferior barriers and protein:protein interactions (Sabeh et al., 2009). Another mechanism by which cells traverse cellular barriers is termed entosis (Overholtzer et al., 2007). Briefly, tumor cells transit through other cells and emerge on the other side. Amazingly, many times neither cell is harmed during the process. Based upon some in vitro estimates, entosis can sometimes be quite common. However, the frequency in vivo has not been well studied.

Each type of motility is governed by a variety of cellular factors. Cellular motility is triggered by autocrine inducers of random movement (Jiang et al., 2006, Silletti et al., 1994). Tumor cells produce lysophospholipase D (autotaxin) which stimulates motility, as does lysophosphatidic acid (LPA; Liu et al., 2009, Stracke et al., 1992). LPA can be produced by autotaxin activity on lysophosphatidylcholine. Likewise, HGF/SF interacts with its receptor, c-met, to induce chemokinetic activity of epithelial cells, resulting in an invasive phenotype (Klominek et al., 1998). In fact, disruption of the HGF axis is currently the target of drug development against metastasis (Cecchi et al., 2010, Eder et al., 2009). Directional motility is a chemotactic (following a soluble concentration gradient) or haptotactic (following an insoluble concentration gradient) effect in response to a gradient of soluble or localized factors, respectively. Chemotaxis is often the result of growth factors such as insulin-like growth factor (IGF) and chemokines of the CCR and CXC families (Mantovani et al., 2010). Among the best studied CCR/CXC interactions in metastasis is cellular response to SDF1 (CXCL-12; stromal derived factor-1) as a ligand for the CXCR4 receptor (Gladson and Welch, 2008, Muller et al., 2001, Teicher and Fricker, 2010). SDF-1 levels are often high in tissues commonly colonized by tumor cells (e.g., lung, bone) that express abundant CXCR4. As with the HGF axis, inhibitors of CXCR4 are being studied in preclinical models and are showing efficacy in multiple tumor types (Kim et al., 2008, Richert et al., 2009). Haptotaxis is characterized as a response to gradients of ECM components such as laminin-5 and fibronectin and can be modulated positively or negatively by proteolysis (McCarthy et al., 1985).

Even cells that have been selected for invasive and metastatic capacity exhibit low efficiency for developing metastasis, seldom exceeding 0.1%. Entry of cells into the blood stream (termed intravasation) is apparently not uncommon. In fact, more than a million cells per gram of tumor can be shed daily (Butler and Gullino, 1975). Tarin and colleagues illustrated metastatic inefficiency of hematogenous metastases using peritovenous (Levine) shunts to palliate ascites burden for patients suffering from various cancer types (Tarin et al., 1984). Although millions of tumor cells were directly deposited into the vena cava daily, the patients did not develop secondary blood-borne tumors with higher frequency.

The fate of already intravasated tumor cells is uncertain because of apparently contradictory experimental evidence. Using radiolabeled cells, Fidler et al. found that most do not survive (Fidler, 1970, Fidler, 1973b, Fidler and Nicolson, 1977) because of hemodynamic sheer (Weiss, 1989, Weiss, 1990, Weiss and Schmid-Schonbein, 1989, Weiss et al., 1985), anoikis (Kim et al., 1999, Phadke et al., 2008, Wong et al., 2001), or immune selection (Fidler, 1974, Gorelik et al., 1980, Hanna, 1985, North and Nicolson, 1985, Van Netten et al., 1993, Young and Newby, 1986). In contrast, using a fluorescent tag Naumov et al., 1999, Naumov et al., 2002 showed that a majority of cells not only survived but also extravasated. Muschel et al. used intravital microscopy in lung and brain metastasis models to show that the majority of cells remained intravascular and began to proliferate (Carbonell et al., 2009, Wong et al., 2001). Their data illustrate how extravasation is not essential for successful establishment of a secondary mass. Plausible explanations for these dichotomous results include different cell monitoring methods (i.e., radiolabeling vs. fluorescent tagging), analysis of tumor cell behavior in two different tissues (i.e., lung vs. liver), and whether the studies were done completely in vivo versus ex vivo.

Critically, all of these observations highlight the importance of tumorā€“stromal interactions in the metastatic process and clearly demonstrate that a ā€œone-size-fits-allā€ description of the metastatic process does not exist. For a cell to accomplish all these ā€œstepsā€ involved in invasion, specific genetic programs must be expressed and functional. Once again, it is stressed that inhibition of any of these requirements would render a cell less metastatic.

Secondary tumors can arise because tumor cells have migrated via lymphatics (i.e., lymph node metastases are extremely common in many carcinomas), the blood vasculature, or across body cavities (e.g., ovarian carcinoma cells most frequently establish secondary tumors by dissemination in the peritoneum while rarely forming metastases via blood-borne routes). Lugassy and colleagues recently documented dissemination of melanoma cells along the space between endothelium and basement membrane (Lugassy et al., 2002, Lugassy et al., 2004, Lugassy et al., 2007). That is, the cells do not appear to enter the vascular lumen per se. The latter route of dissemination is reminiscent of perineural spread, which is common in pancreatic and prostatic carcinomas in which tumor cells migrate along nerve sheaths (Liebig et al., 2009). Thus, the route of dissemination is not inherent to a definition of metastasis. Nonetheless, the varying pathways to metastasis illustrate different barriers which tumor cells must surmount.

English surgeon Stephen Paget asked, ā€œWhat is it that decides what organs shall suffer in a case of disseminated cancer?ā€ (Paget, 1889). Upon reviewing autopsy records from 735 women with breast cancer, he recognized discrepancies between the blood supply going to specific organs and the frequency of metastasis to those organs. For example, despite abundant blood circulation to the heart, spleen, and kidney, breast cancers (indeed most cancers) infrequently colonize these tissues. Paget concluded that unequal distribution of metastases could not be exclusively explained by passive embolus arrest in the first capillaries encountered, as supported by the famed pathologists, Rudolph Virchow (Talmadge and Fidler, 2010, Virchow, 1858), Leonard Weiss (Bross and Blumenson, 1976, Weiss, 1979, Weiss, 1992, Weiss and Ward, 1982), and James Ewing (Ewing, 1919). Autopsy results for patients succumbing to multiple types of cancer indeed show that most metastases are found in the first lymph node or capillary beds encountered by intravasated tumor cells (Gershenwald and Fidler, 2002, Hess et al., 2006, Park et al., 2009). However, there are several well-known examples of metastatic colonization patterns that simply cannot be explained (Table 3.1).

Throughout the latter half of the twentieth century, numerous studies supported a blending of the seed and soil and the mechanical hypotheses. As alluded above, many tumor cells can seed lots of tissues, most commonly at the first lymph node or capillary bed encountered. However, the capacity of cells to proliferate and complete the metastatic process is determined by the ability of tumor cells to respond to growth promoting while avoiding growth inhibitory signals.

Section snippets

Genetic Regulation of Metastasis

The field of metastasis genetics and the very existence of genes that control specifically metastasis have been called into question (Steeg, 2004a). Inarguably, functional data with the metastasis suppressor genes specifically control metastasis, not tumorigenicity. Some array data were interpreted to suggest that metastatic potential is inherent in tumor cells (Bernards and Weinberg, 2002), but the metastasis suppressor data argue against this interpretation (Eccles and Welch, 2007).

BRMS1

Because metastasis requires the coordinated expression of particular genes at multiple steps, a key regulatory molecule would be one that functions by regulating metastasis-associated gene transcription. Breast cancer metastasis suppressor-1 (BRMS1) alters the expression of multiple metastasis-associated genes including osteopontin (OPN; Hedley et al., 2008, Samant et al., 2007, Shevde et al., 2006), uPA (Cicek et al., 2005, Cicek et al., 2009), fascin (Zhang et al., 2006), epidermal growth

Conclusions and Perspectives

The process of metastasis is obviously very complex and involves intrinsic and extrinsic factors. In this chapter, we have focused on genetic changes, specifically metastasis suppressors, in tumor cells, but as the data were collated and as the field matured, awareness that the function of metastasis regulatory genes were not functioning autonomously became acute. Even with incomplete knowledge regarding the interrelationships of tumor cells with their microenvironments, some patterns are

Acknowledgments

We are grateful to many colleagues who have shared insights with us during the writing of this chapter. Although we have attempted to be thorough, there may be some whose work was not cited. For that we apologize. We dedicate this chapter to the memories of our mothers, who died from cancers, and for the inspiration both still give us. Work from the Welch and Hurst laboratories has been generously funded by the National Cancer Institute (CA062168; CA087728, CA134981, CA089019), U.S. Army

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