Abstract
A greater understanding of the processes of tumor invasion and metastasis, the principal cause of death in cancer patients, is essential to determine newer therapeutic targets. Metastasis suppressor genes, by definition, suppress metastasis without affecting tumorigenicity and, hence, present attractive targets as prognostic or therapeutic markers. This short review focuses on those twelve metastasis suppressor genes for which functional data exist. We also outline newly identified genes that bear promising traits of having metastasis suppressor activity, but for which functional data have not been completed. We will also summarize the biochemical mechanism(s) of action (where known), and present a working model assembling potential metastasis suppression pathways.

Keywords: Metastasis; Suppression; Genes; Cancer; Tumor; Tumorigenesis; KISS1; MKK4; BRMS1; CRSP3; TXNIP; E-cadherin; CRMP1; Maspin, CD44; SSeCKS; Nm23; KAI1; TIMPs; DRG-1; Metastin; Multigene; Invasion; Prognosis; Therapeutic; Marker; Target; Anoikis; Proliferation; Apoptosis; Angiogenesis; Neovascularisation

1. Introduction
Despite better local treatments for cancer using surgery and radiotherapy, the clinical challenge remains combating systemic metastatic disease. Metastasis via the lymphatics, hematogenous system, or through the body cavities results in significant morbidity. Not only must cells leave the primary tumor, but they must also proliferate at the secondary site [1 and 2]. Metastasis culminates the evolution of tumor cells whereby a tumor's composition collectively becomes progressively more malignant [3 and 4]. Tumor progression results from genetic instability coupled with selection of subpopulations of cells [3]. Eventually some cells accumulate sufficient capacity to dissociate and spread. Depending on whether the mutations occur early or late in tumor progression determines proportions of metastatic cells within tumors of a given size. This conclusion can be appreciated when interpreted in light of classical studies of Luria and Delbrück [5]. Selection of metastatic cells varies with the nature of a tumor as well as between patients. Although it is generally true that larger tumors are more likely to spread, size does not necessarily correlate with metastatic capacity [6 and 7]. In addition to accumulating mutations, there are exogenous signals that can influence metastatic efficiency.

2. Host–tumor interactions in neoplastic advancement
Tumorigenicity and metastasis are distinct, but interrelated phenotypes. Tumorigenicity is necessary, but not sufficient, for metastasis. In part, metastasis is also determined, to a great extent, by tumor–host interactions. That is, the microenvironment participates in the induction and selective proliferation of malignant cells [8].

How does the host environment at the metastatic site affect the metastatic behavior of cells? The relationship is reciprocal, and reflects both host endocrine and immunologic status. Host physiology can foster or reject neoplastic cells. In response to tumor-secreted cytokines and chemokines, diverse leukocyte populations are recruited including neutrophils, dendritic cells, macrophages, eosinophils, mast cells and lymphocytes. All inflammatory cells can produce a plethora of cytokines, proteases (e.g. MMPs), membrane-perforating agents and soluble cytotoxic mediators (e.g. TNF-, interleukins and interferons) [9]. For example, tumor-associated macrophages, play a dual role in tumor development. They can kill neoplastic cells following activation by IL-2, IL-12 and interferons; but they can also induce angiogenesis by growth factor, cytokine and proteinase secretion [9]. Indeed proteinases in the tumor milieu are largely stroma-derived [10]. Thus, metastatic tumor cells can modify the host environment so that tumor cells are nurtured.

Tumor–host interactions formed the basis of Sir Steven Paget's `seed and soil' theory [11] to explain the predilection of breast cancer spread to bone. He proposed that the tumor cells (seed) are scattered in many directions by the circulatory system, but grow only in response to the microenvironments of specific organs (soil). While this review focuses on metastasis genes (i.e. in the seed), we emphasize that the regulation of those genes by the host cannot be ignored. That is, the context in which the genes function must be considered, even though the details are not yet known.

3. Stochastic and selective aspects of cancer metastasis
In order to metastasize, cells must complete a series of sequential steps, each of which is rate-limiting. Following primary tumor growth (including establishment of neovasculature or primitive vascular channels [12 and 13]), tumor cells detach and enter a circulatory compartment. The tumor vasculature is immature and incontinent [14], providing easier access to the vasculature. Once there, tumor cells can remain as single cells or form homo- or hetero-typic emboli but they must survive shear forces as well. At the secondary site, tumor cells can arrest due to size restriction or become tethered to vascular endothelium using a variety of surface adhesion molecules. In some cases, tumor cells recognize endothelial addressins––surface molecules that designate the cells as from a particular organ, tissue or vessel structure [15, 16, 17 and 18]. Additionally, tumor cells can respond to chemoattractants produced by different tissues [9 and 19]. For the most part, the identity of the attractants are not yet known [20], but recent data implicate chemokines [9, 21, 22 and 23]. Depending upon tumor type and the tissue in which the tumor cells have arrested, cells can begin to proliferate within the vasculature or extravasate before proliferating [24, 25, 26, 27 and 28]. Merely getting to the secondary site does not constitute a metastasis. Metastases are defined as secondary masses.

Overall, the process of metastasis is quite inefficient [29 and 30]. Cells in the vasculature are cleared biphasically [29 and 31]. The initial phase (6–24 h), represents an exponential decline of cell number, presumably due to mechanical trauma, oxygen toxicity, anoikis and immune clearance. A second, more gradual decline, presumably represents cell death at secondary sites [29]. Tumor cells that arrive at a second site do not necessarily proliferate immediately. Some cells may remain `dormant' for extended periods or until conditions become favorable for proliferation [32, 33, 34 and 35].

Dormancy of pre-angiogenic metastases is more accurately described as a balance between proliferation and apoptosis [36]. Wong et al. [37] found that the majority of cells underwent apoptosis within 24 h of intravasation. If apoptosis was inhibited, metastatic potential increased. In contrast, Luzzi et al. [33], and Cameron et al. [38] found that most cells survived, but failed to proliferate. It is not yet possible to reconcile these two apparently conflicting conclusions. However, since the tumor cells and host tissue were not identical and since the data are not mutually exclusive, it is likely that both are correct. It is probable that the rate-limiting steps of metastasis will vary by cell lines and in different tissues, reflecting yet another level of heterogeneity within tumors.

Technical advances have made it possible to detect single cancer cells or microscopic foci in experimental models [39, 40, 41 and 42]. If model data are extrapolated to the clinical setting, diagnosis and treatment decisions become significantly more complex. The issue is whether microscopic foci justify aggressive treatment because of their potential to grow into overt lesions. Or, if the percentage of cells that eventually proliferate is vanishingly small, should patients be spared toxic chemotherapy since the mere detection of cell clusters at a secondary site does not necessarily translate into establishment of macroscopic metastases?

Considerations such as these underscore the need for markers that can be used to accurately and definitively predict metastatic potential (in this case, defined as the possibility of forming macroscopic metastases) [43]. New technologies such as microdissection, microarray, real-time RT-PCR, proteomics and comparative genomic hybridization (CGH) are being evaluated to define and characterize metastatic potential of cancer specimens [44, 45, 46, 47, 48, 49, 50, 51, 52 and 53]. Identifying molecules that are specifically involved in metastasis (as opposed to indirect changes in gene expression due to tumor progression) presents a daunting challenge as well as significant opportunity. The difficulty relates to discriminating between mere association from causality [2, 43, 54, 55, 56 and 57]. Metastasis suppressor genes are attractive candidates for marker development because, by definition, their loss should be associated with the acquisition of metastatic potential [58]. Moreover, they represent potential therapeutic targets.

We emphasize that, while it takes a finely orchestrated set of functions to metastasize, blockage of even one step halts the process. Since the discovery of the first metastasis suppressor gene, nm23, more than a decade ago, the number of metastasis suppressors identified has grown significantly (reviewed in Ref. [2]).

Various studies involving CGH, loss of heterozygosity (LOH) and karyotype analysis identified distinctively altered regions and/or genomic imbalances involving various human chromosomes [55]. Some changes correlated temporally with acquisition of metastatic propensity. By inference, then, those chromosomal regions were thought to predict the location(s) for metastasis-associated genes. In the case of genetic loss, replacement of the chromosomes by microcell-mediated transfer (MMCT) was predicted to suppress metastasis. MMCT has been instrumental in identifying several metastasis suppressor genes.

MMCT of chromosomes 2, 7, 8, 10, 11, 12, 13, 16, 17 and 20 suppressed metastasis of prostate carcinoma cells without blocking tumorigenicity (reviewed in Ref. [59]). By positional cloning regions on chromosome 17 were narrowed to an ~70 cM [60]. Yoshida et al. [34] eventually cloned the MKK4 metastasis suppressor gene. Details regarding individual genes will be provided below. The identities of the invasion-suppressing genes with regard to metastasis suppression have not been as easily forthcoming. Importantly, inhibition of invasion (unless completely inhibited) does not necessarily suppress metastasis. While invasion is required for metastasis, tumor cells must merely be able to accomplish the step [43, 56, 61 and 62]. They do not have to be extraordinarily efficient at component processes.

Structural alterations involving chromosome 6 are frequent in metastatic melanoma [63]. MMCT of full-length human chromosome 6 suppressed metastasis of the human metastatic melanoma cell line C8161 [64 and 65]. Chromosome 6 hybrids were less motile, but just as invasive [66]. Chromosome 6 hybrids engineered to express green fluorescent protein were used to demonstrate that they completed every step of the metastatic cascade except proliferation at the secondary site [67]. Using subtractive hybridization the KISS-1 metastasis suppressor was identified [68]. Also using the C8161 melanoma, MMCT of chromosome 1 suppressed metastasis [69].

Alterations of chromosome 11 in metastatic breast carcinoma are well documented [51]. Following MMCT of chromosome 11 into the metastatic human breast carcinoma cell line, MDA-MB-435, hybrids were significantly suppressed for lung and lymph node metastasis [70].

MMCT has been the most lucrative technique for identifying metastasis suppressors. However, other approaches (subtractive hybridization, differential display and microarrays) have been used successfully and their frequency of identification is rapidly growing.

4. NM23
By screening cDNA libraries of matched metastatic/non-metastatic K1735 murine melanoma cell lines by differential hybridization, `non-metastatic clone 23' gene, was identified as the first metastasis suppressor gene [71]. Enforced expression in cell lines of diverse cellular origin, suppressed metastasis without altering tumor growth (reviewed in Ref. [72]). The product of the human ortholog, NM23-H1, was identified to be a nucleoside diphosphate kinase (NDPK). NDPKs catalyze the transphosphorylation of the -phosphate of a deoxynucleoside triphosphate to a deoxynucleoside diphosphate with the formation of a histidine-phosphorylated intermediate. The Drosophila nm23 ortholog, awd, is required for proper differentiation of tissues of epithelial origin (reviewed in Ref. [73]). To date, eight NM23 family members have been identified, designated NME1 through NME8. Of these, NM23-H1 and NM23-H2 have reported metastasis suppressor activity, but NDPK activity has been dissociated from metastasis suppression [74]. Postel and colleagues identified Nm23-H2 as a PuF, a transcription-promoting factor of the c-myc gene [75].

Protein–protein and other Nm23 interaction studies have been complicated by the `sticky' nature of the molecule, making it difficult to establish specificity [72]. Yet, building upon previous experiments in which histidine kinase activity of NM23 was correlated with reduced metastasis [76], Hartsough et al., showed that Nm23 immunoprecipitated kinase suppressor of Ras (KSR) [77]. KSR is a scaffold protein for the mitogen activated protein kinase (MAPK) cascade. Nm23 phosphorylated KSR at serine 392, a 14-3-3-binding site. This, coupled with observations that Nm23 transfected MDA-MB-435 cells had lower levels of phosphorylated MAPK led to the conclusion that Nm23 signals through the ERK-MAPK pathway [78 and 79]. Numerous papers have documented signaling through the Ras-ERK-MAPK as important in metastasis. Therefore the KSR result is especially intriguing.

Another interesting interaction involving Nm23-H1 was recently described by Fan et al. [80]. They provide evidence that Nm23-H1 interacts with granzyme A in the process of DNA damage induction in cytotoxic T-cell apoptosis. The mechanism has not been demonstrated in tumor cells; however, the association relates to the NDPK activity of Nm23s and may offer an alternative mechanism for metastasis suppression.

Clinical studies assessing Nm23 as a marker for metastasis were recently reviewed [72]. Briefly, decreased expression (as would be expected for a metastasis suppressor) correlated in many, but not in all cancers. Higher expression in neuroblastoma correlated with aggressiveness. A few studies found no correlation with metastasis. Interpretation is sometimes complicated because each study used different antibodies and involved different criteria. Thus, Nm23 has shown promise for some cancer types, but is not yet considered an independent prognostic factor.

5. KAI-1 (CD82)
KAI-1 was identified in prostate cancer cell lines (Dunning rat AT3.1 and AT6.1) that were suppressed for metastasis following introduction of human chromosome 11 [81]. Positional cloning mapped KAI1 to 11p11.2 [82].

KAI-1 is an evolutionarily conserved member of the tetraspanin transmembrane protein family of leukocyte surface glycoproteins. It is the only tetraspanin with an internalization sequence at the C-terminus [83]. Although no allelic losses were seen, expression in the epithelial compartment was consistently down-regulated during prostate cancer progression [84]. Expression also inversely correlated with breast cancer metastasis [85]. Enforced constitutive expression suppressed metastasis of breast cancer [86] and melanoma [87]. Additionally KAI1 inhibited key steps in metastasis (i.e. invasion and motility) of colon cancer cells [88].

There are contradicting reports [89 and 90] regarding interactions between p53 the KAI1 promoter following identification of a p53-consensus binding sequence. There is evidence of KAI1 epigenetic regulation by methylation of CpG islands in the promoter [91]. The mechanism of action is enigmatic, in part, because KAI1 functions as an adhesion molecule on leucocytes, but does dramatically influence adhesion in tumor cells. So, other mechanisms have been proposed. KAI1 directly associates with the EGF receptor and suppresses induced lamellipodia and migration signaling [92]. Attenuation of EGF-induced signaling is accomplished by ligand-induced receptor endocytosis. Thus, KAI1 might suppress metastasis by altering the balance between KAI1 and EGFR, which might affect proliferative and migratory signals delivered. KAI1 also associates with the cytoskeleton promoting phosphorylation and association of both the guanine exchange factor Vav and the adaptor protein SLP76 leading to de novo actin polymerization [93]. Involvement of Rho GTPases in KAI1 signaling brings to the forefront additional pathways in KAI1 signaling.

Immunohistochemical detection of KAI1 correlated inversely with metastasis in many different cancers [59]. Down-regulation of KAI1 was also seen in cancer lines of urogenital, gynecological, and pulmonary origin [94].

6. KISS-1, TXNIP and CRSP3
KISS-1 was identified as a melanoma metastasis suppressor using subtractive hybridization to compare chromosome 6 metastasis-suppressed melanoma hybrids with metastatic parental cells [68 and 95]. Surprisingly, the KISS-1 gene mapped to the long arm of chromosome 1 [68]. Enforced expression of KISS-1 suppressed metastasis of melanoma and breast carcinoma [96]. A deletion variant (neo6qdel; neo6del(q16.3-q23)) of neomycin-tagged human chromosome 6 did not suppress metastasis and did not express KISS1 [97]. Therefore, it was hypothesized that regulators of KISS-1 were encoded on chromosome 6.

Ultimately, the mechanism of action of KISS-1 remains unknown. Research has been stymied by an apparently short protein half-life. However, three groups studying an orphan G-protein coupled receptor (GPR54, hOT7T175, AXOR12) identified a fragment of KISS-1 as the ligand [98, 99 and 100]. KISS-1 fragments were named Metastin [100] and Kisspeptins [98]. The functional peptides were amidated [100]. Ligand binding initiates hydrolysis of (PIP2) and Ca⁺² mobilization and arachidonate release. ERK1/2 and p38^MAPK phosphorylation have also been observed concomitant with cytoskeletal changes [98, 99, 100, 101 and 102]. Boyd and colleagues showed that constitutive up-regulation of KISS-1 in HT10810 cells resulted in decreased NFB activation which, in turn, led to diminution of MMP-9 transcription [103].

While Ohtaki and colleagues showed elegant data showing that exogenous Metastin/Kisspeptin treatment of receptor-transfected B16–BL6 melanoma reduced metastasis and anchorage-independent growth [100], activity of the endogenous receptor has not been demonstrated to date in cancer cells. Likewise, endogenous receptor expression and mutation analysis still need to be done to firmly establish a connection with melanoma metastasis.

The normal physiological function(s) of KISS-1 (and its receptor) are only recently becoming elucidated. KISS-1 levels are higher in early placenta and molar pregnancies and are reduced in choriocarcinoma cells, favoring a predominant role in the control of the invasive and migratory properties of trophoblast cells [104].

A clinical role for KISS-1 was inferred by the experimental studies showing metastasis suppression. The following issues have made it difficult to complete a detailed study-lack of antibodies/antisera recognizing KISS-1 or Metastin/Kisspeptin; lack of reagents recognizing receptor; and short life span of the nascent protein. Nonetheless, Shirasaki and colleagues used in situ hybridization to examine KISS-1 expression in clinical melanoma samples [105]. As expected, an inverse correlation of KISS-1 with malignancy were found. While carefully performed, information regarding KISS-1 processing or the receptors was not possible in those studies. Importantly, the studies compared LOH on 6q loci with KISS-1 expression [105]. The clinical studies corroborated the experimental MMCT data linking loci between 6q16.3-q23. Murine orthologs of metastin and GPR54 were used to demonstrate activation of phospholipase C following ligand binding [102].

Recently, Goldberg et al., identified two molecules (TXNIP and CRSP3) that appear to function upstream of KISS-1 [53]. Briefly, paired microarrays compared metastatic C8161 and non-metastatic neo6/C8161 cells. Also, metastatic neo6qdel/C8161 cells were compared to neo6/C8161. The gene with greatest differential expression in both arrays was VDUP1 (Vitamin D3 upregulated protein 1). VDUP1 was first identified in HeLa cells by differential display following treatment with 1,25-dihydroxyvitamin-D3 [106]. Subsequently it was identified as an interactor of thioredoxin (TRN) in a yeast two-hybrid screen and is also known as TBP2 (TRN binding protein 2) and TXNIP (TRN-interacting protein, preferred name). TRN is a redox- signal regulating protein [107] and regulates stress-response MAPK signaling via suppression of the apoptosis signal-regulating kinase 1 (ASK1) activation and also activation of transcription factors. TXNIP binds to the reduced form of TRN to inhibit function and expression [108 and 109]. TXNIP also regulates stress-response apoptosis signal transduction [110 and 111]. Concomitant with increased TXNIP expression is decreased expression of TRN and arrest of cell growth [112]. Based upon trends toward increased TRN in many tumors and cell lines, TXNIP may have tumor suppressor effects as well.

CRSP3 encodes a co-factor required for SP1-mediated activation of transcription. Sp1 (Specificity protein 1) is a general transcription factor that binds to and acts through GC-boxes, widely distributed promoter elements [113 and 114]. CRSP3 has no known yeast or murine orthologs [115]. Definitive clinical studies have not yet been done, but CRSP3 and TXNIP expression, generally inversely correlate with melanoma progression. Additionally, sequence tagged sites adjacent to CRSP3 in patient samples [105] suggest that the gene may indeed show changes associated with clinical outcome.

7. TIMPs
Tissue inhibitors of metalloproteinases (TIMPs) are a family of secreted proteins that selectively, but reversibly, inhibit metalloproteinases (MMPs) with 1:1 stoichiometry [10, 116 and 117]. Modulation of MMP and TIMP levels is critical to the control of extravasation and tumor-induced angiogenesis, processes that involve basement membrane degradation. Paradoxically, TIMP-1, 2 and 4 have an anti-apoptotic effect, while TIMP-3 induces apoptosis. TIMP-2, in concert with MT1-MMP can bind to and activate proMMP-2 (reviewed in Ref. [116]). Although there are no known TIMP-specific receptors, membrane-bound molecules such as MT-MMPs and metalloproteinase disintegrins (ADAMs) serve as TIMP-binding molecules at the cell surface [117].

TIMPs are expressed in tumor tissues and are present in the sera of cancer patients, raising the possibility that TIMP levels could predict clinical outcome and risk of metastasis [118, 119, 120 and 121]. But results are complicated because the ratio of TIMPs to MMPs is the crucial parameter. Nonetheless, the possibility that serum TIMP levels could be useful in a clinical setting remains. Gene therapy studies for local or systemic delivery of TIMPs are in an exploratory phase (reviewed in Ref. [122]).

8. Cadherins
Cadherins are transmembrane glycoproteins responsible for Ca⁺²-dependent cell adhesion. Although the family is widely expressed, E-cadherin (gene designation CAD1) is expressed on epithelial cells. A precursor protein (135 kDa) is processed to a mature 120 kDa form. The extracellular N-terminus is critical for homophilic Ca⁺²-dependent cell–cell adhesion. The C-terminus interacts with -catenin to mediate actin binding. E-cadherin/-catenin binding sequesters the latter, blocking nuclear translocation and transcription of c-myc and cyclin D1.

Defining a role for E-cadherin as a metastasis suppressor is complicated. Over-expression decreases motility and invasiveness [123]. Mutations of CAD1 and -catenin have been associated with invasion [124]. High E-cadherin levels inhibit shedding of tumor cells from the primary tumor; thus, CAD1 is a metastasis-suppressor [124, 125 and 126]. However, CAD1 can also be a tumor suppressor [124, 125 and 127]. Loss of expression occurs in many tumors and is directly associated with loss of differentiation (reviewed in Ref. [128]). Mechanisms of reduced expression include: reduction or loss of E-cadherin expression (by LOH or epigenetic silencing [129]), redistribution to different sites within the cell, shedding of E-cadherin and competition from other proteins (reviewed in [130]). Stimulation of the EGFR by EGF, TGF- or PP2 brings about phosphorylation of E-cadherin and -catenin resulting in dissociation of the complex [131 and 132]. Other than breast and gastric cancers, with nearly 50% of the tumors affected, mutations of CAD1 appear to be infrequent [133]. Evidence supports a role of E-cadherin in tumor suppression rather than just being an epiphenomenon of the tumor cells' phenotypic changes [134]. Since loss of E-cadherin alone, leading to decreased cell–cell adhesion is insufficient for the tumor cells to invade, it appears more than likely that down-regulation actively transduces specific signals that support tumor invasion.

Recently, Kashima et al., showed that N-cadherin and cadherin-11 (osteoblast cadherin), which are both highly expressed in osteoblasts (bone forming cells), reduce metastasis to lungs without negatively affecting tumorigenicity [135]. Reduced motility was presumably the mechanism responsible for diminished metastasis. Curiously, N-cadherin and cadherin-11 are frequently over-expressed in many metastatic breast and prostatic carcinoma cells [136, 137 and 138]. Moreover, transfection and over-expression promotes invasion and metastasis in breast and melanoma cells [136, 139 and 140]. These results highlight the complexities of interpretation because of cell origin. They further reinforce the point raised above––gene context is important.

9. MKK4
MKK4/JNKK1/SEK1 is a mitogen-activated protein kinase, which transduces signals from MEKK1 to stress-activated protein kinase/JNK1 and p38^MAPK [59]. MKK4 transmits stress signals to nuclear transcription factors that mediate proliferation, apoptosis and differentiation. Portions of the MKK4 gene (on chromosome 17) were deleted or altered in cancer cell lines that displayed defects in signal transduction from MEKK1 [141]. Suppression of prostate cancer cell metastasis was brought about by over-expressed MKK4 [142]. An inverse relationship between Gleason score and MKK4 staining was established in prostate tumors [143]. MKK4 is also a metastasis suppressor in ovarian carcinomas [144].

10. BRMS1
Following upon MMCT studies, Seraj et al., performed differential display to identify the gene(s) responsible for chromosome 11 suppression of breast cancer metastasis. Three novel cDNAs were identified. BRMS1 suppressed metastasis in MDA-MB-231 and MDA-MB-435 [145] breast carcinomas in addition to two human melanoma (C8161 and MelJuSo, [146]) and two murine mammary carcinoma cell lines (4T1 and 66cl4 [147]). BRMS1 transfectants were not suppressed for growth in vitro or in vivo; adhesion to extracellular components (LN, FN, collagens I or IV, Matrigel); expression of gelatinases (MMP-2, MMP-9) or heparanase, or invasion in vitro [148].

The BRMS1 gene mapped to human chromosome 11q13.1-q13.2, a region frequently altered in metastatic breast cancer. Expression of other metastasis suppressors (i.e. NM23, KAI-1, KISS-1, CAD1) did not correlate with BRMS1. Motility was moderately reduced in wound assays as was the ability to grow in soft agar. The most striking change amongst transfectants was restoration of gap junctional intercellular communications (GJIC) [148 and 149], accompanied by increased expression of connexin (Cx) 43 and decreased expression of Cx32 [150]. Connexins are the protein subunits of gap junctions and the expression pattern in BRMS1 transfectants more closely mimics normal breast tissue. Using real time RT-PCR, BRMS1 expression inversely correlated with metastasis in human melanoma cells [146]. Expression of BRMS1 also reduced metastasis of T24T, a metastatic variant of the human bladder carcinoma cell line, T24 [151]. Although a role in normal physiology has not been determined, BRMS1 does not appear to regulate invasive and/or migratory properties of trophoblast cells [104]. BRMS1 RNA expression was detected in villous cytotrophoblasts, but the level in invasive cytotrophoblasts, the subclass of trophoblast cells that invades into the decidua was not examined, thus warranting prudence in interpreting the data.

Hunter and colleagues [152 and 153] using a genetic approach to identify factors predisposing to metastatic disease, co-localized the Brms1 gene with the Mtes1 (Metastasis Efficiency Suppressor 1) locus on chromosome 19 (orthologous to human chromosome 11). Later studies utilizing comparative sequence analysis, however, suggest that Brms1 is not likely Mtes1 [152 and 154].

11. SSeCKS
SSeCKS (pronounced essex) for Src-suppressed C kinase substrate expression is down-regulated in src-and ras-transformed rodent fibroblasts [155 and 156]. It is the likely rodent ortholog of human Gravin/AKAP12, a cytoplasmic scaffolding protein for protein kinases A and C [157], concentrating at the cell edge and podosomes. In response to phorbol esters, SSeCKS controls elaboration of a cortical cytoskeletal matrix. Over-expression suppresses v-src-induced morphological transformation and tumorigenesis. ERK2 activity was induced 5- to 10-fold in presence of v-src [158], resulting in decreased cyclin D1 expression and pRb phosphorylation, thereby playing a role cell cycle progression [158 and 159]. While SSeCKS/Gravin protein is detected in untransformed rat and human prostate epithelial cell lines, expression is severely reduced in metastatic prostate carcinoma cell lines. Re-expression significantly decreased lung metastases, induced filopodia-like projections and decreased anchorage-independent growth [160] in vitro.

12. RhoGDI2
Rho GTPases are guanine nucleotide binding proteins, which cycle between active GTP-bound state and inactive GDP-bound state. RhoGDI (Rho GDP dissociation inhibitors) stabilize the GDP-bound form and sequester them in an inactive non-membrane localized, cytoplasmic compartment [161]. In an earlier bladder carcinoma study, RNA expression of RhoGDI2 was associated with decreased metastatic potential [151]. Transfection and enforced expression suppressed metastasis of T24 human bladder carcinoma variants [162]. Gene expression profiling of 105 bladder carcinomas, corroborated the expression pattern––i.e. RhoGDI2 expression correlated inversely with the invasive phenotype of tumors.

13. Drg-1
Drg-1 (a.k.a. RTP, cap43 and rit42) was identified as a differentiation-associated gene in colon carcinomas by differential display [163]. It is orthologous to mouse TDD45 and Ndr1 and rat Bdm1. Kurdistani and colleagues showed that introduction of Drg-1 cDNA suppressed tumorigenicity of human bladder carcinoma cells, suggesting that Drg-1 is a tumor suppressor gene [164]. However, in vitro invasion and liver metastases are inhibited from colorectal carcinomas when expression is restored either by inhibiting DNA methylation or by transfection [165]. Likewise, Bandopadhyay et al., recently showed that prostate carcinoma cells are suppressed for metastasis, but not tumorigenicity, when Drg-1 is over-expressed [166]. The latter studies support the contention that Drg-1 is a metastasis suppressor.

Drg-1 expression inversely correlated with Gleason score in human prostate cancer specimens [166]. While the mechanism of action of Drg-1 is unknown, it is up-regulated by PTEN and p53 and phosphorylated by Protein Kinase A [167]. It is postulated that Drg-1 might function downstream of MKK4, since it is induced similarly to the stress activated protein kinases (JNK/SAPK) [168] via MKK4, itself a metastasis-suppressor.

14. Metastasis suppressors without functional portfolio
The above genes have functional evidence supporting classification as metastasis suppressors. We will briefly describe below several others whose evidence is suggestive, but the data are deficient with regard to classification as metastasis suppressors for two reasons. First, the data are at this time correlative, not functional. Second, functional suppression of metastasis occurs concurrent with diminished tumorigenicity. In the absence of experimental arms to accommodate differential growth rates and detailed analysis to verify expression, designation as metastasis suppressors by the strict definition is not possible.

Responding to environmental and growth stimuli, axons extend growth cones in several directions. Semaphorins, a large family of secreted and membrane-bound proteins participate in a repulsive (collapse) process [169 and 170]. CRMP proteins aid intracellular transduction of collapse signals [171]. CRMP-1, for Collapsin Response Mediator Protein-1, is one of five proteins in the CRMP family, whose molecular mechanisms have not yet been characterized, although recent literature implicates involvement in controlling cell movement (reviewed in Ref. [172]). Recently, CRMP-1 was shown to reduce invasion of lung cancer cells [51]. Shih et al., demonstrated that CRMP-1 expression was inverse to lung carcinoma grade. Expression correlated directly with survival and time to relapse.

Gelsolin modulates actin assembly and disassembly to regulate motility. It also inhibits apoptosis [173]. Gelsolin decreases colonization in soft agar, retards spread, reduces chemotaxis to fibronectin and suppresses both tumorigenicity and metastasis of melanoma [174], bladder carcinoma [175] and lung carcinoma [176].

Following identification by DD-RT-PCR comparing normal mammary epithelium and invasive mammary carcinoma cells, maspin (mammary serine protease inhibitor) was reported to suppress invasion and metastasis (but no metastasis data was shown in the original paper). Complicating interpretation, tumorigenicity and growth were also reduced. [177]. The gene, SERPINB5, is a member of the serine protease inhibitor (serpin) gene cluster on chromosome 18q21.3. Maspin transgenic mice show attenuated tumor progression and metastasis, supporting its role against tumor spread [178]. Mechanistically, maspin also sensitizes cells to induced apoptosis [179] and reduces angiogenesis [180]. Expression of maspin is controlled at several levels. Futscher et al. [181] showed that cell-type specific expression of maspin inversely correlated with methylation of SERPINB5. SERPINB5 expression can be surmounted by treatment with 5-aza-2'-deoxycytidine [182]. Regulation of maspin by p53 has also been reported using EMSA [183].

Heterochromatin-associated protein 1 (HP1^HS) expression is down-regulated in highly invasive metastatic cells compared to non-metastatic cells where it is predominantly localized in the nucleus. Although the clinical correlations show promise as a metastasis suppressor HP1 in breast carcinoma [184], no data functional evidence for metastasis suppression are yet available.

Data for CD44 as a metastasis suppressor are controversial. Gao et al., showed CD44 to have metastasis suppressor activity in AT3.1 prostate carcinoma cells, without altering tumorigenicity [185]. Complexity exists because CD44, which encodes a membrane protein that binds the extracellular membrane components hyaluronic acid and osteopontin exists in multiple isoforms. The standard isoform, CD44-s, significantly (>60%) reduces lung metastases, but it is still not certain which are the most relevant isoforms for cancer and metastasis. Reagents to study the role(s) of particular isoforms in tumorigenicity and/or metastasis are under development. Until then, CD44 data should be interpreted cautiously.

SHP-2 is a widely expressed cytoplasmic tyrosine phosphatase that is believed to participate in signal relay downstream of growth factor receptors. SHP-2 impairs spreading of fibroblasts on fibronectin and migration (in vitro) [186]. Cells expressing mutant SHP-2 display reduced focal adhesion kinase de-phosphorylation as well as decreased association with paxillin. In vivo demonstration of metastasis suppression remains to be done.

15. Remaining questions and perspectives
The critical clinical threshold for any cancer is development of metastasis. Diagnosis occurring prior to the establishment of secondary lesions means favorable prognosis and more effective treatment. As a result, earlier, more effective diagnosis has been instrumental in improving cure rates for cancer.

Unfortunately, there are many cases in which there is no evidence of cancer spread at the time of diagnosis. Treatment plans are usually based upon somewhat subjective morphologic criteria in tissue specimens submitted to the pathologist. In the case of breast cancer, approximately 25% of node-negative patients develop metastases despite being designated `metastasis negative' at the time of diagnosis. What can be done to identify the patients whose cancers are likely to spread and those whose cancers are unlikely to form secondary lesions? The answer depends upon a thorough understanding of the underlying genetic and biochemical basis of metastasis.

While it is not yet known how, or whether, metastasis suppressor genes will play a role in predicting the propensity to metastasize in clinical cancer, information gained by understanding the mechanisms of action of the metastasis suppressors is providing insight into the fundamental mechanisms controlling cancer spread. The metastasis suppressors identified in Table 1 and Fig. 1 were discovered in several laboratories, using different model systems, and tested using distinct experimental systems. There is variability in terms of understanding mechanism and with regard to clinical evaluation. Nonetheless, the pieces to a complex jigsaw puzzle are beginning to take form. Pathways are beginning to emerge that connect heretofore independent metastasis suppressors. The picture is still sketchy; but some common elements are apparent.

Table 1. Characteristics of metastasis suppressor genes

^a The method of discovery is abbreviated: Clin, clinical correlation; DD, differential display; MA, microarray; MMCT/DD, microcell-mediated chromosome transfer + differential display; or SH, subtractive hybridization.
^b Arrows depict direction of change in behavior or expression (in clinical samples). () depicts no consistent change. Fields left blank indicate that the experiments have not yet been done or have not been reported.

Fig. 1. Proposed model of pathways of metastasis suppression. Metastasis suppressor genes are featured in solid black shapes. Solid lines indicate pathways for which biochemical evidence has been provided. The dotted lines represent inferred/implied pathways. Putative metastasis suppressors are stippled. If the location of the proteins are known, they are placed. If the location or functional subcellular compartment are not definitively known, the proteins are placed in the box at the left. Some connections are omitted to simplify the figure. The shadowed boxes positioned within the nucleus highlight the existence of pro- and anti-metastatic genes involved in transcription. Two molecules are highlighted with regard to promoting metastasis, Id1 [199 and 200] and MTA1 [201 and 202].

First, many metastasis suppressors have functions that amplify `signals' (i.e. there are several branches downstream in each signaling arbor). This situation is highly desirable for controlling complex, multigenic phenotypes like metastasis. Second, metastasis suppressors exist within all cellular compartments. The situation is reminiscent of the genes controlling cell cycle, apoptosis, and differentiation. The expectation (hope?) is that, like the cell cycle genes, some higher order will become evident as the regulatory molecules are put into pathways. Moreover, it is hoped that key rate-limiting steps will be identified. Third, many metastasis suppressors function in diverse cell types (i.e. genes discovered in one tumor type also suppress metastasis in cells of other origins). Fourth, despite use of a strict definition of metastasis suppression (i.e. demonstration of a functional suppression of metastasis without inhibition of tumor formation), the number of metastasis suppressor genes is continuing to grow. How many metastasis suppressor genes are there? We do not know. Based upon similarly highly regulated phenotypes, we would predict that the number is limited within the core regulatory pathway(s). The complexity is daunting if alterations downstream are also counted.

The field of metastasis genetics and the existence of genes that specifically control metastasis has been called into question by some [6 and 7]. Yet, functional data with the metastasis suppressor genes strongly argue that there are specific genes controlling metastasis.

Our colleague, Kent Hunter has collected some very important data that support the existence of metastasis genes using breeding strategies in mice. Using a transgene-induced mouse mammary tumor model (MMTV-PyMT), mice were crossed with mice of varying genetic backgrounds. Significant differences in metastasis were found despite failure to alter tumor initiation or growth kinetics in some strains. Since all of the mouse tumors were initiated by the same oncogenic event, the differences in metastasis and gene expression are most likely due to genetic background. His data reinforce a notion that we introduced earlier––gene context is an important parameter in determining metastatic potential.

Further contributing to the argument that microenvironment is important are observations from multiple laboratories showing that many metastasis suppressors act at the terminal steps of the metastatic cascade, i.e. proliferation at the secondary site [34, 67 and 187]. In studies from our laboratory, we have showed, that tumor cells proliferated in some sites (i.e. orthotopic) but not others (i.e. metastatic). Furthermore, we have preliminary evidence that some metastasis suppressor genes suppress colonization in some organs, but not others (J.F. Harms and D.R. Welch unpublished). Much more work will be required to understand the interplay between metastasis-controlling genes and microenvironment; however, the importance of cellular context cannot be overstated.

An issue that has stymied the field for several years is the imprecise use of terminology. Even a cursory look at the literature finds numerous papers that claim suppression of metastasis. Many claims are unfounded because there is no biological data to support them. Metastasis is an in vivo phenotype and, quite simply, in vitro assays are not always predictive of in vivo behavior. In short, many labs suppressed steps of metastasis (i.e. invasion, motility, adhesion, resistance to apoptosis, growth) without testing the impact of changes using in vivo metastasis assays. Correlative studies are often related to promises unfulfilled. Nonetheless, we are encouraged by the emergence of new researchers in the metastasis field and the breadth of expertise that they bring. More common are claims that a gene blocks metastasis when it blocks growth––tumorigenicity. The issue was addressed above. However, the field must address the paradox that emerges when metastasis is suppressed in one cell type but tumorigenicity is suppressed in another (as for E-cadherin and DRG-1).

What do the data summarized in this review tell us about the clinical control of metastasis? Readers are cautioned to note that reliable antibodies/antisera recognizing many of the metastasis suppressors do not yet exist. As a result, many of the correlations presented are measured using RNA. While proportional expression of RNA and protein is anticipated for most, data are not yet available to definitively conclude such. Likewise, it is not known whether some metastasis suppressors are post-translationally modified. Ultimately, interpretation will depend upon identifying the functional protein responsible for metastasis suppression.

Another area of active research relates to the mechanisms responsible for loss of metastasis suppressor gene expression. Both anecdotal and published data suggest that many metastasis suppressor genes are not mutated, but are differentially expressed (reviewed in Ref. [188]). While not described in detail here, there are several levels at which expression could be regulated––protein translation [189 and 190], methylation [191 and 192], histone acetylation [192, 193, 194 and 195], mRNA protein stability [196 and 197]. Pat Steeg and colleagues have been pioneering the notion that metastasis suppressor genes may be re-expressed in a clinical setting. Recent data from her laboratory show that dexamethasone and medroxyprogesterone acetate can enhance expression of Nm23 [198]. They have also presented evidence that hypomethylation by 5-azacytidine can restore Nm23 expression as well [79]. While data were not collected for the other metastasis suppressors, their data support the possibility of pharmacologic regulation of metastasis via metastasis suppressor genes. Given that the drugs used for their experiments are first line, the possibility for therapeutic intervention in the near term is very real.

Acknowledgements
Work from the Welch lab has been supported by grants from the NIH (CA62168; CA88728, and CA89109), the US Department of Defense Medical Research and Materiel Command (DAMD-17-96-1-6152 and DAMD17-02-1-0541) and the National Foundation for Cancer Research Center for Metastasis Research. L.R.S. is the recipient of a Susan G. Komen Postdoctoral Fellowship (PDF-2000-218). We also appreciate our colleagues Pat Steeg, Carrie Rinker-Schaeffer and Kent Hunter for stimulating conversations and inspiration. Finally, we ask the forbearance of authors whose work was not cited for space considerations.

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