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R.E. Mansel et al. (eds.), Metastasis of Breast Cancer, 47-75. © 2007 Springer.

possible only if cancer cells can move actively and passively along certain routes. Yet, the relation between motility and metastasis formation is not always straightforward. Normal cells with a stable tissue position, can become highly motile when isolated and brought in culture, but do not metastasize when reinoculated into the host organism. Normal cells sometimes do "metastasize", like leukocytes which can migrate from the bone marrow towards their homing or inflammation sites, or like trophoblast cells towards the lungs of certain rodents during pregnancy (1). The common denominator in the motility by these normal cells is the restrict-tion in time and space: sensitivity to contact inhibition and the switching off by an "internal clock" appear to be mechanisms that are deficient in cancer cells. Again, this should not be taken too strictly, since cancer cell motility is a transient phenomenon, which can be switched off spontaneously and often temporarily in cells once they have established a metastasis. Aware of this complexity, we should consider motility as a necessary, but not as a sufficient condition for metastasis, and thus not conceive it as a functional marker of metastatic capability.

The content of this chapter is strictly related to the contribution of breast cancer motility to metastasis. Cell motility covers a number of aspects we will deal with separately for didactic reasons (Figure 1). First, breast cancer cells dispose of highly dynamic structures like actin filaments and a cytoplasmic microtubular complex, for which the traditional term "cytoskeleton" probably is a misnomer because it is too static (2). This intrinsic motility machinery can be considered as both the engine and the steering wheel of the cell, since it allows directional migration. This implicates that motility is not random, but that moving breast cancer cells are always on their way to form metastases. Second, motility is only one prerequisite for invasion, and is influenced by transient adhesive interactions with the cell's microenvironment. So, homotypic (between cells of the same type) cell-cell adhesions usually keep the cells in contact with each other, and serve an invasion suppressive aim. Heterotypic (between cells of different types) cell-cell adhesions, however, can help the invading cancer cell to use neighbouring stromal cells as a "grip". For cell-matrix adhesions the role in invasion is dual: some extracellular matrix structures, such as the basement membrane, can act as barriers or anchors for the cancer cells, while others rather offer tracks for the moving cell, such as interstitial type I collagen fibres. Third, extracellular proteases continuously help to remodel the cancer cell's microenvironment by disrupting cell-cell and cell-matrix adhesion proteins temporarily, and by dissolving extracellular matrix structures to facilitate cell displacement. For these reasons extracellular proteolysis is an important activity in invasion, not at least because it can generate chemotactic and angiogenic peptide fragments (3).

Figure 1. Schematic overview of the different aspects implicated in cancer cell motility and the first steps of metastasis formation. Normal epithelial cells (EP) transform into cancer cells (CA), where different components of the cytoskeleton (CS) organize to provide the machinery for directional migration (DM, dotted arrow). Integrins (INT) on normal and cancer cells can interact with components of the extracellular matrix (ECM), such as laminin in the basement membrane (BM). Cadherins (CAD) are implicated in cell-cell adhesion, while proteolysis (PL) by proteinases dissolves the matrix. Host cells have a molecular cross-talk with normal and cancer cells (full arrows). Chemotactic ligands (L) and receptors (R) guide the cancer cells towards the vessels for intravasation (EC: endothelial cell).

Figure 1. Schematic overview of the different aspects implicated in cancer cell motility and the first steps of metastasis formation. Normal epithelial cells (EP) transform into cancer cells (CA), where different components of the cytoskeleton (CS) organize to provide the machinery for directional migration (DM, dotted arrow). Integrins (INT) on normal and cancer cells can interact with components of the extracellular matrix (ECM), such as laminin in the basement membrane (BM). Cadherins (CAD) are implicated in cell-cell adhesion, while proteolysis (PL) by proteinases dissolves the matrix. Host cells have a molecular cross-talk with normal and cancer cells (full arrows). Chemotactic ligands (L) and receptors (R) guide the cancer cells towards the vessels for intravasation (EC: endothelial cell).

The old "seed and soil" hypothesis by Paget (4), stating that organ-specific metastasis from the primary tumour depends on the right combination of tumour cell and host organ factors, is still valid. For motility and breast cancer metastasis we will rephrase this hypothesis in terms of ligands and receptors as much as possible. Not only can these lead to a more complete picture of molecular interactions in metastasis, they may also indicate new targets for anti-metastatic therapeutic strategies in oncology. The latter concern is inspired by recent statistics telling that meta-

stases are by far the major cause of death in cancer patients, including breast cancer.

In this chapter we will apply three major restrictions. First, the data will only relate to breast cancer cells, which does not exclude that they may be relevant for other cancer types as well. Second, only cell motility information is included in order to avoid overlap with the other chapters of this book. Third, the motility data have been related to breast cancer invasion and metastasis, and are relevant to the general theme of this book.

2. THE DYNAMICS OF THE CYTOSKELETON IN BREAST CANCER CELLS: THE DRIVING FORCE OF THE CANCER CELL ON ITS WAY TO METASTASIS

The dynamic assembly/disassembly of the cytoplasmic actin microfilament complex is based on the rapid and reversible polymerisation of monomelic globular G-actin monomers into polymeric filamentous F-actin. Actin microfilament formation is typically observed in membrane protrusions coined lamellipodia and invadopodia. Analyses based on micro-arrays and proteomics have shown that the dynamics of actin polymerisation/depolymerisation reactions are controlled by actin-binding proteins. In cancer cells the expression of these proteins can be aberrant: some are downregulated like gelsolin (5,6), while others like fascin are upregulated (7,8). More recent data have modified our thinking of how actin-binding proteins regulate cancer cell motility: their localisation inside the cell appears to be more important than their gross general concentration. Some of them, like the LIM-and-SH3 protein (LASP1), are phosphorylated upon stimulation by external signals, relocate in the cytoplasm of the leading edge of the cancer cell and locally associate with actin to build up focal adhesion complexes in a dynamic way (9). Remarkably, actin and actin-binding proteins also co-exist in the nucleus. How this finding relates to cell motility is not always clear, but for the actin-capping protein CapG for example, it was shown that transport from the cytoplasm to the nucleus stimulates cell invasion (10). If this phenol-menon proves to be clinically relevant, the import receptor importin p, which is responsible for this nuclear relocation of CapG, may become an interesting therapeutic target for anti-invasive and anti-metastatic agents.

An underestimated actin-binding protein with relation to metastasis is probably myosin (11). In breast cancer cells in vitro non-muscle myosin II A and B are localised both in the rear end of the moving cell, where they are crucial for the retraction of the posterior cell part, and in the leading edge, where they associate with S100A4 (also known as metastasin-1). The A4 isoform of S100 is a motogenic molecule, that can induce the epithelioid-to-mesenchymal transition (EMT) in cancer cells, and its expression has been related with the metastatic phenotype repeatedly (12-14). We have shown that a non-invasive cell line variant, derived from a human breast cancer, did only weakly express the heavy chains of non-muscle myosins II A and B, as compared to an invasive variant from the same tumour, expressing high levels of these molecules. Attempts in our laboratory to downregulate the expression of non-muscle myosin II A and B in invasive and metastatic breast cancer cells are currently in progress, and try to confirm our hypothesis that these myosins are targets for anti-invasive agents.

While actin and non-muscle myosin II are the motor of the cell, the function of the cytoplasmic microtubular complex has been referred to as a steering wheel, because it is instrumental for direction-finding during cell movement. One strong indication for this idea was provided by experiments in vitro with cancer cells on glass, in which different classes of microtubule inhibitors all blocked directional migration, but not random motility nor cell ruffling. Treated cells lost their elongated shape, and became flattened and disclike with intense membrane ruffling all over the perimeter of the cell. Moreover, this inhibition was sufficient to block invasion in different assays in cell and organ culture (15), and offered an explanation for the anti-metastatic effect of chemotherapy regimens containing microtubule inhibitors, such as vinca-alkaloids (16). Indeed, the microtubule inhibitors not only block the cancer cell cycle in the M-phase, but also prevent dissemination to locoregional tissues and distant organs.

Rho, Rac, and Cdc42, three small Rho GTPases, control signal trans-duction pathways linking membrane receptor signals to the assembly and disassembly of the actin cytoskeleton. Rho regulates stress fiber and focal adhesion assembly, Rac regulates the formation of lamellipodia and membrane ruffles and Cdc42 triggers filopodial extensions at the cell periphery. These observations have led to the suggestion that, wherever filamentous actin is used to drive a cellular process, e.g., cell movement, axon guidance, phagocytosis, or cytokinesis, the Rho GTPases may play an important regulatory role. Furthermore, Rho, Rac, and Cdc42 have been reported to control other cellular activities, including regulation of the JNK and p38 MAP kinase cascades. So, all three GTPases have been implicated in growth control and although mutations at the gene loci of these molecules have not been found in human cancers, experiments suggest that Rac in particular might play an important role in invasion and metastasis (17). As a rule, activation of Rac and Cdc42 and inactiva-tion of Rho lead to increased motility, invasion and metastasis. Yet, some considerations have to be added to this generalisation. First, the family of

Rho GTPases is continuously increasing with new members (e.g., Tiam-1 (18) and Deleted in Liver Cancer-1 (19), both motogenic factors) and isoforms. These new members add a new level of complexity to our general rules. So, Rac1 is associated with lamellipodia formation, while Rac3 is not, but activation of either isoform increases motility and invasion (20,21). RhoC overexpression is positively correlated with breast cancer metastasis formation (22). Moreover, the balance between Rho GTPases and guanine nucleotide dissociation inhibitors also appears to regulate the metastatic capability of breast cancer cells (23). Second, the effects of Rho on motility, invasion, and metastasis may be influenced by the cellular context: effects of Tiam-1 on cell motility for instance depend on the cell type under study (24).

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