Adhesion Cadherins As Ligands And As Receptors

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Integrins are the sensors of the cancer cells for their surrounding ECM, and participate in the molecular translation of the "seed and soil" hypothesis by Paget. As mentioned before, the integrin-basement membrane interaction can help in maintaining positional stability in normal epithelia, but in carcinoma many clues indicate that integrins promote motility, invasion, and hence, metastasis. Many data from the recent literature point toward the relation between (over)expression of certain types of integrins, cell motility, and breast cancer metastasis (38). Examples of integrin subunits related to motility and metastasis are a3 and a4 (both inducing MMP-9 secretion) (39,40), a5, a6 (via the "nuclear factor of activated T-cells" mediators, abbreviated as NFAT1 and NFAT5) (41), a8 (interacting with tenascin V, an ECM molecule thought to be involved in motogenic and invadogenic effects by the ECM) (42), av (often related to breast cancer metastasis) (26,43,44), p1 (activated by phosphatidyl inositol-3 kinase, abbreviated as PI3K (45), and Akt2 (46)), and p4. One indication of the importance of integrins in metastasis is the anti-metastatic effect of drugs designed to act as inactivating ligands for the integrins. Some of these drugs are referred to as the RGD peptidomimetics S137 and S246, and provide a proof of principle of the integrin implication (47). Yet and again, the integrin repertoire expressed by the cancer cells is not explaining all aspects of motility and metastasis formation. Also important appears to be the composition of the ECM in the tumor microenvironment, and a seminal paper by Barsky et al. (48) revealed ECM differences between normal breast and cancer. More recent papers seem to confirm that subtle ECM differences can have an impact on cancer cell motility and invasion. This was shown for type I collagen (telopeptide-free, invasion-permissive versus normal, invasion-resistant) (49), fibronectin (involuting breast-derived, invasion-permissive versus nulliparous breast-derived, invasion-resistant) (50), laminin (catechin-pretreated, invasion resistant versus native, invasion-permissive) (51) and hyaluronate (hyaluronate synthase 2 antisense-treated, invasion, resistant versus parental, invasion-permissive) (52).

One important cytoplasmic signalling molecule transducing inhibitory integrin signals to the effector machineries of breast cancer cell motility, invasion, and metastasis, is integrin-linked kinase (ILK) (53). Eliminating chromosome 11, where the ILK gene locus is situated, induces invasion and metastasis, and has led to consider ILK as a metastasis suppressor. Another metastasis suppressor gene Kiss-1, coding for kisspeptin (54) (metastin), was shown to exert its inhibitory effect on breast cancer metastasis via increased adhesion to collagen type IV, which is a constituent of basement membrane (54,55).

Homotypic cell-cell interaction mediated by homophilic cadherin recognitions is a crucial regulator of cell motility, invasion, and metastasis. In this chapter the discussion will be restricted to type I (also called "classical") cadherins, which are characterised by a typical homophilic recognition, histidine-alanine-valine (HAV) amino acid sequence in their extracellular part. However, this does not imply an underestimation of the role of other cadherins, such as cadherin-11, in breast cancer motility. The three cadherins of interest here are epithelial (E), neural (N), and placental (P) cadherin, which consist of a highly conserved cytoplasmic, a membrane-spanning part, and an ectodomain composed of five calcium-binding protomers, which marks the identity of each cadherin type. The role of these cadherins in breast cancer motility and invasion is not entirely elucidated, but available data indicate that E-cadherin is a motility/invasion suppressor, while the effects of N- and P-cadherin are in line with motility/invasion promotion.

E-cadherin is present in all normal epithelia, promotes cell-cell adhesion in the adherens junctions and plays a role as master molecule during the organisation of the other epithelial cell junction types. In epithelial layers E-cadherin is responsible for contact inhibition of motility. When the expression of this molecule is downregulated in an epithelioid background, such as in certain embryonic stages or in invasive carcinomas, the cells become more motile and start to occupy the neighbouring stromal tissues (56,57). Evidence that this downregulation is indeed causally related to invasion not only stems from correlation studies in breast cancer between the immunohistochemical E-cadherin expression level/pattern and pathology grade: experimental manipulation of E-cadherin expression via antisense technology has confirmed the invasion suppressor function of this molecule in mammary cells in vitro (58), and subsequently in transgenic rat pancreas in vivo (59), and the metastasis suppressor function in drosophila larva eye disk (60). Hence, one important question in oncology is: what are the downregulating mechanisms of E-cadherin in breast cancer, and can targets for therapy be discovered among them? Mutations in the E-cadherin gene are not a frequent cause of human cancers. They occur incidentally in the germ-line of some families in New Zealand and Portugal, and lead to the development of breast and other cancers with an early age onset (61). Somatic mutations of E-cadherin appear to be a frequent phenomenon in lobular breast carcinoma, but a rare one in ductal carcinoma. These mutations are detectable early at the very stage of carcinoma in situ, and often create premature stop codons giving rise to truncated versions of the molecule (62,63). Theoretical considerations predict that many of these truncated forms lack the membrane-spanning part, and are not anchored in the cell membrane. So, if they are secreted, they may diffuse into the extracellular fluids and in blood, and can be developed as a circulating tumor marker for lobular breast carcinoma patients. While mutations are rare, downregulation of E-cadherin expression can often be traced back to promoter silencing. The list of negative transcription factors for E-cadherin is increasing steadily: slug, snail, twist, SIP1, 5EF1, E47 (64,65). Another mechanism of silencing may be the result of methylation of promoter DNA bases. Post-translational modifications of the E-cadherin molecule have been shown to be crucial in the regulation of epithelial cell-cell adhesion, motility, and invasion as well. These modifications such as extracellular domain N-glycosylation and cytoplasmic tail serine/threonine/tyrosine phosphorylation have to be described within a broader intracellular signalling context. The cytoplasmic tail of E-cadherin associates in a non-covalent way with a group of catenin molecules (a-, P-, y-catenin, -the latter one being identical to plakoglobin-, and p120 catenin), which form a link with the actin cytoskeleton.

While serine/threonine phosphorylation of E-cadherin and P-catenin are implicated in the regulation of cell-cell adhesion, it is the tyrosine phosphorylation of P-catenin that has gained major attention. The latter phenomenon has been related to a dissociation of P-catenin from the E-cadherin/catenin complex, and to inactivation of the cell-cell adhesion structures. Moreover, tyrosine-phosphorylated P-catenin becomes resistant to proteasome degradation, and diffuses into the nucleus to activate protein transcription of, among others, MMP-7 and myc genes. This is an illustration of how an invasion suppressor molecule, provided it is integrated in the E-cadherin/catenin complex, can adopt the role of an oncogene and invasion promoter after the proper post-translational modifications have occurred. Tyrosine phosphorylation of P-catenin can result from activation of cell surface peptide receptors. Relevant to breast cancer cell motility and metastasis here are the receptor families for epidermal growth factor (EGF), insulin-like growth factor I (IGF-1) (66), and nerve growth factor (NGF) (67). Heregulin, a ligand of the EGF receptor-3, was shown to increase E-cadherin-mediated breast cancer cell-cell adhesion, and to inhibit their invasion (68). Similarly, IGF-I and insulin were anti-invasive in organotypic confronting cultures by improving the function of the E-cadherin/catenin complex. Our team has gathered indications that these ligands trigger rapid (within 10 minutes) exocytosis of a pool of subcortically stored E-cadherin in human MCF-7/6 breast adenocarcinoma cells (69). The E-cadherin/catenin complex is also amenable to modulation by estrogens and anti-estrogens. The selective estrogen receptor modulator (SERM) tamoxifen, which has been administered successfully to breast carcinoma patients as an adjuvant therapy for three decades, was shown to activate the complex and to inhibit motility and invasion. A potent natural phyto-estrogen from hops coined 8-prenylnaringenin (8-PN) or hopein, was also shown to increase E-cadherin-mediated cell-cell adhesion between MCF-7/6 cells (70). For a number of empirical stimulators of E-cadherin cell-cell adhesion no molecular target has been found yet. Using MCF-7/6 cells as a model in vitro, a stimulation of cell-cell adhesion and an inhibition of invasion was described for: the citrus methoxyflavone tangeretin (71), the hops prenylated chalcone xanthohumol (72), the vitamin A analog retinoic acid (73) and a number of related polyphenols. A large group of closely related congeners of these polyphenols were tested for potentially anti-invasive effects on MCF-7/6 cells in confronting cultures with embryonic chick heart fragments (74). The degree of anti-invasive activity of these compounds was related to their three-dimensional features by means of the QSAR software, and predictions on the characteristics of optimally anti-invasive compounds are expected to be available soon (75).

N-cadherin is functionally the opponent of E-cadherin in many aspects (76). While E-cadherin is a suppressor of epithelioid motility and invasion, N-cadherin is an activator of both activities, and hence a factor of bad prognosis (77). Simultaneous expression of E- and N-cadherin in human breast cancer cells showed that the function of N-cadherin dominates the one of E-cadherin with respect to cell motility, invasion, and metastasis formation (78). In malignant tumours, including breast cancers, downregulation of E-cadherin is accompanied or followed by upregulation of N-cadherin. Aberrant expression studies of the transcript-tion factors snail and SIP 1 show that these phenomena may be elements of a broader dedifferentiation program observed in a number, but not in all cancers, namely the epithelial-to-mesenchymal transition (EMT) (79,80). In EMT epithelial cells not only resemble mesenchymal fibroblasts morphologically, but also express mesenchymal markers (e.g., N-cadherin and vimentin) (81). Recent data indicate that EMT is a reversible process (as sometimes observed at metastatic sites), and that the maintenance of the epithelial state is one of the multiple functions of p53. So, this cell cycle gatekeeper is no longer implicated in proliferation and apoptosis only, but has to be considered an important EMT suppressor and hence a major motility and metastasis suppressor (82).

Upregulation of N-cadherin leads to enzymatic cleavage of the ecto-domain close to the plasma membrane (Figure 2). Several proteinases have been shown to perform this cleavage: while ADAM 10 appears to be the main actor, other enzymes such as plasmin and the membranetype matrix metalloproteinases MT1-MMP and MT5-MMP can be implicated as well. All this results in shedding of a 90 kD soluble N-cadherin fragment (sN-CAD) into the extracellular fluid. This sN-CAD was shown to stimulate cancer cell motility and angiogenesis, and future experiments will show whether or not this fragment contributes to metastasis formation in vivo (83). Using an ELISA assay, we were able to detect sN-CAD in a number of biological fluids, such as blood and semen (84). For several tumour types, including breast cancer, the patient's serum concentration of sN-CAD was higher than in a reference population with no evidence of disease (median value 584 versus 99 ng/ml respectively). We are currently evaluating the potential value of serum sN-CAD as a tumour marker in cancer patients.

For P-cadherin, data on its role in motility and invasion of breast cancer are still sparse, but the list is growing steadily (Table 1). P-cadherin is expressed in the normal human breast by the myoepithelial cells. It is implicated in growth and differentiation, as evidenced by knockout mice displaying precocious differentiation of the mammary gland, and is aberrantly expressed in mammary carcinomas of high histological grade and poor prognosis. It has been suggested that suppression of the P-cadherin gene is lost during carcinogenesis, but the nature of this mechanism and the biological role of the newly acquired P-cadherin still remain interesting areas of research. In one study, blocking the estrogen receptor with the pure antagonist ICI 182,780 induced the expression of P-cadherin in MCF-7/AZ cells, which coincided with the acquisition of invasive capacity and the loss of cell-cell adhesion in vitro. Retroviral transduction in MCF-7/AZ cells confirmed the pro-invasive activity of P-cadherin, which required the juxtamembrane, p120 catenin-binding domain of its cytoplasmic tail. The effect of P-cadherin on cell-cell adhesion, motility, and invasion are clearly dependent on the cell context, since opposite effects were obtained in melanoma cells (85). Here, P-cadherin expression suppresses invasion in different assays, and may be a target for future gene therapy in this tumour.

Figure 2. Schematic overview of the enzymatic cleavage of the N-cadherin ectodomain (sN-CAD). Epithelial cadherin (E-CAD) is normally present on the breast epithelia (EP) while the cancer cell (CA) often express Neural cadherin (N-CAD). N-cadherin is also present on stromal cells (SC), like myofibroblast and endothelial cells (EC). Plasmin, AD AMI 0, MT1-MMP, and MT5-MMP are responsible for the proteolysis (PL) of N-cadherin. sN-CAD can be present in the extracellular matrix (ECM) but also in the blood.

Figure 2. Schematic overview of the enzymatic cleavage of the N-cadherin ectodomain (sN-CAD). Epithelial cadherin (E-CAD) is normally present on the breast epithelia (EP) while the cancer cell (CA) often express Neural cadherin (N-CAD). N-cadherin is also present on stromal cells (SC), like myofibroblast and endothelial cells (EC). Plasmin, AD AMI 0, MT1-MMP, and MT5-MMP are responsible for the proteolysis (PL) of N-cadherin. sN-CAD can be present in the extracellular matrix (ECM) but also in the blood.

5. THE INTERACTION OF THE BREAST CANCER CELL WITH SOLUBLE AND CELL-ASSOCIATED SIGNALS: INVASION WITHIN A MICRO-ECOSYSTEM

In accordance with the Paget hypothesis, breast cancer cells express different types of receptors, which make them sensitive to extracellular signals. Some of these signals are secreted, others are cell-bound, but all of them contribute to the organ-specific nature of metastasis. The chemo-kine with C-X-C motif ligand 12 (CXCL12) is secreted by hepatocytes and is recognized by the CXC receptor 4 (CXCR4) on circulating breast cancer cells (105-107). The receptor is absent on normal breast epithelial cells, but is expressed on ductal cells at very early stages of tumori-genesis such as in atypical hyperplasia or carcinoma in situ (108), and this receptor is upregulated by heregulin and by adenosine in the hypoxic tumor micro-environment (109). The molecular interaction between the ligand and the receptor triggers a number of signalling pathways in the cancer cell. The G-protein coupled CXCR4 receptor recrutes the a-, P-, and y-subunits (110), and activates the following pathways: (a) phosphatidyl inositide-3 kinase (PI3K), Akt1, and focal adhesion kinase (FAK), (b) p-arrestin and erk (111), and (c) epidermal growth factor receptor (EGFR) (112). These signals result in effects that eventually prepare the cancer cell for extravasation into the organ of metastasis: apoptosis is inhibited, endothelial transmigration is promoted, and motility/invasion is induced through an reorganisation of the actin cytoskeleton. The CXC example has at least two merits. First, it translates the Paget hypothesis on seed-and-soil into CXCR4 and CXCL12 respectively, and, second, it appears to be a useful target for anti-invasive treatments, as will be outlined in section 6. Recently, an opposite ligand/receptor interaction effect was published on cancer cell motility and metastasis: the tetra-spanin KAI1/CD82 on cancer cells interacts with the Duffy antigen receptor for cytokines (DARC). The interaction suppresses metastasis by inhibiting extravasation of circulating cancer cells (113).

The CXCR4/CXCL12 system is only one example of molecular interactions between breast cancer cells and distant organs that trigger motility and determine organ-specific metastasis formation. Table 2 lists a number of interactions affecting both breast cancer motility and metastasis.

Table 1. Studies concerning the expression and function of P-cadherin in neoplastic breast tissues and their physiological counterparts

Author

Methods

Findings1

Radice et al., 1997 (87) Deugnier et al., 1999 (88)

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