Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membrane-distal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains. Co-expression of a transmembrane ALCAM deletion mutant, which lacks the ligand binding domain, and endogenous wild-type ALCAM inhibited homophilic cell-cell interactions by interference with ALCAM avidity, while homophilic, soluble ligand binding remained unaltered. The extracellular structures of ALCAM thus provide two structurally and functionally distinguishable modules, one involved in ligand binding and the other in avidity. Functionality of both modules is required for stable homophilic ALCAM-ALCAM cell-cell adhesion.     

Molecular model of the N-terminal receptor-binding domain of the human CD6 ligand ALCAM.

CD6-ligand interactions have been implicated in the regulation of T-cell adhesion and activation. CD6 is a member of the scavenger receptor family, whereas its human ligand (ALCAM) belongs to the immunoglobulin superfamily. The extracellular region of ALCAM includes five immunoglobulin-like domains. As a fusion protein, the N-terminal extracellular domain of ALCAM (ALCAMD1) binds specifically to CD6. We report the construction, assessment, and analysis of a molecular model of ALCAMD1. The model defines the CDR-analogous loops, the location of N-linked glycosylation sites, and residues that form the beta-sheet faces of the immunoglobulin-like domain. Predicted structural characteristics of the A'GFCC'C" face of the model are consistent with the presence of monomeric and dimeric forms of ALCAMD1, which has implications for the receptor-ligand interactions.     

Dual knockdown of Galectin-8 and its glycosylated ligand,the activated leukocyte cell adhesion molecule (ALCAM/CD166), synergistically delays in vivo breast cancer growth

Galectin-8 (Gal-8), a ‘tandem-repeat’-type galectin, has been described as a modulator of cellular functions including adhesion, spreading, growth arrest, apoptosis, pathogen recognition, autophagy, and immunomodulation. We have previously shown that activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, serves as a receptor for endogenous Gal-8. ALCAM is a member of the immunoglobulin superfamily involved in cell-cell adhesion through homophilic (ALCAM-ALCAM) and heterophilic (i.e. ALCAM-CD6) interactions in different tissues. Here we investigated the physiologic relevance of ALCAM-Gal-8 association and glycosylation-dependent mechanisms governing these interactions. We found that silencing of ALCAM in MDA-MB-231 triple negative breast cancer cells decreases cell adhesion and migration onto Gal-8-coated surfaces in a glycan-dependent fashion. Remarkably, either Gal-8 or ALCAM silencing also disrupted cell-cell adhesion, and led to reduced tumor growth in a murine model of triple negative breast cancer. Moreover, structural characterization of endogenous ALCAM N-glycosylation showed abundant permissive structures for Gal-8 binding. Importantly, we also found that cell sialylation controls Gal-8-mediated cell adhesion. Altogether, these findings demonstrate a central role of either ALCAM or Gal-8 (or both) in controlling triple negative breast cancer.     

Activated leukocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration

Transendothelial leukocyte migration is a major aspect of the innate immune response. It is essential in repair and regeneration of damaged tissues and is regulated by multiple cell adhesion molecules (CAMs) including members of the immunoglobulin (Ig) superfamily. Activated leukocyte cell adhesion molecule (ALCAM/CD166) is an Ig CAM expressed by activated monocytes and endothelial cells. Hitherto, the functional relevance of ALCAM expression by endothelial cells and activated monocytes remained unknown. In this report, we demonstrate soluble recombinant human ALCAM significantly inhibited the rate of transendothelial migration of monocyte cell lines. Direct involvement of ALCAM in transendothelial migration was evident from the robust inhibition of this process by ALCAM blocking antibodies. However, soluble recombinant ALCAM had no impact on monocyte migration or adhesion to endothelium. Localization of ALCAM specifically at cell-cell junctions in endothelial cells supported its role in transendothelial migration. This study is the first to localize ALCAM to endothelial cell junctions and demonstrate a functional relevance for co-expression of ALCAM by activated monocytes and endothelial cells.     

Activated leukocyte cell adhesion molecule expression and shedding in thyroid tumors

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology.     

Activated leukocyte cell adhesion molecule modulates neurotrophin signaling

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.     

Three-Dimensional Analysis of CD6 Site-Directed Mutagenesis and Monoclonal Antibody Binding Studies Using the X-ray Structure of Mac-2 Binding Protein and a Molecular Model of the CD6 Ligand Binding Domain

The extracellular region of CD6 consists of three scavenger receptor cysteine-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues important for the CD6-ALCAM interaction have previously been identified by mutagenesis. A total of 22 CD6 residues were classified according to their importance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The three-dimensional structure of the SRCR domain of Mac-2 binding protein has recently been determined, providing a structural prototype for the SRCR protein superfamily. This has made a thorough three-dimensional analysis of CD6 mutagenesis and mAb binding experiments possible. Mutation of buried residues compromised both mAb and ALCAM binding, consistent with the presence of structural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were found to map to the surface in the same region of the domain. This suggests that the CD6 ligand binding site and epitopes of tested mAbs overlap and provides an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the ALCAM binding site.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050263Abbreviations ALCAM activated leukocyte cell adhesion molecule - CD6D3 third (membrane-proxi-mal) extracellular domain of CD6 - IgSF immunoglobulin superfamily - mAb monoclonal antibody - M2BP Mac-2 binding protein - SRCR scavenger receptor cysteine-rich domain - SRCRSF scavenger receptor cysteine-rich protein superfamily     

Collective cell migration in morphogenesis and cancer

The movement of cells that maintain cell-cell junctions yet protrude along or within tissues is an important mechanism for cell positioning in morphogenesis, tissue repair and cancer. Collective cell migration shares similarities but also important differences to individually migrating cells. Coherent groups of cells are arranged and held together by cell-cell adhesion molecules, including cadherins, integrins, ALCAM and NCAM. Integrins of the beta 1 and beta 3 families further provide polarized interactions with the extracellular tissue environment, while matrix-degrading proteases become focalized to substrate contacts to widen tissue space for the advancing cell mass. By generating one functional unit, in contrast to individual cell migration, collective migration provides the active and passive translocation of mobile and non-mobile cells, respectively. This review highlights cellular and molecular principles of collective migration in the context of morphogenic tissue patterning and tumor cell invasion.     

Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.     

A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion.

Protein tyrosine phosphatases (PTPases), such as SHP-1 and SHP-2, that contain Src homology 2 (SH2) domains play important roles in growth factor and cytokine signal transduction pathways. A protein of approximately 115 to 120 kDa that interacts with SHP-1 and SHP-2 was purified from v-src-transformed rat fibroblasts (SR-3Y1 cells), and the corresponding cDNA was cloned. The predicted amino acid sequence of the encoded protein, termed SHPS-1 (SHP substrate 1), suggests that it is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SH2-domain binding sites, in its cytoplasmic region. Various mitogens, including serum, insulin, and lysophosphatidic acid, or cell adhesion induced tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2 in cultured cells. Thus, SHPS-1 may be a direct substrate for both tyrosine kinases, such as the insulin receptor kinase or Src, and a specific docking protein for SH2-domain-containing PTPases. In addition, we suggest that SHPS-1 may be a potential substrate for SHP-2 and may function in both growth factor- and cell adhesion-induced cell signaling.     

ALCAM/CD166: A pleiotropic mediator of cell adhesion,stemness and cancer progression

Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166) is a glycoprotein involved in homotypic and heterotypic cell adhesion. ALCAM can be proteolytically cleaved at the cell surface by metalloproteases, which generate shedding of its ectodomain. In various tumors, ALCAM is overexpressed and serves as a valuable prognostic marker of disease progression. Moreover, CD166 has been identified as a putative cancer stem cell marker in particular cancers. Herein, we summarize biochemical aspects of ALCAM, including structure, proteolytic shedding, alternative splicing, and specific ligands, and integrate this information with biological functions of this glycoprotein including cell adhesion, migration and invasion. In addition, we discuss different patterns of ALCAM expression in distinct tumor types and its contribution to tumor progression. Finally, we highlight the role of ALCAM as a cancer stem cell marker and introduce current clinical trials associated with this molecule. Future studies are needed to define the value of shed ALCAM in biofluids or ALCAM isoform expression as prognostic biomarkers in tumor progression.     

Specific syndecan-1 domains regulate mesenchymal tumor cell adhesion, motility and migration

Background

Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression.

Methodology/Principal Findings

We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration.

Conclusions/Significance

Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.     

The ALCAM shedding by the metalloprotease ADAM17/TACE is involved in motility of ovarian carcinoma cells

Previous findings indicated that the activated leukocyte cell adhesion molecule (ALCAM) is expressed by tumors and plays a role in tumor biology. In this study, we show that ALCAM is shed from epithelial ovarian cancer (EOC) cells in vitro, leading to the generation of a soluble ALCAM (sALCAM), consisting of most of the extracellular domain. A similar sALCAM molecule was also found in the ascitic fluids and sera from EOC patients, suggesting that this process also occurs in vivo. sALCAM is constitutively produced by EOC cells, and this process can be enhanced by cell treatment with pervanadate, phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF), a known growth factor for EOC. Pharmacologic inhibitors of matrix metalloproteinases (MMP) and of a disintegrin and metalloproteases (ADAM), and the tissue inhibitor of metalloproteinase-3, significantly inhibited sALCAM release by EOC cells. The ADAM17/TACE molecule was expressed in EOC cell lines and ADAM17/TACE silencing by specific small interfering RNA-reduced ALCAM shedding. In addition, inhibitors of ADAM function blocked EOC cell motility in a wound-healing assay. Conversely, a recombinant antibody blocking ALCAM adhesive functions and inducing ALCAM internalization enhanced EOC cell motility. Altogether, our data suggest that the disruption of ALCAM-mediated adhesion is a relevant step in EOC motility, and ADAM17/TACE takes part in this process, which may be relevant to EOC invasive potential.     

Structure of the extracellular domain of Tie receptor tyrosine kinases and localization of the angiopoietin-binding epitope

Angiogenesis is essential for tissue repair and regeneration during wound healing but also plays important roles in many pathological processes including tumor growth and metastasis. The receptor protein tyrosine kinase Tie2 and its ligands, the angiopoietins, have important functions in the regulation of angiogenesis. Here, we report a detailed structural and functional characterization of the extracellular region of Tie2. Sequence analysis of the extracellular domain revealed an additional immunoglobulin-like domain resulting in a tandem repeat of immunoglobulin-like domains at the N terminus of the protein. The same domain organization was also found for the Tie1 receptor that shares a high degree of homology with Tie2. Based on structural similarities to other receptor tyrosine kinases and cell adhesion molecules, we demonstrate that the N-terminal two immunoglobulin-like domains of Tie2 harbor the angiopoietin-binding site. Using transmission electron microscopy we furthermore show that the extracellular domain of Tie receptors consists of a globular head domain and a short rod-like stalk that probably forms a spacer between the cell surface and the angiopoietin-binding site. Mutational analysis demonstrated that the head domain consists of the three immunoglobulin-like domains and the three epidermal growth factor-like modules and that the stalk is formed by the three fibronectin type III repeats. These findings might be of particular interest for drug development because Tie receptors are potential targets for treatment of angiogenesis-associated diseases.     

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control.     

Distinct cellular functions mediated by different VLA integrin alpha subunit cytoplasmic domains.

To characterize VLA alpha subunit cytoplasmic domain functions, unaltered alpha 2 cDNA (called X2C2) and two chimeric cDNAs (called X2C5 and X2C4) were constructed with extracellular alpha 2 domains and cytoplasmic alpha 2, alpha 5, and alpha 4 domains respectively. Upon transfection into rhabdomyosarcoma (RD) cells, each construct yielded comparable expression levels, immunoprecipitation profiles, and avidity for collagen and laminin. However, while RDX2C2 and RDX2C5 transfectants mediated collagen gel contraction, RDX2C4 and a mock transfectant (RDpF) did not. Conversely, only RDX2C4 cells (but not RDX2C2 or RDX2C5) showed enhanced cell migration on collagen and laminin compared with RDpF cells. This indicates markedly differing roles for integrin alpha subunit cytoplasmic domains in post-ligand binding events. Furthermore, stable exertion of physical force (collagen gel contraction) may involve fundamentally different cellular machinery than the transient adhesion occurring during cell migration. Finally, these findings provide insight into a functional flexibility perhaps resulting from multiple integrins binding to identical ligands.     

Calcium-sensing receptor modulates cell adhesion and migration via integrins

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins. Immunopurification and mass spectrometry, co-immunoprecipitation, and co-localization studies showed that the CaSR and β1-containing integrins are components of a macromolecular protein complex. In fibronectin-based cell adhesion and migration assays, the CaSR-positive allosteric modulator NPS R-568 induced a concentration-dependent increase in cell adhesion and migration; both of these effects were blocked by a specific CaSR-negative allosteric modulator. These effects were mediated by integrins because they were blocked by a peptide inhibitor of integrin binding to fibronectin and β1 knockdown experiments. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium. These results demonstrate for the first time that an ion-sensing G protein-coupled receptor functionally couples to the integrins and, in conjunction with intracellular calcium release, promotes cellular adhesion and migration in tumor cells. The significance of this interaction is further highlighted by studies implicating the CaSR in cancer metastasis, axonal growth, and stem cell attachment, functions that rely on integrin-mediated cell adhesion.     

Sphingomyelin metabolites in vascular cell signaling and atherogenesis

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.