Superfamily Cell Adhesion Molecules Research Articles

The Regulatory Roles of Cerebellar Glycosphingolipid Microdomains/Lipid Rafts.

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed.

Bma-LAD-2, an Intestinal Cell Adhesion Protein, as a Potential Therapeutic Target for Lymphatic Filariasis.

ABSTRACTLymphatic filariasis is a debilitating disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Despite substantial success, efforts to eliminate LF will likely require more time and resources than predicted. Identifying new drug and vaccine targets in adult filariae could help elimination efforts. This study’s aim was to evaluate intestinal proteins in adult Brugia malayi worms as possible therapeutic targets. Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was determined to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1 day post-siRNA incubation and an 87.02% reduction in protein expression 2 days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6 days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult Brugia malayi and may be an effective therapeutic target.

Adhesion molecules in multiple myeloma oncogenesis and targeted therapy.

Every day we march closer to finding the cure for multiple myeloma. The myeloma cells inflict their damage through specialized cellular meshwork and cytokines system. Implicit in these interactions are cellular adhesion molecules and their regulators which include but are not limited to integrins and syndecan-1/CD138, immunoglobulin superfamily cell adhesion molecules, such as CD44, cadherins such as N-cadherin, and selectins, such as E-selectin. Several adhesion molecules are respectively involved in myelomagenesis such as in the transition from the precursor disorder monoclonal gammopathy of undetermined significance to indolent asymptomatic multiple myeloma (smoldering myeloma) then to active multiple myeloma or primary plasma cell leukemia, and in the pathological manifestations of multiple myeloma.

Affinity requirements for control of synaptic targeting and neuronal cell survival by heterophilic IgSF cell adhesion molecules.

SUMMARYNeurons in the developing brain express many different cell adhesion molecules (CAMs) on their surfaces. CAM-binding affinities can vary by more than 200-fold, but the significance of these variations is unknown. Interactions between the immunoglobulin superfamily CAM DIP-α and its binding partners, Dpr10 and Dpr6, control synaptic targeting and survival of Drosophila optic lobe neurons. We design mutations that systematically change interaction affinity and analyze function in vivo. Reducing affinity causes loss-of-function phenotypes whose severity scales with the magnitude of the change. Synaptic targeting is more sensitive to affinity reduction than is cell survival. Increasing affinity rescues neurons that would normally be culled by apoptosis. By manipulating CAM expression together with affinity, we show that the key parameter controlling circuit assembly is surface avidity, which is the strength of adherence between cell surfaces. We conclude that CAM binding affinities and expression levels are finely tuned for function during development.

Sticks and Stones, a conserved cell surface ligand for the Type IIa RPTP Lar, regulates neural circuit wiring in Drosophila.

Type IIa receptor-like protein tyrosine phosphatases (RPTPs) are essential for neural development. They have cell adhesion molecule (CAM)-like extracellular domains that interact with cell-surface ligands and coreceptors. We identified the immunoglobulin superfamily CAM Sticks and Stones (Sns) as a new partner for the Drosophila Type IIa RPTP Lar. Lar and Sns bind to each other in embryos and in vitro, and the human Sns ortholog, Nephrin, binds to human Type IIa RPTPs. Genetic analysis shows that Lar and Sns function together to regulate larval neuromuscular junction development, axon guidance in the mushroom body (MB), and innervation of the optic lobe (OL) medulla by R7 photoreceptors. In the neuromuscular system, Lar and Sns are both required in motor neurons, and may function as coreceptors. In the MB and OL, however, the relevant Lar-Sns interactions are in trans (between neurons), so Sns functions as a Lar ligand in these systems.

Synapse formation in the visual cortex

Neurodevelopment Synapses between neurons depend on cell-adhesion molecules for their formation. In the visual cortex, synapses are built and refined in response to visual experience. Nectin-3, one of a group of immunoglobulin superfamily cell-adhesion molecules, can shake hands with its binding partner nectin-1 across the synaptic cleft to stabilize a developing synapse. Inferring synapse numbers from dendritic spine density, Tomorsky et al. studied the function of nectin-3 in the development of the mouse visual cortex. In normal development, the number of dendritic spines increases once neonatal mice open their eyes. The authors found that overexpression of nectin-3 locked in too much stability and that not enough new dendritic spines formed for normal neural migration. Underexpression of nectin-3 allowed too many new dendritic spines to form, which was also harmful to ocular development. As in much of life, somewhere precisely in between seems to work best. Neural Dev. 15 , 13 (2020).

An Axon-Pathfinding Mechanism Preserves Epithelial Tissue Integrity

Epithelial tissues form the boundaries of organs, where they perform a range of functions, including secretion, absorption, and protection. These tissues are commonly composed of discrete cell layers-sheets of cells that are one-cell thick. In multiple systems examined, epithelial cells round up and move in the apical direction before dividing, likely in response to neighbor-cell crowding [1-6]. Because of this movement, daughter cells may be born displaced from the tissue layer. Reintegration of these displaced cells supports tissue growth and maintains tissue architecture [4]. Two conserved IgCAMs (immunoglobulin superfamily cell adhesion molecules), neuroglian (Nrg) and fasciclin 2 (Fas2), participate in cell reintegration in the Drosophila follicular epithelium [4]. Like their vertebrate orthologs L1CAM and NCAM1/2, respectively, Nrg and Fas2 are cell adhesion molecules primarily studied in the context of nervous system development [7-10]. Consistent with this, we identify another neural IgCAM, Fasciclin 3 (Fas3), as a reintegration factor. Nrg, Fas2, and Fas3 are components of the insect septate junction, the functional equivalent of the vertebrate tight junction, but proliferating follicle cells do not have mature septate junctions, and we find that the septate junction protein neurexin IV does not participate in reintegration [11, 12]. Here, we show that epithelial reintegration works in the same way as IgCAM-mediated axon growth and pathfinding; it relies not only on extracellular adhesion but also mechanical coupling between IgCAMs and the lateral spectrin-based membrane skeleton. Our work indicates that reintegration is mediated by a distinct epithelial adhesion assembly that is compositionally and functionally equivalent to junctions made between axons.

Drosophila R8 photoreceptor cell subtype specification requires hibris.

Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. We conducted a genetic screen to identify additional regulators of this process and identified a novel mutation in the hibris gene, which encodes an irre cell recognition module protein (IRM). These immunoglobulin super family cell adhesion molecules include human KIRREL and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptor cell diversity. We demonstrate that hibris is required within the retina, and that hibris over-expression is sufficient to disrupt normal photoreceptor cell patterning. These findings suggest an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells.

Neuronal immunoglobulin superfamily cell adhesion molecules in epithelial morphogenesis: insights from Drosophila.

In this review, we address the function of immunoglobulin superfamily cell adhesion molecules (IgCAMs) in epithelia. Work in the Drosophila model system in particular has revealed novel roles for calcium-independent adhesion molecules in the morphogenesis of epithelial tissues. We review the molecular composition of lateral junctions with a focus on their IgCAM components and reconsider the functional roles of epithelial lateral junctions. The epithelial IgCAMs discussed in this review have well-defined roles in the nervous system, particularly in the process of axon guidance, suggesting functional overlap and conservation in mechanism between that process and epithelial remodelling. We expand on the hypothesis that epithelial occluding junctions and synaptic junctions are compositionally equivalent and present a novel hypothesis that the mechanism of epithelial cell (re)integration and synaptic junction formation are shared. We highlight the importance of considering non-cadherin-based adhesion in our understanding of the mechanics of epithelial tissues and raise questions to direct future work. This article is part of the discussion meeting issue 'Contemporary morphogenesis'.

Abstract 1129: CADM1 is a novel serum marker for small cell lung cancer

Abstract Small cell lung cancer (SCLC) accounts for about 15% of lung cancer. Although SCLC often responds favorably to combined-modality chemotherapy at the initial treatment, resistant tumors develop rapidly, which makes SCLC one of the representative cancers refractory to any therapeutic approaches. Moreover, molecular targeting therapy has not been developed for SCLC so far. Therefore, novel approaches to the diagnosis and treatment of SCLC on the basis of molecular understanding would be prerequisite to control this refractory cancer. One of the most critical issues of SCLC is its early detection in the initial screening and after chemotherapy. For this purpose, pro-gastrin releasing peptide (ProGRP) and neuron specific enolase (NSE) are widely used for serum markers for detection of SCLC, although combination of ProGRP and NSE can detect at most 60% of SCLC. We have previously demonstrated that CADM1, a member of the immunoglobulin superfamily cell adhesion molecules, is highly expressed in around 75% of SCLC. In addition, SCLC expresses a splicing variant, CADM1v8/9, which is observed specifically in normal testis. Here, we report that the extracellular fragment of CADM1v8/9 is digested by ADM17 and released into cell medium or human serum. So, we generated specific monoclonal antibody against CADM1v8/9 using Cadm1-deficient mice and developed a serum diagnostic marker system for SCLC. Preliminary study shows that CADM1v8/9 detects 47% of SCLC with specificity of 92%, which is independent of and partly overlapped with the cases detected by ProGRP or NSE. CADM1v8/9 can detect 25% of patients with limited disease and 77% of patients with extensive disease of SCLC. Furthermore, the amount of CADM1v8/9 fragments well correlates with the disease activity of SCLC before and after chemotherapy. These findings indicate that detection of CADM1v8/9 in serum from patients is a novel and promising approach to detect and follow up SCLC patients. Since CADM1 is highly expressed in cell membrane of SCLC, CADM1 would also provide a promising target for the treatment of SCLC. Citation Format: Takeshi Ito, Toko Funaki, Goh Tanaka, Takahide Nagase, Yoshinori Murakami. CADM1 is a novel serum marker for small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1129.

B22 Development of a Novel Serum Marker for Detecting Small-Cell Lung Cancer by Targeting a Cell Adhesion Molecule 1 (CADM1)

Small-cell lung cancer (SCLC) accounts for about 15% of lung cancer. Although SCLC often responds favorably to combined-modality chemotherapy at the initial treatment, resistant tumors develop rapidly, which makes SCLC one of the representative cancers refractory to any therapeutic approaches. Moreover, molecular targeting therapy has not been developed for SCLC so far. Therefore, novel approaches to the diagnosis and treatment of SCLC on the basis of molecular understanding would be prerequisite to control this refractory cancer. One of the most critical issues of SCLC is its early detection in the initial screening and after chemotherapy. For this purpose, progastrin-releasing peptide (ProGRP) and neuron specific enolase (NSE) are widely used for serum markers for detection of SCLC, although combination of ProGRP and NSE can detect at most 60% of SCLC. We have previously demonstrated that CADM1, a member of the immunoglobulin superfamily cell adhesion molecules, is highly expressed in around 75% of SCLC. In addition, SCLC expresses a splicing variant, CADM1v8/9, which is observed specifically in normal testis. Here, we report that the extracellular fragment of CADM1v8/9 is digested by ADM17 and released into cell medium or human serum. Then, we generated specific monoclonal antibody against CADM1v8/9 using Cadm1-deficient mice and developed a serum diagnostic marker for SCLC. Preliminary study shows that CADM1v8/9 detects 47% of SCLC, which is independent of and partly overlaps with the cases detected by ProGRP. CADM1v8/9 can also detect a significant portion of patients with limited disease of SCLC. Furthermore, the amount of CADM1v8/9 fragments correlates well with the disease activity of SCLC before and after the chemotherapy. These findings indicate that detection of CADM1v8/9 in serum from patients is a novel and promising approach to detect and follow up SCLC patients. CADM1 would also provide a promising target for the treatment of SCLC.

Abstract 4509: Overexpression of CADM1 enhances malignant features of small-cell lung cancer

Abstract Small-cell lung cancer (SCLC) is one of the representative intractable cancers with the 5-year survival of less than 10 %. Novel approaches to the treatment of SCLC are required because recurrence arises inevitably after the initial chemotherapy. CADM1 was originally identified as a tumor suppressor of non-small cell lung cancer (NSCLC) which encodes a member of immunoglobulin superfamily cell adhesion molecules. CADM1 consists of an extracellular domain with three immunoglobulin-like loops, a trans-membrane domain and a short cytoplasmic domain containing a protein 4.1-binding motif (4.1BM) and a PDZ-II-binding motif. We have reported that CADM1 is highly expressed in 90% of human SCLC cell lines and promotes subcutaneous tumor formation of SCLC cells in nude mice. In this study, we performed clinicopathological evaluation of CADM1 in SCLC and analyzed the molecular mechanisms of malignant progression of SCLC by overexpressing CADM1. Firstly, we performed immunohistochemical analysis of CADM1 expression in high-grade neuroendocrine tumor (HGNEC) of the lung, including SCLC and large cell neuroendocrine carcinoma (LCNEC). We found that CADM1 protein was expressed in 75% (33/44) of SCLC tumors and correlated with the nodal involvement in lung HGENC. To understand pathological significance of CADM1 overexpression in SCLC, we introduced a full-length CADM1 and its mutants in the extracellular or intracellular domain into SCLC cells, SBC5, lacking endogenous CADM1 expression and assessed their malignant features in comparison with control SBC5 cells. CADM1 expression significantly enhanced colony formation in soft agar and subcutaneous tumor formation in nude mice of SBC5, whereas a mutant in 4.1BM did not. Then, we screened chemical library of 170 small molecules of known function to identify compounds which inhibit anchorage-independent growth of SCLC cells enhanced by CADM1. Inhibitors of PI3K, Akt and PKA significantly suppressed cell growth of SBC5, suggesting that CADM1 activates PI3K/Akt and PKA signaling pathways through its 4.1BM and promotes anchorage-independent growth of SCLC cells. We also found that orthotopic injection of NCI-N417 cells overexpressing CADM1 into the lung of nude mice significantly enhanced their metastasis to mediastinal lymph nodes. These results suggest that CADM1 enhances tumor growth and metastasis of SCLC cells probably by activating PI3K/Akt and PKA pathways through its binding proteins at 4.1BM. Citation Format: Toko Funaki, Takeshi Ito, Yoshinori Murakami. Overexpression of CADM1 enhances malignant features of small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4509.

Long-term endurance running activity causes pulmonary changes depending on the receptor for advanced glycation end-products

The receptor for advanced glycation end-products (RAGE) is an immunoglobulin superfamily cell adhesion molecule predominantly expressed in the lung, but its pulmonary importance is incompletely understood. Since RAGE alters the respiratory mechanics, which is also challenged by endurance running activity, we studied the RAGE-dependent effect of higher running activity on selected lung parameters in a long-term animal model using wild-type (WT) and RAGE knockout (RAGE-KO) mice. Higher long-term running activity of mice was ensured by providing a running wheel for 8months. Recording the running activity revealed that RAGE-KO mice are more active than WT mice. RAGE-KO caused an increased lung compliance which additionally increased after long-term running activity with minor limitation of the expiratory flow, whereas the respiratory mechanics of WT mice remained constant. Although RAGE-KO mice had a less dense alveolar-capillary barrier for immune cells, higher long-term running activity led only in WT mice to more leukocyte infiltrations in the lung tissue and aggregations of lymphoid cells in the airways. In this regard, WT mice of the activity group were also more sensitive to ventilation-mediated airway damages. In contrast to RAGE-KO mice of the activity group, lungs of WT mice did not show an increase in the cAMP response element-binding protein, a transcription factor regulating many pro-survival genes. Our findings suggest an important role of RAGE in the physical capability due to its effect on the lung compliance as well as RAGE as a mediator of airway damages caused by higher long-term running activity.

Microarray Analysis of Gene Expression Changes in Neuroplastin 65-Knockout Mice: Implications for Abnormal Cognition and Emotional Disorders

Neuroplastin 65 (Np65) is an immunoglobulin superfamily cell adhesion molecule involved in synaptic formation and plasticity. Our recent study showed that Np65-knockout (KO) mice exhibit abnormal cognition and emotional disorders. However, the underlying mechanisms remain unclear. In this study, we found 588 differentially-expressed genes in Np65-KO mice by microarray analysis. RT-PCR analysis also revealed the altered expression of genes associated with development and synaptic structure, such as Cdh1, Htr3a, and Kcnj9. In addition, the expression of Wnt-3, a Wnt protein involved in development, was decreased in Np65-KO mice as evidenced by western blotting. Surprisingly, MRI and DAPI staining showed a significant reduction in the lateral ventricular volume of Np65-KO mice. Together, these findings suggest that ablation of Np65 influences gene expression, which may contribute to abnormal brain development. These results provide clues to the mechanisms underlying the altered brain functions of Np65-deficient mice.

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

Glycosylphosphatidylinositol-Anchored Immunoglobulin Superfamily Cell Adhesion Molecules and Their Role in Neuronal Development and Synapse Regulation.

Immunoglobulin superfamily (IgSF) cell adhesion molecules (CAMs) are cell surface glycoproteins that not only mediate interactions between neurons but also between neurons and other cells in the nervous system. While typical IgSF CAMs are transmembrane molecules, this superfamily also includes CAMs, which do not possess transmembrane and intracellular domains and are instead attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In this review, we focus on the role GPI-anchored IgSF CAMs have as signal transducers and ligands in neurons, and discuss their functions in regulation of neuronal development, synapse formation, synaptic plasticity, learning, and behavior. We also review the links between GPI-anchored IgSF CAMs and brain disorders.

Functions and Dysfunctions of Neuroplastin65 in the Nervous System

The enormous number and complexity of connections in the nervous system depend on many families of adhesion proteins Cell adhesion molecules are extensively distributed among cells in the nervous system and their functions are indispensable for the formation of intercellular connections both during development and in the adult Neuroplastin NP a member of immunoglobulin superfamily cell adhesion molecules has been shown to be important for neuritogenesis synaptogenesis and the regulation of synaptic plasticity Recent advances suggest it may involve in the maintenance of excitatory inhibitory balance and dysfunctions of NP could probably affect several cognitive and emotional processes Here we present the functions of NP and provide potential evidences for dysfunctions of neruoplastin in neuropsychiatric disorders of the nervous system

Immunohistochemical Expression of MCAM/CD146 in Canine Melanoma

MCAM/CD146 (melanoma cell adhesion molecule/CD146) is a transmembrane immunoglobulin superfamily cell adhesion molecule involved in transendothelial migration and signal transduction. It is expressed in melanoma, squamous cell carcinoma, prostatic, ovarian, cervical and endometrial cancers and promotes tumour growth, angiogenesis and metastasis. Melanoma is the most common malignant oral tumour of dogs and also arises in the skin, nail bed and footpad. The aim of this study was to investigate the immunohistochemical expression of MCAM/CD146 in 51 canine melanomas, including oral, cutaneous and ocular tumours. Seventeen of the 51 (33.3%) cases were negative, eight (15.7%) were weakly positive, seven (13.7%) were moderately positive and 19 (37.3%) were strongly positive. MCAM/CD146 was expressed by both oral and cutaneous melanomas; however, the intensity and the extent of the immunoreactivity was higher in oral (P=0.009) than in cutaneous tumours (P=0.058). Most ocular melanomas did not express MCAM/CD146 (P=0.256). Expression of MCAM/CD146 by canine melanomas may suggest the molecule as a target for treatment, especially in oral melanomas.

Nectin spot: a novel type of nectin-mediated cell adhesion apparatus.

Nectins are Ca(2+)-independent immunoglobulin (Ig) superfamily cell adhesion molecules constituting a family with four members, all of which have three Ig-like loops at their extracellular regions. Nectins play roles in the formation of a variety of cell-cell adhesion apparatuses. There are at least three types of nectin-mediated cell adhesions: afadin- and cadherin-dependent, afadin-dependent and cadherin-independent, and afadin- and cadherin-independent. In addition, nectins trans-interact with nectin-like molecules (Necls) with three Ig-like loops and other Ig-like molecules with one to three Ig-like loops. Furthermore, nectins and Necls cis-interact with membrane receptors and integrins, some of which are associated with the nectin-mediated cell adhesions, and play roles in the regulation of many cellular functions, such as cell polarization, movement, proliferation, differentiation, and survival, co-operatively with these cell surface proteins. The nectin-mediated cell adhesions are implicated in a variety of diseases, including genetic disorders, neural disorders, and cancers. Of the three types of nectin-mediated cell adhesions, the afadin- and cadherin-dependent apparatus has been most extensively investigated, but the examples of the third type of apparatus independent of afadin and cadherin are recently increasing and its morphological and functional properties have been well characterized. We review here recent advances in research on this type of nectin-mediated cell adhesion apparatus, which is named nectin spot.

The role of ICAM-2 in neuroblastoma

The role of immunoglobulin superfamily cell adhesion molecules (CAMs) in facilitating immune responses in normal and tumor cells is well established. Cell adhesion molecule-1 (CADM1), for example, suppresses development of mouse mammary tumor cell metastasis by interacting with CD8+ T cells in immune-competent hosts [1]. Similarly, co-expression of Intercellular Adhesion Molecule-2 (ICAM-2) and chemokine C-X-C motif ligand 17 (CXCL17) elicits anti-tumor immune responses and suppresses tumor growth [2]. Although controversial, current literature suggests that proteins of the immunoglobulin superfamily CAMs also have functions that may well be distinct from their roles in mediating immune responses. Junctional adhesion molecule-A (JAM-A), for example, negatively regulates breast cancer cell invasiveness by disrupting tight junctions [3]; and a member of the B7 family of the immunoglobulin superfamily proteins, B7-H3, impairs osteogenic differentiation in vitro and in vivo [4]. Our lab demonstrated that ICAM-2 inhibited the development of disseminated neuroblastoma tumors in a preclinical model of metastatic neuroblastoma [5-7]. This inhibition depended on the interaction of ICAM-2 with the actin cytoskeletal linker protein α-actinin, an interaction that inhibited cell motility [7]. Ectopic expression of ICAM-2 did not affect the tumorigenic potential of neuroblastoma cells [7]. Importantly, immunohistochemical analyses of primary neuroblastoma tumor specimens demonstrated that neuroblastoma cells expressing ICAM-2 are phenotypically and histologically those recognized clinically to have limited metastatic potential [5]. Since metastatic disease is responsible for >90% of cancer-related deaths for multiple types of solid tumors, we suggest that elucidation of the molecular mechanism by which ICAM-2 suppresses the metastatic potential of neuroblastoma cells would identify proteins or pathways that might be exploited therapeutically to prevent metastatic disease progression. In normal tissues ICAM-2 is expressed predominantly by neovascular endothelial cells, and at lower levels by established vasculature and some leukocytes. The 202 amino acids comprising its extracellular domain mediate binding of ICAM-2 on endothelial cells to β2-integrins on the surface of leukocytes, to facilitate migration of neutrophils through the vascular endothelium as a component of immune reactions [8]. In neuroblastoma cells ICAM-2 inhibits cell motility independent of immune response, as we observed this inhibition in wound healing and modified Boyden chamber assays in vitro, assays that clearly lack an immune component [5,7]. In silico modeling indicated that ICAM-2 with mutations in the proposed α-actinin binding domain had a more ‘closed’ configuration than the wild type protein, and predicted that these ICAM-2 mutants would not interact with α-actinin [7]. Co-immunoprecipitation experiments confirmed in silico predictions [7]. In support of these findings, the interaction of ICAM-2 with α-actinin was essential to inhibit development of disseminated tumors in vivo [7]. As mentioned, ICAM-2 did not affect tumorigenic potential, and unpublished data showed no effect of ICAM-2 on expression of epithelial-mesenchymal transition (EMT) or stemness markers. Although we regard the inhibition of tumor cell motility by ICAM-2 in vitro to be compelling evidence that ICAM-2 participates in cell functions distinct from immune responses, we acknowledge the complex nature of metastatic tumor progression and propose that our data suggest multiple hypotheses with respect to the mechanism by which ICAM-2-inhibits neuroblastoma cell motility. The first hypothesis is that the intracellular interaction of ICAM-2 with α-actinin initiates ‘inside out’ signaling, and causes conformational changes in the extracellular domain of ICAM-2 that facilitate (or inhibit) interactions with specific extracellular matrix proteins that play an integral role in cell motility. The second is that the interaction of ICAM-2 with α-actinin alters the conformation of α-actinin to facilitate (or inhibit) the association of this actin cytoskeletal protein with alternative binding partners, with ICAM-2 acting as an ‘activator’ (or inhibitor) of α-actinin rather than as a membrane anchor protein. The third hypothesis, based on unpublished microarray data, is that ICAM-2 expression indirectly up-regulates protein tyrosine phosphatases (PTPs) involved in the formation and maintenance of focal adhesions such as focal adhesion kinase (FAK), Src, or Rac and Rho GTPases, each of which has crucial role in tumor cell motility. Our data show that ICAM-2 inhibits tumor cell motility and suppresses the metastatic potential of neuroblastoma cells. We propose that elucidating molecular events associated with ICAM-2 expression will identify key protein interactions that regulate the metastatic process in this cell type.