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Cell adhesion molecule 1 (CADM1), a single-pass transmembrane protein, is involved in oncogenesis. We previously demonstrated the therapeutic efficacy of anti-CADM1 ectodomain monoclonal antibodies against mesothelioma; however, the underlying mechanism is unclear. In the present study, we explored the molecular behavior of anti-CADM1 antibodies in CADM1-expressing tumor cells. Sequencing analyses revealed that the anti-CADM1 chicken monoclonal antibodies 3E1 and 9D2 are IgY and IgM isotype antibodies, respectively. Co-administration of 3E1 and 9D2 altered the subcellular distribution of CADM1 from the detergent-soluble fraction to the detergent-resistant fraction in tumor cells. Using recombinant chicken-mouse chimeric antibodies that had been isotype-switched from IgG to IgM, we demonstrated that the combination of the variable region of 3E1 and the constant region of IgM was required for CADM1 relocation. Cytochemical studies showed that 3E1 colocalized with late endosomes/lysosomes after co-administration with 9D2, suggesting that the CADM1-antibody complex is internalized from the cell surface to intracellular compartments by lipid-raft mediated endocytosis. Finally, 3E1 was conjugated with the antimitotic agent monomethyl auristatin E (MMAE) via a cathepsin-cleavable linker. Co-administration of 3E1-monomethyl auristatin E and 9D2 suppressed the growth of multiple types of tumor cells, and this anti-tumor activity was confirmed in a syngeneic mouse model of melanoma. 3E1 and 9D2 are promising drug delivery vehicles for CADM1-expressing tumor cells., Competing Interests: Declaration of competing interest This study was partially funded by Pharma Foods International Corporation., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), is a heterogeneous disease with respect to clinicopathological features. Cell adhesion molecule 1 (CADM1) has been reported to be ectopically expressed in adult T cell leukaemia/lymphoma (ATLL). However, the frequency of CADM1 expression remains unknown in peripheral T cell lymphomas. In the current study, CADM1 expression was analysed in 88 PTCL-NOS patients. CADM1 was expressed in 14 of 88 (15.9%) PTCL-NOS cases, and its expression was associated with C-C chemokine receptor type 4 (CCR4) expression and nuclear atypia. CADM1-positive PTCL-NOS cases (10/74) had a significantly poorer prognosis than CADM1-negative cases (64/74) (P = 0.001). Multivariate analysis confirmed that CADM1 expression was an independent prognostic factor in PTCL-NOS. These findings suggest that CADM1 expression is a novel prognostic factor for PTCL-NOS.

Cancer stem cells (CSCs), which are highly differentiated and self-renewing, play an important role in the occurrence, therapeutic resistant and metastasis of hepatacellular carcinoma (HCC). Oncolytic adenoviruses have targeted killing effect on tumor cells, and are invoked as candidate drugs for cancer treatment. We designed a dual-regulated oncolytic adenovirus Ad.wnt-E1A(△24bp)-TSLC1 that targets Wnt and Rb signaling pathways respectively, and carries the tumor suppressor gene, TSLC1. Previous studies have demonstrated that oncolytic adenovirus mediated TSLC1can target liver cancer and exhibit significant cytotoxicity. However, whether Ad.wnt-E1A(△24bp)-TSLC1 can effectively eliminate liver CSCs remains to be explored. We first used the spheroid culture to enrich the liver CSCs-like cells, and detected the self-renewal capacity, differentiation, drug resistance and tumorigenicity. The results showed that Ad-wnt-E1A(△24bp)-TSLC1 could effectively lead to autophagic death. In addition, recombinant adenovirus effectively induced the apoptosis, inhibit metastasis of hepatic CSCs-like cells in vivo. Further animal experiments indicated that Ad-wnt-E1A(△24bp)-TSLC1could effectively inhibit the growth of transplanted tumor of hepatic CSCs and prolong the survival time of mice. Therefore, the novel oncolytic adenovirus Ad.wnt-E1A(△24bp)-TSLC1 has potential application as a therapeutic target for HCC stem cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

Solid tumors are increasingly recognized as a systemic disease that is manifested by changes in DNA, RNA, proteins and metabolites in the blood. Whereas many studies have reported gene mutation events in the circulation, few studies have focused on epigenetic DNA methylation markers. To identify DNA methylation biomarkers in peripheral blood for ovarian cancer, we performed a two-stage epigenome-wide association study. In the discovery stage, we measured genome wide DNA methylation for 485 000 CpG sites in peripheral blood in 24 epithelial ovarian cancer (EOC) cases and 24 age-matched healthy controls. We selected 96 significantly differentially methylated CpG sites for validation using Illumina's Custom VeraCode methylation assay in 206 EOC cases and 205 controls and 46 CpG sites validated in the independent replication samples. A set of 6 of these 46 CpG sites was found by the receiver operating characteristic analysis to have a prediction accuracy of 77.3% for all EOC (95% confidence interval: 72.9-81.8%). Pathway analysis of the genes associated with the 46 CpG sites revealed an enrichment of immune system process genes, including LYST (cg16962115, FDR = 1.24E-04), CADM1 (cg21933078, FDR = 1.22E-02) and NFATC1 (cg06784563, FDR = 1.46E-02). Furthermore, DNA methylation status in peripheral blood was correlated with platelet parameters/coagulation factor levels. This study discovered a panel of epigenetic liquid biopsy markers closely associated with overall immunologic conditions and platelet parameters/coagulation systems of the patients for detection of all stages and subtypes of EOC., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cell adhesion molecule 1 (CADM1) is a cell adhesion molecule that is expressed in brain, liver, lung, testis, and some kinds of cancer cells including adult T-cell leukemia/lymphoma (ATLL). Recent studies have indicated the involvement of CADM1 in cell-cell contact between cytotoxic T-lymphocytes and virus infected cells. We previously reported that cell-cell interaction between lymphoma cells and macrophages induces lymphoma cell proliferation. In the present study, we investigated whether CADM1 is associated with cell-cell interaction between several human lymphoma cell lines and macrophages.CADM1 expression was observed in the ATLL cell lines, ATN-1, ATL-T, and ATL-35T, and in the B cell lymphoma cell lines, TL-1, DAUDI, and SLVL, using western blotting. Significant cell-cell interaction between macrophages and ATN-1, ATL-T, ATL-35T and MT-2, DAUDI, and SLVL cells, as assessed by induction of cell proliferation, was observed. Immunohistochemical analysis of human biopsy samples indicated CADM1 expression in 10 of 14 ATLL cases; however, no case of follicular lymphoma or diffuse large B-cell lymphoma was positive for CADM1. Finally, the interaction of macrophages with cells of the CADM1-negative ED ATLL cell line and CADM1-transfected ED cells was tested. However, significant cell-cell interaction between macrophage and CADM1-transfected ED cells was not observed. We conclude that CADM1 was not associated with cell-cell interaction between lymphoma cells and macrophages, although CADM1 may be a useful marker of ATLL for diagnostic procedures.

Epithelial carcinomas occasionally have sarcomatous components that consist primarily of spindle and cuboidal cells, which often resemble osteoblasts. Sarcomatoid carcinomas consist of similar cells. Recent studies have characterized these phenomena as a manifestation of epithelial-mesenchymal transition in carcinoma cells, but the mesenchymal phenotypes that manifest in sarcomatous cells of epithelial carcinomas are not well understood. Here, we examined the expression profiles of four osteoblastic differentiation biomarkers in the sarcomatous components of multiple carcinoma types, including five renal clear cell, four breast invasive ductal, two esophageal, one maxillary squamous cell, three larynx, three lung, one liver, and one skin sarcomatoid carcinoma. Expression was analyzed by immunohistochemistry using antibodies against cell adhesion molecule 1, a member of the IgCAM superfamily, osterix transcription factor (Osterix), cluster of differentiation 151, a transmembrane 4 superfamily member, and alkaline phosphatase. Immunostaining intensity was rated in scale 0 (negative), 0.5 (weak), and 1 (strong) for each marker, and the four scale values were summed to calculate osteoblastic scores. In all, 10 cases had a osteoblastic score ≥3, and all of these 10 cases were cell adhesion molecule 1- and Osterix-positive. Eight and five of the nine samples with a osteoblastic score <3 were negative for cell adhesion molecule 1 ( p < 0.0001) and Osterix ( p = 0.006), respectively. The other markers showed no statistical significance. These results indicate that osteoblastic differentiation can occur in carcinoma cells and that cell adhesion molecule 1 could be a useful marker for identifying this phenomenon in carcinoma tissues.

The mRNA expression of cell adhesion molecule 1 (CADM1) and its clinicopathological significance in esophageal squamous cell carcinoma (ESCC) tissues were investigated. CADM1 mRNA and protein expression were detected in tissue samples from 50 patients with ESCC by reverse transcription-polymerase chain reaction (RT-PCR) and streptavidin-peroxidase (SP) immunohistochemistry; adjacent tissues served as controls. The average CADM1 mRNA expression was significantly downregulated in the cancer tissues (0.522 ± 0.247) than in the controls (0.871 ± 0.192), (t = 7.882, P < 0.05). CADM1 mRNA expression was significantly downregulated in ESCC patients with positive lymph node metastasis than in those with negative lymph node metastasis (t = 3.207, P < 0.05). There was a correlation between CADM1 mRNA expression and tumor-node-metastasis (TNM) stage (t = 2.673, P < 0.050), but not with age, gender, and histological grade (P > 0.05). The positive expression rate of CADM1 protein in the 50 cases of ESCC was significantly lower than that of the control group (χ 2 = 29.87, P < 0.01). Out of 28 patients with non-lymph node metastasis, 20 (71.43%) positively expressed CADM1; out of 22 patients with lymph node metastasis, only 7 (31.82%) positively expressed CADM1. There was a significant difference in the positive protein expression rates of CADM1 between the two groups (χ 2 = 7.782, P < 0.01). CADM1 mRNA expression was highly upregulated in normal tissues compared to ESCC tissues, indicating that the loss of CADM1 expression influenced the pathogenesis, invasion, and metastasis of ESCC, and allowing for the prognosis of the disease in patients with ESCC after treatment.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.

Background: Cardiac fibrosis play a key role in the atrial fibrillation pathogenesis but the underlying potential molecular mechanism is still understood. However, potential mechanisms for miR-21 upregulation and its role in cardiac fibrosis remain unclear. The controls cell proliferation and processes fundamental to disease progression., Methods: In this study, immunohistochemistry, real-time RT-PCR, cell transfection, cell cycle, cell proliferation and Western blot were used, respectively., Results: Here we have been demonstrated that the tumor suppressor cell adhesion molecule 1 (CADM1) is the potential target of miR-21. Our study revealed that miR-21 regulation of CADM1 expression, which was decreased in cardiac fibroblasts and fibrosis tissue. The cardiac fibroblasts transfected with miR-21 mimic promoted miR-21 overexpression enhanced STAT3 expression and decreased CADM1 expression. Nevertheless, the cardiac fibroblasts transfected with miR-21 inhibitor obtained the opposite expression result. Furthermore, downexpression of miR-21 suppressed cardiac fibroblast proliferation., Conclusions: These results suggested that miR-21 overexpression promotes cardiac fibrosis via STAT3 signaling pathway by decrease CADM1 expression, indicating miR-21 as an important signaling molecule for cardiac fibrotic remodeling and AF.

Chemo-resistance is still a major obstacle in successful cancer treatment. Previously, we found that miR-21 (miR-21-5p) was upregulated in drug-resistant tongue cancer (TC) cell line Tca8113/PYM. However, the mechanisms for miR-21 upregulation and its role in chemo-resistance in TC remain unclear. Here, we demonstrated that functional inhibition of miR-21 sensitized TC cells to chemotherapy. In agreement, overexpressed miR-21 enhanced chemo-resistance in TC cells. We found that miR-21 directly targeted CADM1 expression, which was downregulated in drug-resistant TC cells. Restored CADM1 expression sensitized TC cells to chemotherapy, but CADM1 knockdown induced chemo-resistance. Mechanically, CADM1 interacted with BMI1 to inhibit its nuclear translocation. Moreover, MYCN which was overexpressed in drug-resistant TC cells directly bound to the miR-21 promoter to upregulated miR-21 expression in TC cells. Importantly, the expression levels of miR-21 and CADM1 negatively correlated, but MYCN and miR-21 positively correlated in TC tissues. High levels of miR-21 and MYCN and low level of CADM1 were associated with poor prognosis in TC patients. In conclusion, our study suggests an important role of the MYCN/miR-21/CADM1 axis in chemo-resistance in TC patients and may lead to promising prognostic biomarkers and novel treatment strategies to improve the chemotherapeutic efficacy for TC patients., Key Messages: MiR-21 enhances chemo-resistance via targeting CADM1 in tongue cancer cells. CADM1 sensitizes tongue cancer cells to chemotherapy. CADM1 interacts with BMI1 to inhibit its nuclear translocation. MYCN transcriptionally regulates miR-21 expression. Dysregulated MYCN/miR-21/CADM1 axis associates with poor prognosis in TC patients.

Introduction: Recent studies have shown that CADM1/MAL-methylation testing detects high-grade CIN lesions with a high short-term progression risk for cervical cancer. Women treated for CIN2/3 are at risk of post-treatment disease, representing either persistent (incompletely treated) or incident (early onset) lesions. Here, we evaluated CADM1/MAL-methylation analysis as potential tool for detecting recurrent high-grade CIN lesions (rCIN2/3)., Methods and Materials: A multicenter prospective clinical cohort study was conducted among 364 women treated for CIN2/3. Cervical scrapes were taken prior to treatment, and six and 12months post-treatment and tested for cytology, hrHPV (plus genotype) and CADM1/MAL-methylation. When at six months either of these tests was positive, a colposcopy-directed biopsy was obtained. At 12months, all women underwent an exit-colposcopy with biopsy. In case of rCIN2/3, re-treatment was done., Results: We found 28 rCIN2 (7.7%) and 14 rCIN3 (3.8%), resulting in a total recurrence rate of 11.5%. All 14 women with rCIN3 and 15/28 (54%) with rCIN2 showed hrHPV type-persistence. Of these, 9/14 (64%) rCIN3 and 8/15 (53%) rCIN2 were CADM1/MAL-methylation positive. All incident rCIN2, characterized by hrHPV genotype-switch, were CADM1/MAL-methylation negative. All three carcinomas found after re-treatment were CADM1/MAL-methylation positive. CADM1/MAL-methylation positivity at both baseline and follow-up significantly increased the risk of ≥rCIN3 (from 0.7% to 18.4%), and ≥rCIN2 (from 8.2% to 36.8%), compared to a consistently CADM1/MAL-methylation negative result (p-value: <0.001)., Conclusion: Post-treatment monitoring by CADM1/MAL-methylation analysis identifies women with an increased risk of rCIN2/3. Our results confirm previous data indicating that CADM1/MAL-methylation analysis provides a high reassurance against cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

Unlabelled: Select adhesion proteins control the development of synapses and modulate their structural and functional properties. Despite these important roles, the extent to which different synapse-organizing mechanisms act across brain regions to establish connectivity and regulate network properties is incompletely understood. Further, their functional roles in different neuronal populations remain to be defined. Here, we applied diffusion tensor imaging (DTI), a modality of magnetic resonance imaging (MRI), to map connectivity changes in knock-out (KO) mice lacking the synaptogenic cell adhesion protein SynCAM 1. This identified reduced fractional anisotropy in the hippocampal CA3 area in absence of SynCAM 1. In agreement, mossy fiber refinement in CA3 was impaired in SynCAM 1 KO mice. Mossy fibers make excitatory inputs onto postsynaptic specializations of CA3 pyramidal neurons termed thorny excrescences and these structures were smaller in the absence of SynCAM 1. However, the most prevalent targets of mossy fibers are GABAergic interneurons and SynCAM 1 loss unexpectedly reduced the number of excitatory terminals onto parvalbumin (PV)-positive interneurons in CA3. SynCAM 1 KO mice additionally exhibited lower postsynaptic GluA1 expression in these PV-positive interneurons. These synaptic imbalances in SynCAM 1 KO mice resulted in CA3 disinhibition, in agreement with reduced feedforward inhibition in this network in the absence of SynCAM 1-dependent excitatory drive onto interneurons. In turn, mice lacking SynCAM 1 were impaired in memory tasks involving CA3. Our results support that SynCAM 1 modulates excitatory mossy fiber inputs onto both interneurons and principal neurons in the hippocampal CA3 area to balance network excitability., Significance Statement: This study advances our understanding of synapse-organizing mechanisms on two levels. First, the data support that synaptogenic proteins guide connectivity and can function in distinct brain regions even if they are expressed broadly. Second, the results demonstrate that a synaptogenic process that controls excitatory inputs to both pyramidal neurons and interneurons can balance excitation and inhibition. Specifically, the study reveals that hippocampal CA3 connectivity is modulated by the synapse-organizing adhesion protein SynCAM 1 and identifies a novel, SynCAM 1-dependent mechanism that controls excitatory inputs onto parvalbumin-positive interneurons. This enables SynCAM 1 to regulate feedforward inhibition and set network excitability. Further, we show that diffusion tensor imaging is sensitive to these cellular refinements affecting neuronal connectivity., (Copyright © 2016 the authors 0270-6474/16/367465-12$15.00/0.)

Contact between β-cells is necessary for their normal function. Identification of the proteins mediating the effects of β-cell-to-β-cell contact is a necessary step toward gaining a full understanding of the determinants of β-cell function and insulin secretion. The secretory machinery of the β-cells is nearly identical to that of central nervous system (CNS) synapses, and we hypothesize that the transcellular protein interactions that drive maturation of the two secretory machineries upon contact of one cell (or neural process) with another are also highly similar. Two such transcellular interactions, important for both synaptic and β-cell function, have been identified: EphA/ephrin-A and neuroligin/neurexin. Here, we tested the role of another synaptic cleft protein, CADM1, in insulinoma cells and in rat and human islet β-cells. We found that CADM1 is a predominant CADM isoform in β-cells. In INS-1 cells and primary β-cells, CADM1 constrains insulin secretion, and its expression decreases after prolonged glucose stimulation. Using a coculture model, we found that CADM1 also influences insulin secretion in a transcellular manner. We asked whether extracellular CADM1 interactions exert their influence via the same mechanisms by which they influence neurotransmitter exocytosis. Our results suggest that, as in the CNS, CADM1 interactions drive exocytic site assembly and promote actin network formation. These results support the broader hypothesis that the effects of cell-cell contact on β-cell maturation and function are mediated by the same extracellular protein interactions that drive the formation of the presynaptic exocytic machinery. These interactions may be therapeutic targets for reversing β-cell dysfunction in diabetes., (Copyright © 2016 the American Physiological Society.)

Background: Increasing evidence indicates that downregulation of cell adhesion molecule 1 (CADM1) contributes to tumorigenesis in various cancers. The present study was undertaken to investigate the CADM1 expression pattern in human hepatocellular carcinoma (HCC), and to elucidate the mechanism underlying CADM1-mediated tumor suppression., Methods: CADM1 expression in HCC cell lines was measured by quantitative real-time PCR. The function of CADM1 in the context of tumor suppression in HCC cells was determined using proliferation assays, cell cycle analysis, EdU incorporation assays, in vitro colony formation analysis, and in vivo tumorigenicity assays. The mechanism by which CADM1 acts as a tumor suppressor gene in HCC was investigated using Western blotting analysis., Results: Downregulation of CADM1 expression is frequently detected in both HCC cells and clinical samples. Restoration of CADM1 expression in HCC cell lines significantly inhibits cell growth and negatively regulates the G1/S transition. CADM1 overexpression can inhibit the tumorigenicity of HCC cells both in vitro and in vivo. Western blotting analysis revealed that ectopic expression of CADM1 in HCC cells is associated with increased expression of Retinoblastoma (Rb) protein., Conclusions: Our results showed that suppression of tumorigenesis by CADM1 may be mediated by the Rb-E2F pathway, involving upregulation of Rb protein levels. This pathway could therefore represent an attractive target for HCC therapy.

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax-at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax-HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax-CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1- cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax-CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus.

Formation and stability of synapses are required for proper brain function. While it is well established that synaptic adhesion molecules are important regulators of synapse formation, their specific role during different phases of synapse development remains unclear. To investigate the function of the synaptic cell adhesion molecule SynCAM 1 in the formation, stability, and maintenance of spines we used 2-photon in vivo imaging to follow individual spines over a long period of time. In SynCAM 1 knockout mice the survival rate of existing spines was reduced and fewer filopodia-like structures were converted into stable spines. SynCAM 1(flag) overexpression resulted in more stable spines and fewer filopodia-like structures. When SynCAM 1(flag) overexpression is turned on the spine density rapidly increases within a few days. Interestingly, the spine density stayed at an elevated level when SynCAM 1(flag) overexpression was turned off. Our data indicate that the SynCAM 1 induced altered spine density is not caused by the formation of newly emerging protrusions, instead SynCAM 1 stabilizes nascent synaptic contacts which promotes their maturation. Concomitant with the synaptic stabilization, SynCAM 1 generally prolongs the lifetime of spines. In summary, we demonstrate that SynCAM 1 is a key regulator of spine stability.

Background: The immune response plays a significant role in pericarditis, but the mechanisms of disease are poorly defined. Further, efficient monitoring and predictive clinical tools are unavailable. Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is an immune-inhibitory protein, while MHC class I chain related protein A (MICA) and B (MICB) have an immune-stimulating function., Methods and Results: Serum CEACAM1, MICA and MICB concentrations were measured by ELISA in ~50 subjects of each group: acute pericarditis (AP), recurrent pericarditis (RP) and lupus (SLE) patients, metastatic melanoma patients as well as healthy donors. Serum CEACAM1 was dramatically elevated in AP and RP patients, but not in SLE patients, and displayed a highly accurate profile in ROC curve analyses. MICA and MICB were elevated in some pericarditis patients. All markers were enhanced in metastatic melanoma patients irrespective of neoplastic pericardial involvement. Etiology-guided analysis of RP patients showed that very low MICA levels were associated with idiopathic RP, while high MICA was associated with autoimmune and post-operative RP. Importantly, MICA was significantly associated with recurrences, independently of other potentially confounding parameters such as age, time of follow up or treatment modality., Conclusions: Here we report for the first time on CEACAM1 as a potentially novel biomarker for pericarditis, as well as on MICA as an innovative prognostic marker in these patients. Determination of the roles of these immune factors, as well as their diagnostic and prognostic values should be determined in future prospective studies.

Although squamous cell carcinomas (SqCCs) of the lungs, head and neck, oesophagus, and cervix account for up to 30% of cancer deaths, the mechanisms that regulate disease progression remain incompletely understood. Here, we use gene transduction and human tumor xenograft assays to establish that the tumour suppressor Cell adhesion molecule 1 (CADM1) inhibits SqCC proliferation and invasion, processes fundamental to disease progression. We determine that the extracellular domain of CADM1 mediates these effects by forming a complex with HER2 and integrin α6β4 at the cell surface that disrupts downstream STAT3 activity. We subsequently show that treating CADM1 null tumours with the JAK/STAT inhibitor ruxolitinib mimics CADM1 gene restoration in preventing SqCC growth and metastases. Overall, this study identifies a novel mechanism by which CADM1 prevents SqCC progression and suggests that screening tumours for loss of CADM1 expression will help identify those patients most likely to benefit from JAK/STAT targeted chemotherapies.

The survival of adult-born dentate gyrus granule cells critically depends on their synaptic integration into the existing neuronal network. Excitatory inputs are thought to increase the survival rate of adult born neurons. Therefore, whether enhancing the stability of newly formed excitatory synapses by overexpressing the synaptic cell adhesion molecule SynCAM 1 improves the survival of adult-born neurons was tested. Here it is shown that overexpression of SynCAM 1 improves survival of adult-born neurons, but has no effect on the proliferation rate of precursor cells. As expected, overexpression of SynCAM 1 increased the synapse density in adult-born granule neurons. While adult-born granule neurons have very few functional synapses 15 days after birth, it was found that at this age adult-born neurons in SynCAM 1 overexpressing mice exhibited around three times more excitatory synapses, which were stronger than synapses of adult-born neurons of control littermates. In summary, the data indicated that additional SynCAM 1 accelerated synapse maturation, which improved the stability of newly formed synapses and in turn increased the likelihood of survival of adult-born neurons., (© 2015 Wiley Periodicals, Inc.)

Constitutional 11q interstitial deletion syndrome presents with congenital anomalies including microcephaly with craniostenosis, minor dysmorphic features, vitreoretinopathy, and renal anomalies. This syndrome is occasionally associated with neuroblastoma (NB) as a life-threatening complication, which is important for clinical care. Although the corresponding locus to NB has been predicted to exist in 11q22-23 by previous deletion studies related to NB, the causative haploinsufficient genes have not yet been identified. We herein reported for the first time the simultaneous coexistence of adrenal NB and abdominal prevertebral ganglioneuroma in a 6-year-old girl with a constitutional hemizygous 11q14.1-23.3 deletion. Of the 11 haploinsufficient genes predicted with an in silico database, we focused on NCAM1 and CADM1 as the genes accountable for NB and ganglioneuroma. The deletion range, especially the 11q22.3 involvement, needs to be determined in 11q deletion cases in order to predict susceptibility to peripheral nerve tumors involving NB and ganglioneuroma., (© 2015 Wiley Periodicals, Inc.)

Recent studies have shown that CADM1/MAL methylation levels in cervical scrapes increase with severity and duration of the underlying cervical intraepithelial neoplasia (CIN) lesion. Multiple lesions of different histological grades and duration are frequently present on the cervix. To gain more insight into the possible epigenetic heterogeneity and its consequences for the methylation status in cervical scrapes, we performed an exploratory study of CADM1/MAL methylation in different grades of CIN lesions present in women with multiple cervical biopsies. CADM1-M18 and MAL-M1 methylation was assessed using a standardised, multiplex, quantitative methylation specific PCR on 178 biopsies with various grades of CIN in 65 women, and in their corresponding cervical scrapes. CADM1/MAL methylation positivity increased with disease severity, from 5.5% in normal biopsies to 63.3% and 100% in biopsies with CIN3 and cervical cancer, respectively. In the majority (8/9) of women where besides a CIN2/3 lesion a biopsy from normal cervical tissue was present, the CIN2/3 biopsy was CADM1/MAL methylation positive and the normal biopsy was CADM1/MAL methylation negative. A good concordance (78%) was found between CADM1/MAL methylation results on the scrapes and the biopsy with the worst diagnosis, particularly between samples of women with CIN3 and cervical cancer (92% and 100% concordance, respectively). Thus, in women with multiple cervical biopsies, CADM1/MAL methylation increases with severity of the lesion and is lesion-specific. CADM1/MAL methylation status in cervical scrapes appears to be representative of the worst underlying lesion, particularly for CIN3 and cervical cancer., (© 2015 UICC.)

Objectives: Loss of Tumor Suppressor in Lung Cancer 1(TSLC1) was observed in many different cancers, but there were only limited research on TSLC1 gene and its roles in cancer suppression in ovarian cancer. This study explores the relationship between TSLC1 gene expression and the ovarian epithelial cancer., Materials and Methods: Expression levels of TSLC1 were detected by immunohistochemical staining on formal-fixed paraffin embedded pathological specimen. A total of 259 samples were collected, including 24 benign ovarian tumor and 235 malignant ovarian cancers among them., Results: Suppressed expression of TSLC1 protein was observed in 87.8% of poorly differentiated, 85.1% of moderately differentiated, and 46% of well differentiated ovarian epithelial cancers, respectively. There were none suppressed TSLC1 expression in benign ovarian tumor. Kruskal-Wallis test showed a significant association between the expression levels of TSLC1 gene and the degree of ovarian cancer differentiation (P < 0.001)., Conclusions: Decreased expression of TSLC1 is associated with the differentiation in ovarian epithelial cancer. TSLC1 might be used as a molecular marker of severity in early stage ovarian cancer, and to help differentiating benign and malignant ovarian tumors.

Objective: To investigate the relationship between the expression of tumor suppressor in lung cancer-1 (TSLC1) and clinicopathological characteristics of patients with non-muscle-invasive bladder cancer (NMIBC) and evaluate the prognostic significance of TSLC1., Methods: TSLC1 expression in 241 specimens of NMIBC was examined by immunohistochemistry. The correlation between TSLC1 expression and clinicopathological characteristics was evaluated using the chi-square test. The prognostic significance of TSLC1 was analyzed by univariate, multivariate analysis and Kaplan-Meier survival curves., Results: The total negative rate of TSLC1 expression was 47.3% in NMIBC. Decreased expression of TSLC1 was correlated with a higher clinical stage, higher pathological grade, the number of tumors, lager tumor size, tumor recurrence and progression. TSLC1 expression was an independent risk factor for predicting tumor recurrence (p = 0.005) and progression (p < 0.001)., Conclusion: Decreased expression of TSLC1 is correlated with the malignancy of NMIBC tissues and low TSLC1 expression may serve as a predictor for bladder cancer recurrence and progression., (© 2015 S. Karger AG, Basel.)

The cleft is an integral part of synapses, yet its macromolecular organization remains unclear. We show here that the cleft of excitatory synapses exhibits a distinct density profile as measured by cryoelectron tomography (cryo-ET). Aiming for molecular insights, we analyzed the synapse-organizing proteins Synaptic Cell Adhesion Molecule 1 (SynCAM 1) and EphB2. Cryo-ET of SynCAM 1 knockout and overexpressor synapses showed that this immunoglobulin protein shapes the cleft's edge. SynCAM 1 delineates the postsynaptic perimeter as determined by immunoelectron microscopy and super-resolution imaging. In contrast, the EphB2 receptor tyrosine kinase is enriched deeper within the postsynaptic area. Unexpectedly, SynCAM 1 can form ensembles proximal to postsynaptic densities, and synapses containing these ensembles were larger. Postsynaptic SynCAM 1 surface puncta were not static but became enlarged after a long-term depression paradigm. These results support that the synaptic cleft is organized on a nanoscale into sub-compartments marked by distinct trans-synaptic complexes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Even though a large proportion of patients with acute myeloid leukemia (AML) achieve a complete remission upon initial therapy, the majority of them eventually relapse with resistant disease. Overexpression of the gene coding for the transcription factor Ecotropic Virus Integration site 1 (EVI1) is associated with rapid disease recurrence and shortened survival. We therefore sought to identify EVI1 target genes that may play a role in chemotherapy resistance using a previously established in vitro model system for EVI1 positive myeloid malignancies. Gene expression microarray analyses uncovered the Cell Adhesion Molecule 1 (CADM1) gene as a candidate whose deregulation by EVI1 may contribute to drug refractoriness. CADM1 is an apoptosis inducing tumor suppressor gene that is inactivated by methylation in a variety of tumor types. In the present study we provide evidence that it may play a role in chemotherapy induced cell death in AML: CADM1 was induced by drugs used in the treatment of AML in a human myeloid cell line and in primary diagnostic AML samples, and its experimental expression in a cell line model increased the proportion of apoptotic cells. CADM1 up-regulation was abolished by ectopic expression of EVI1, and EVI1 expression correlated with increased CADM1 promoter methylation both in a cell line model and in primary AML cells. Finally, CADM1 induction was repressed in primary samples from AML patients at relapse. In summary, these data suggest that failure to up-regulate CADM1 in response to chemotherapeutic drugs may contribute to therapy resistance in AML., (© 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.)

Objectives: To evaluate the value of cervical cell methylation markers in screening HIV-infected women also positive for high-risk human papillomavirus (hrHPV)., Design: Cross-sectional and prospective., Methods: Two hundred forty-eight HIV-infected hrHPV-positive women enrolled in a cervical cancer screening study in Nairobi, Kenya, had colposcopy-directed biopsy and histological diagnoses. Exfoliated cervical cells were used to measure methylation levels of the CADM1, MAL, and MIR124-2 genes using quantitative methylation-specific polymerase chain reaction. Methylation levels were summarized as cycle threshold (Ct) ratios compared with the β-actin gene. Median Ct ratios were compared across histological diagnoses, with 95% confidence intervals calculated by bootstrapping. Methylation levels at 6 months were assessed in 128 women who remained hrHPV positive., Results: All 3 methylation markers showed significantly (P < 0.001) raised median Ct ratios in women with cervical intraepithelial neoplasia (CIN) grade 3 compared with women with a normal cervix. When markers were combined into a single test, the area under the receiver operating characteristic curve for prediction of CIN2 or worse (CIN2+) was 0.80. When the test was calibrated to have similar specificity, sensitivity of the combined tri-marker test for CIN2+ was comparable with cytology [atypical squamous cells of undetermined significance or worse] (89% and 95%, respectively) and superior to visual inspection with acetic acid (85% vs 70%) and HPV16/18 genotyping (65% vs 40%). Among women with no CIN2+ at baseline and persistent hrHPV at 6-month follow-up, MAL-m1 and MIR124-2 Ct ratios increased significantly., Conclusions: Methylation markers in combination with HPV testing may offer a full molecular screening strategy to the many HIV-infected women who are also hrHPV positive.

Background: Pulmonary emphysema is characterized histologically by destruction of alveolar walls and enlargement of air spaces due to lung epithelial cell apoptosis. Cell adhesion molecule 1 (CADM1) is an immunoglobulin superfamily member expressed in lung epithelial cells. CADM1 generates a membrane-associated C-terminal fragment, αCTF, through A disintegrin- and metalloprotease-10-mediated ectodomain shedding, subsequently releasing the intracellular domain (ICD) through γ-secretase-mediated intramembrane shedding of αCTF. αCTF localizes to mitochondria and induces apoptosis in lung epithelial cells. αCTF contributes to the development and progression of emphysema as a consequence of increased CADM1 ectodomain shedding. The purpose of this study was to examine whether the ICD makes a similar contribution., Results: The ICD was synthesized as a 51-amino acid peptide, and its mutant was synthesized by substituting seven amino acids and deleting two amino acids. These peptides were labeled with fluorescein isothiocyanate and were introduced into various cell lines. ICD peptide-derived fluorescence was well visualized in lung epithelial cells at the site of Mitotracker mitochondrial labeling, but was detected in locations other than mitochondria in other cell types. Mutant peptide-derived fluorescence was detected in locations other than mitochondria, even in lung epithelial cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays revealed that transduction of the ICD peptide increased the proportion of apoptotic cells 2- to 5-fold in the lung epithelial cell lines, whereas the mutant peptide did not. Abundance of the ICD was below the Western blot detection limit in emphysematous (n = 4) and control (n = 4) human lungs. However, the ICD was detected only in emphysematous lungs when it was immunoprecipitated with anti-CADM1 antibody (4/4 vs. 0/4, P = 0.029)., Conclusions: As the abundance of ICD molecules was sparse but present, increased CADM1 shedding appeared to contribute to the development of emphysema by generating αCTF and the ICD in lung epithelial cells.

Objective: The aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of gastric cancer (GC). We here aimed to investigate the mechanism of microRNAs in the regulation of GC pathogenesis., Methods: Transwell chambers (8-μM pore size; Costar) were used in the in vitro migration and in vision assay. Dual luciferase reporter gene construct and dual luciferase reporter assay to identify the target of miR-126. CADM1 expression was evaluated by immunohistochemical staining. The clinical manifestations, treatments and survival were collected for statistical analysis., Results: Inhibition of miR-126 effectively reduced migration and invasion of gastric cancer cell lines. Bioinformatics and luciferase reporter assay revealed that miR-126 specifically targeted the 3'UTR of cell adhesion molecule 1 (CADM1) and regulated its expression. Down-regulation of CADM1 enhanced migration and invasion of GC cell lines. Furthermore, in tumor tissues obtained from gastric cancer patients, the expression of miR-126 was negatively correlated with CADM1 and the high expression of miR-126 combined with low expression of CADM1 might serve as a risk factor for stage1 gastric cancer patients., Conclusions: Our study showed that miR-126, by down-regulation CADM1, enhances migration and invasion in GC cells.

Background: Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, αCTF, through protease-mediated ectodomain shedding, termed α-shedding. Increased CADM1 α-shedding contributes to AEC apoptosis in emphysematous lungs., Methods: Formalin-fixed, paraffin-embedded lung lobes (n = 39) from 36 autopsied patients with IIP were classified as acute IIP (n = 10), fibrosing-type nonspecific IIP (f-NSIP, n = 10), cryptogenic organizing IIP (n = 9), and usual IIP (n = 10). CADM1 expression in the lung sections was examined by western blotting and compared with control lungs (n = 10). The rate of CADM1 α-shedding was calculated as the relative amount of αCTF to full-length CADM1, and the full-length CADM1 level was estimated per epithelial cell by normalization to cytokeratin 7, a lung epithelial marker. Apoptotic AECs were detected by immunohistochemistry for single-stranded DNA (ssDNA). NCI-H441 and A549 human lung epithelial cells were transfected with small interfering RNA (siRNA) to silence CADM1 expression and analyzed by terminal nucleotide nick end labeling assays., Results: The rate of CADM1 α-shedding was higher in all IIP subtypes than in the control (P ≤ 0.019), and the full-length CADM1 level was lower in f-NSIP (P = 0.007). The α-shedding rate and full-length CADM1 level were correlated with each other (P = 0.015) and with the proportion of ssDNA-positive AECs (P ≤ 0.024). NCI-H441 cells transfected with siRNA exhibited a 61 % lower rate of expression of full-length CADM1 and a 17-fold increased proportion of apoptotic cells. Similar results were obtained with A549 cells., Conclusions: CADM1 α-shedding appeared to be increased in all four IIP subtypes and consequently contributed to AEC apoptosis by decreasing the full-length CADM1 level. This mechanism particularly impacted f-NSIP. The molecular mechanism causing AEC apoptosis may be similar between IIP and emphysema.

NG2 cells comprise a heterogeneous precursor population but molecular markers distinguishing between the assumed NG2 cell subpopulations are lacking. Previously, we described that a subfraction of the synaptic cell adhesion molecule SynCAM 1 is modified with the glycan polysialic acid (polySia) in NG2 cells. As for its major carrier, the neural cell adhesion molecule NCAM, polySia attenuates SynCAM 1 adhesion. Functions, as well as cellular and subcellular distribution of polySia-SynCAM 1 are elusive. Using murine glial cultures we now demonstrate that polySia-SynCAM 1 is confined to the Golgi compartment of a subset of NG2 cells and transiently recruited to the cell surface in response to depolarization. NG2 cells with Golgi-confined polySia were NCAM-negative, but positive for markers of oligodendrocyte precursor cells (OPCs). Consistent with previous data on polySia-SynCAM 1, polySia in Ncam(-/-) NG2 cells was exclusively attached to N-glycans and synthesized by ST8SIA2, one out of two mammalian polysialyltransferases. Unexpectedly, Golgi-confined polySia was also detected in Ncam(-/-) microglia, but this fraction resided on O-glycans and was produced by the second polysialyltransferase, ST8SIA4, indicating the presence of yet another polySia carrier in microglia. Searching for this carrier, we identified polysialylated neuropilin-2, so far only known from dendritic cells and exudate macrophages. Microglia activation by LPS, but not interleukin-4, caused a transient translocation of Golgi-localized polySia to the cell surface, resulting in complete depletion. Finally, NO-production of LPS-stimulated microglia was attenuated by addition of polySia suggesting that the observed loss of polySia-neuropilin-2 is involved in negative feedback regulation of pro-inflammatory microglia polarization., (© 2015 Wiley Periodicals, Inc.)

Oligodendrocyte precursor cells (OPCs) are the progenitors of myelinating oligodendrocytes in brain development and repair. Successful myelination depends on the control of adhesiveness during OPC migration and axon contact formation. The decoration of cell surface proteins with the glycan polysialic acid (polySia) is a key regulatory element of OPC interactions during development and under pathological conditions. By far the major protein carrier of polySia is the neural cell adhesion molecule NCAM, but recently, polysialylation of the synaptic cell adhesion molecule SynCAM 1 has been detected in the developing mouse brain. In mice, polySia-SynCAM 1 is associated with cells expressing NG2, a marker of a heterogeneous precursor cell population, which is the primary source for oligodendrocytes in development and myelin repair but can also give rise to astrocytes and possibly neurons. It is not yet clear if polySia-SynCAM 1 is expressed by OPCs and its occurrence in humans is elusive. By generating uniform human embryonic stem cell-derived OPC cultures, we demonstrate that polySia is present on human OPCs but down-regulated during differentiation into myelin basic protein-positive oligodendrocytes. PolySia on NCAM resides on the isoforms NCAM-180 and NCAM-140, and SynCAM 1 is identified as a novel polySia acceptor in human OPCs., (Copyright © 2015. Published by Elsevier B.V.)

We previously reported that the cell adhesion molecule 1 (CADM1) versus CD7 plot in flow cytometry reflects disease progression in human T-cell leukemia virus type 1 (HTLV-1) infection. In CD4(+) cells from peripheral blood, CADM1(-) CD7(+) (P), CADM1(+) CD7(dim) (D) and CADM1(+) CD7(-) (N) subpopulations are observed. The D and N subpopulations increase as asymptomatic HTLV-1 carriers (AC) progress to indolent adult T-cell leukemia-lymphoma (ATL) and the N subpopulation then expands in aggressive ATL. In the present study we examined whether the analysis can estimate the risk of developing ATL in advanced AC. Peripheral blood samples from AC (N = 41) and indolent ATL patients (N = 19) were analyzed by flow cytometry using the CADM1 versus CD7 plot for CD4(+) cells and inverse long PCR (clonality analysis) of FACS-sorted subpopulations. Almost all AC with a high HTLV-1 proviral load (>4 copies/100 cells) had a CADM1(+) (D + N) frequency of >10%. AC with 25% < CADM1(+) ≤ 50% contained expanded clones similar to smoldering-type ATL. In many patients in the 25% < CADM1(+) ≤ 50% group, the proportion of abnormal lymphocytes was distributed around the 5% line, which divides AC and smoldering-type ATL in Shimoyama's classification. In conclusion, the CADM1 versus CD7 plot is useful for selection of putative high-risk AC. The characteristics of some AC and smoldering ATL are said to be similar; however, long-term follow up is required and the clinical outcome (e.g. rate of transformation) of these cases should be used to determine whether to include them in the same clinical category., (© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Objective: To study the expression and significance of tumor suppressor in lung cancer 1 (TSLC1) gene methylation, the expression of TSLC1 protein in cervix cancer and precancerous lesions as well as their relationship with HR-HPV DNA infection., Methods: The clinicopathological data of 92 cases of different cervical lesions during March 2011 to August 2012 treated in our hospital were collected. There were pathologically confirmed 10 cases of normal cervix, 26 cases of cervical intraepithelial neoplasia (CIN) I, 20 cases of CIN II, 15 cases of CIN III, and 21 cases of cervical cancer. Methylation-specific polymerase chain reaction (MSP) was used to detect the TSLC1 gene methylation status in cervical lesions, immunohistochemistry (SP) was used to detect the expressions of TSLC1 protein in cervical lesions, and the second generation hybrid capture (HC2) method was used to detect the high-risk HPV in cervical lesions., Results: The expression rate of TSLC1 gene methylation in normal cervical tissue, CIN I, CIN II, CIN III and SCC were 10.0%, 30.8%, 55.0%, 60.0%, 66.7%, respectively, showing a statistically significant difference (P = 0.004). The positive expression rate of TSLC1 protein in normal cervical tissue, CIN I, CIN II, CIN III and SCC were 100.0%, 80.8%, 65.0%, 33.3%, and 23.8%, respectively, with a significant difference (P = 0.004). In the progression from CIN to invasive cervical cancer, there was no significant correlation between TSLC1 gene methylation and HR-HPV DNA infection (P = 0.919), TSLC1 protein expression and HR-HPV DNA infection (P = 0.664). The correlation analysis showed a negative correlation between TSLC1 gene methylation and TSLC1 protein expression (r = -0.674, P < 0.001)., Conclusions: TSLC1 gene promoter methylation may be an early event in the cervical carcinogenesis, become an early sensitive marker, and serve the early prevention and prognostic prediction for cervical cancer.

Objectives: Triage of HPV screen-positive women is needed to identify those with underlying cervical intraepithelial neoplasia grade 2/3 or worse (CIN2/3+). Presently, cytology on a physician-taken cervical scrape is mostly accepted as triage test, but needs follow-up testing in order not to miss severe disease. Here, we evaluated the performance of combined cytology and bi-marker CADM1/MAL-methylation analysis as triage test on physician-taken cervical scrapes of HPV positive women., Methods: In this post-hoc analysis, we used 364 left-over HPV positive cytology triage samples of participants of a randomized controlled trial (PROHTECT-3: n=46,001) performed in population-based cervical screening. Study endpoints were CIN2+ and CIN3+ detection. Cytology testing with and without methylation marker analysis was evaluated with regard to sensitivity, specificity, positive and negative predictive value, and referral rate., Results: Bi-marker CADM1/MAL-methylation positivity increased proportionally with severity of underlying lesions. Overall, cytology and bi-marker CADM1/MAL-methylation analysis yielded similar performances with regard to CIN3+ detection, yet in combination a significantly higher sensitivity for CIN3+ (88.7%) was obtained at a specificity of 53.6% and a colposcopy referral rate of 53.6%. The combined strategy detected all six cervical cancers, whereas triage by cytology alone failed to detect two of them., Conclusions: Cytology and bi-marker CADM1/MAL-methylation analysis perform complementary for CIN2+/CIN3+ detection when used as triage tool on cervical scrapes of HPV positive women. This approach not only results in a higher CIN3+ sensitivity than cytology triage with an acceptable referral rate, but also seems to reduce the risk of missing cervical cancers and advanced high-grade lesions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10 min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation.

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.

Importance: Human papillomavirus (HPV) is a known causative agent for oropharyngeal squamous cell carcinoma (OPSCC). Whereas it is becoming more firmly established that HPV-positive head and neck squamous cell carcinoma is associated with better survival outcomes, believed to be because of better response to chemoradiation therapy, the specific mechanisms for these improved survival outcomes remain underexplored., Objective: To examine the relationship between HPV status and promoter methylation in an OPSCC cohort., Design, Setting, and Participants: Real-time quantitative polymerase chain reaction was used to examine oncogenic HPV type 16 in a retrospective cohort of 121 patients with primary OPSCC. Aberrant promoter methylation of IGSF4, DAPK1, and ESR1 genes, known to be methylated in head and neck squamous cell carcinoma, including OPSCC, was examined by means of quantitative methylation-specific polymerase chain reaction., Interventions: Patients received standard therapy., Main Outcomes and Measures: Univariate associations between HPV and methylation were analyzed using Fisher exact tests followed by multivariable logistic regression. Cox proportional-hazards regression was used to model the risk of death given age, race, sex, HPV status, methylation, stage, smoking, and treatment., Results: In univariate logistic regression analyses, HPV-positive status was significantly associated with Caucasian race (P = .02), treatment (radiotherapy only, P = .01; chemoradiotherapy, P = .007), and IGSF4 methylation (P = .005). The final multivariate logistic model, after controlling for patient characteristics (sex, age, smoking, race, and treatment) with backward variable selection among genes, retained IGSF4 methylation (OR, 4.5 [95% CI, 1.6-12.8]; P = .005), Caucasian race (OR, 2.9 [95% CI, 1.0-8.3]; P = .053), treatment (radiotherapy only vs neither: OR, 11.62 [95% CI, 2.02-66.82]; P = .02; chemoradiotherapy vs neither: OR, 11.15 [95% CI, 1.92-64.65]; P = .01), male sex (OR, 4.7 [95% CI, 1.3-17.0]; P = .02), and younger age (OR, 0.9 [95% CI, 0.90-1.0]; P = .008) as independent predictors of HPV-positive status. Cox regression modeling indicated HPV-negative status, age, male sex, smoking, and radiation treatment as independent predictors of mortality., Conclusions and Relevance: Methylation of IGSF4 is an independent predictor of HPV-positive status. DNA methylation in conjunction with HPV infection appears to play a role in OPSCC.

Epigenetic modification at CpG islands located on the promoter regions of tumor-suppressor genes has been associated with tumor development in many human cancers. Our study showed that the cell adhesion molecule 1 (CADM1) is downregulated in human papillomavirus (HPV)-infected cervical cancer cell lines via its hypermethylation and demethylation using 5-aza-2'-deoxycyticine (5-aza-dC) restored the expression of CADM1 protein. Overexpression of CADM1 inhibited cell proliferation. p53 was involved in the regulation of CADM1. Our results demonstrate that epigenetic alteration of CADM1 was more frequent in HPV-positive cervical cancers and that restoration of CADM1 expression may be a potential strategy for cervical cancer therapy.

Aims: Gene promoter hypermethylation is recognised as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1, MAL and miR124-2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer., Methods: A series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, cervical intraepithelial neoplasia grade 3 (CIN3) (n=16) or CIN2 (n=32), and women without evidence of CIN2 or worse (n=120) were assessed for methylation of CADM1, MAL and miR124-2. Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR., Results: All samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI 58.0% to 94.4%) of women with endometrial cancer scored positive for DNA methylation for at least one of the three genes. In women without cancer, methylation frequencies increased significantly with severity of disease from 19.2% (23/120; 95% CI 12.1% to 26.2%) in women without CIN2 or worse to 37.5% (12/32; 95% CI 20.7% to 54.3%) and 68.8% (11/16; 95% CI 46.0% to 91.5%) in women with CIN2 and CIN3, respectively. Overall methylation positivity and the number of methylated genes increased proportionally to the lesion severity., Conclusions: DNA methylation analysis of CADM1, MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: In a protein C deficient family, we recently identified a candidate gene, CADM1, which interacted with protein C deficiency in increasing the risk of venous thrombosis (VT). This study aimed to determine whether CADM1 variants also interact with protein C pathway abnormalities in increasing VT risk outside this family., Materials and Methods: We genotyped over 300 CADM1 variants in the population-based MEGA case-control study. We compared VT risks between cases with low protein C activity (n=194), low protein S levels (n=23), high factor VIII activity (n=165) or factor V Leiden carriers (n=580), and all 4004 controls. Positive associations were repeated in all 3496 cases and 4004 controls., Results: We found 22 variants which were associated with VT in one of the protein C pathway risk groups. After mutual adjustment, six variants remained associated with VT. The strongest evidence was found for rs220842 and rs11608105. For rs220842, the odds ratio (OR) for VT was 3.2 (95% CI 1.2-9.0) for cases with high factor VIII activity compared with controls. In addition, this variant was associated with an increased risk of VT in the overall study population (OR: 1.5, 95% CI 1.0-2.2). The other variant, rs11608105, was not associated with VT in the overall study population (OR: 1.0, 95% CI 0.8-1.1), but showed a strong effect on VT risk (OR: 21, 95% CI 5.1-88) when combined with low protein C or S levels., Conclusions: In a population-based association study, we confirm a role for CADM1 variants in increasing the risk of VT by interaction with protein C pathway abnormalities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Increasing evidence has demonstrated that cell adhesion molecule 1/tumor suppressor in lung cancer 1 (CADM1/TSLC1) is crucially implicated in various biological processes, including proliferation, apoptosis, and invasion during tumorigenesis and development. However, the mechanism underlying its suppression of tumor growth and metastasis in melanoma remains elusive. The aim of the present study was to examine if CADM1/TSLC1 was able to induce growth suppression in melanoma. The plasmid pcDNA3.1‑CADM1/TSLC1 was transfected into A375 cells (a human melanoma cell line). The expression of CADM1/TSLC1 in the transfected cells was determined by RT‑PCR and western blotting analysis. Cell growth was measured by an 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyl‑tetrazolium bromide assay and cell apoptosis was determined by flow cytometry, while a transwell assay was utilized to measure the ability of invasion. The expression of MMP‑2 and MMP‑9 in the transfected cells was determined by western blotting analysis. RT‑PCR and western blotting revealed that in pcDNA3.1‑CADM1/TSLC1 the protein expression of CADM1/TSLC1 protein was higher than in the pcDNA3.1 and A375 cells. The expression of MMP‑2 and MMP‑9 was lower in the pcDNA3.1‑CADM1/TSLC1 than that in the pcDNA3.1 and A375 cells. The growth of CADM1/TSLC1‑transfected cells was significantly suppressed in vitro and the ability of invasion was also reduced, CADM1/TSLC1 was able to induce cell apoptosis. Furthermore, CADM1/TSLC1 was an anti‑invasive gene, the overexpression of which inhibited the invasion of A375 cells. This inhibition may be due to the suppression of the MMP‑2 and MMP‑9 expression, which is relative to tumor metastasis and progression.

Oropharyngeal squamous cell carcinoma (OPSCC) is associated with human papillomavirus (HPV) in a proportion of tumors. HPV-positive OPSCC is considered a distinct molecular entity with a prognostic advantage compared to HPV-negative cases. Silencing of cancer-related genes by DNA promoter hypermethylation may play an important role in the development of OPSCC. Hence, we examined promoter methylation status in 24 common tumor suppressor genes in a group of 200 OPSCCs to determine differentially methylated genes in HPV-positive versus HPV-negative primary OPSCC. Methylation status was correlated with HPV status, clinical features, and patient survival using multivariate methods. Additionally, methylation status of 16 cervical squamous cell carcinomas (SCC) was compared with HPV-positive OPSCC. Using methylation-specific probe amplification, HPV-positive OPSCC showed a significantly higher cumulative methylation index (CMI) compared to HPV-negative OPSCC (P=0.008). For the genes CDH13, DAPK1, and RARB, both HPV-positive and HPV-negative OPSCC showed promoter hypermethylation in at least 20% of the tumors. HPV status was found to be an independent predictor of promoter hypermethylation of CADM1 (P < 0.001), CHFR (P = 0.027), and TIMP3 (P < 0.001). CADM1 and CHFR showed similar methylation patterns in OPSCC and cervical SCC, but TIMP3 showed no methylation in cervical SCC in contrast to OPSCC. Methylation status of neither individual gene nor CMI was associated with survival. These results suggest that HPV-positive tumors are to a greater extent driven by promotor hypermethylation in these tumor suppressor genes. Especially CADM1 and TIMP3 are significantly more frequently hypermethylated in HPV-positive OPSCC and CHFR in HPV-negative tumors., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

CADM1 (Cell adhesion molecule 1), a cell adhesion molecule belonging to the immunoglobulin superfamily, is involved in cell-cell interaction and the formation and maintenance of epithelial structure. Expression of CADM1 is frequently downregulated in various tumors derived from epithelial cells. However, the intracellular signaling pathways activated by CADM1-mediated cell adhesion remain unknown. Here, we established a cell-based spreading assay to analyze the signaling pathway specifically activated by the trans-homophilic interaction of CADM1. In the assay, MDCK cells expressing exogenous CADM1 were incubated on the glass coated with a recombinant extracellular fragment of CADM1, and the degree of cell spreading was quantified by measuring their surface area. Assay screening of 104 chemical inhibitors with known functions revealed that LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), efficiently suppressed cell spreading in a dose-dependent manner. Inhibitors of Akt and Rac1, downstream effectors of PI3K, also partially suppressed cell spreading, while the addition of both inhibitors blocked cell spreading to the same extent as did LY294002. Furthermore, MPP3 and Dlg, membrane-associated guanylate kinase homologs (MAGuK) proteins, connect CADM1 with p85 of PI3K by forming a multi-protein complex at the periphery of cells. These results suggest that trans-homophilic interaction mediated by CADM1 activates the PI3K pathway to reorganize the actin cytoskeleton and form epithelial cell structure.

Primary screening for high-risk human papillomavirus (hrHPV) requires a triage protocol. Repeat cytology testing at baseline and after 6 to 12 months has emerged as a reasonable triage approach, but carries the risk of loss to follow-up. Repeat cytology testing may be omitted if cytology is supplemented with another, complementary triage test at baseline. In this study, the performance of combined triage by cytology and DNA methylation analysis was assessed. In hrHPV-positive cervical scrapes (n = 250), cytology [threshold: atypical squamous cells of undetermined significance (ASCUS)], bi-marker CADM1/MAL methylation testing (at different assay thresholds), and combinations of both were evaluated for endpoints cervical intraepithelial neoplasia grade 2 or worse (CIN2(+)) and grade 3 or worse (CIN3(+)). At a predefined methylation threshold of 70% specificity for CIN3(+), combined triage revealed a CIN3(+) sensitivity of 86.8% [95% confidence interval (CI), 76.1-97.6] compared with 65.8% (95% CI, 50.7-80.9) for sole cytology triage testing. Corresponding CIN3(+) specificity was 64.8% (95% CI, 58.1-71.5) for combined triage and 78.6% (95% CI, 72.8-84.3) for sole cytology triage testing. For CIN2(+), the sensitivity of combined triage testing was 84.5% (95% CI, 75.2-93.8) compared with 65.5% (95% CI, 53.3-77.7) for sole cytology triage, with corresponding specificities of 69.9% (95% CI, 63.1-76.6) and 83.5% (95% CI, 78.0-89.0), respectively. In conclusion, combined triage reached substantially higher CIN2(+)/3(+) sensitivities compared with sole cytology at a slight drop in specificity. Therefore, it is an attractive triage strategy for colposcopy of hrHPV-positive women with a high reassurance for cervical cancer and advanced CIN lesions., (©2014 American Association for Cancer Research.)

Background: The aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of hepatocarcinoma. miR-1246 expression in High invasive ability cell line than significantly higher than that in low invasive ability cell line., Methods: Transwell chambers (8-uM pore size; Costar) were used in the in vitro migration and invison anssay. Dual luciferase reporter gene construct and Dual luciferase reporter assay to identify the target of miR-1246. CADM1 expression was evaluated by immunohistochemistric staining. The clinical manifestations, treatments and survival were collected for statistical analysis., Results: Inhibition of miR-1246 effectively reduced migration and invasion of hepatocellular carcinoma cell lines. Bioinformatics and luciferase reporter assay revealed that miR-1246 specifically targeted the 3'-UTR of Cell adhesion molecule 1 and regulated its expression. Down-regulation of CADM1 enhanced migration and invasion of HCC cell lines. Furthermore, in tumor tissues obtained from liver cancer patients, the expression of miR-1246 was negatively correlated with CADM1 and the high expression of miR-1246 combined with low expression of CADM1 might serve as a risk factor for stage1 liver cancer patients., Conclusions: Our study showed that miR-1246, by down-regulation CADM1, enhances migration and invasion in HCC cells.

Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.

Purpose: Cell adhesion molecule 1 (CADM1), initially identified as a tumor suppressor gene, has recently been reported to be ectopically expressed in primary adult T-cell leukemia-lymphoma (ATL) cells. We incorporated CADM1 into flow-cytometric analysis to reveal oncogenic mechanisms in human T-cell lymphotrophic virus type I (HTLV-I) infection by purifying cells from the intermediate stages of ATL development., Experimental Design: We isolated CADM1- and CD7-expressing peripheral blood mononuclear cells of asymptomatic carriers and ATLs using multicolor flow cytometry. Fluorescence-activated cell sorted (FACS) subpopulations were subjected to clonal expansion and gene expression analysis., Results: HTLV-I-infected cells were efficiently enriched in CADM1(+) subpopulations (D, CADM1(pos)CD7(dim) and N, CADM1(pos)CD7(neg)). Clonally expanding cells were detected exclusively in these subpopulations in asymptomatic carriers with high proviral load, suggesting that the appearance of D and N could be a surrogate marker of progression from asymptomatic carrier to early ATL. Further disease progression was accompanied by an increase in N with a reciprocal decrease in D, indicating clonal evolution from D to N. The gene expression profiles of D and N in asymptomatic carriers showed similarities to those of indolent ATLs, suggesting that these subpopulations represent premalignant cells. This is further supported by the molecular hallmarks of ATL, that is, drastic downregulation of miR-31 and upregulation of abnormal Helios transcripts., Conclusion: The CADM1 versus CD7 plot accurately reflects disease progression in HTLV-I infection, and CADM1(+) cells with downregulated CD7 in asymptomatic carriers have common properties with those in indolent ATLs., (©2014 American Association for Cancer Research.)

Breast cancer brain metastases (BCBM) are detected with increasing incidence. In order to detect potential genes involved in BCBM, we first screened for genes down-regulated by methylation in cell lines with site-specific metastatic ability. The expression of five genes, CADM1, SPARC, RECK, TNFAIP3 and CXCL14, which were also found down-regulated in gene expression profiling analyses of BCBM tissue samples, was verified by qRT-PCR in a larger patient cohort. CADM1 was chosen for further down-stream analyses. A higher incidence of CADM1 methylation, correlating with lower expression levels, was found in BCBM as compared to primary BC. Loss of CADM1 protein expression was detected most commonly among BCBM samples as well as among primary tumors with subsequent brain relapse. The prognostic role of CADM1 expression was finally verified in four large independent breast cancer cohorts (n=2136). Loss of CADM1 protein expression was associated with disease stage, lymph node status, and tumor size in primary BC. Furthermore, all analyses revealed a significant association between loss of CADM1 and shorter survival. In multivariate analyses, survival was significantly shorter among patients with CADM1-negative tumors. Loss of CADM1 expression is an independent prognostic factor especially associated with the development of brain metastases in breast cancer patients.