In the last decade, optimized treatment for non-small cell lung cancer had lead to improved prognosis, but the overall survival is still very short. To further understand the molecular basis of the disease we have to identify biomarkers related to survival. Here we present the development of an online tool suitable for the real-time meta-analysis of published lung cancer microarray datasets to identify biomarkers related to survival. We searched the caBIG, GEO and TCGA repositories to identify samples with published gene expression data and survival information. Univariate and multivariate Cox regression analysis, Kaplan-Meier survival plot with hazard ratio and logrank P value are calculated and plotted in R. The complete analysis tool can be accessed online at: www.kmplot.com/lung. All together 1,715 samples of ten independent datasets were integrated into the system. As a demonstration, we used the tool to validate 21 previously published survival associated biomarkers. Of these, survival was best predicted by CDK1 (p<1E-16), CD24 (p<1E-16) and CADM1 (p = 7E-12) in adenocarcinomas and by CCNE1 (p = 2.3E-09) and VEGF (p = 3.3E-10) in all NSCLC patients. Additional genes significantly correlated to survival include RAD51, CDKN2A, OPN, EZH2, ANXA3, ADAM28 and ERCC1. In summary, we established an integrated database and an online tool capable of uni- and multivariate analysis for in silico validation of new biomarker candidates in non-small cell lung cancer.

Genetic variation in both innate and adaptive immune systems is associated with Crohn's disease (CD) susceptibility, but much of the heritability to CD remains unknown. We performed a genome-wide association study (GWAS) in 896 CD cases and 3204 healthy controls all of Caucasian origin as defined by multidimensional scaling. We found supportive evidence for 21 out of 40 CD loci identified in a recent CD GWAS meta-analysis, including two loci which had only nominally achieved replication (rs4807569, 19p13; rs991804, CCL2/CCL7). In addition, we identified associations with genes involved in tight junctions/epithelial integrity (ASHL, ARPC1A), innate immunity (EXOC2), dendritic cell biology [CADM1 (IGSF4)], macrophage development (MMD2), TGF-beta signaling (MAP3K7IP1) and FUT2 (a physiological trait that regulates gastrointestinal mucosal expression of blood group A and B antigens) (rs602662, P=3.4x10(-5)). Twenty percent of Caucasians are 'non-secretors' who do not express ABO antigens in saliva as a result of the FUT2 W134X allele. We demonstrated replication in an independent cohort of 1174 CD cases and 357 controls between the four primary FUT2 single nucleotide polymorphisms (SNPs) and CD (rs602662, combined P-value 4.90x10(-8)) and also association with FUT2 W143X (P=2.6x10(-5)). Further evidence of the relevance of this locus to CD pathogenesis was demonstrated by the association of the original four SNPs and CD in the recently published CD GWAS meta-analysis (rs602662, P=0.001). These findings strongly implicate this locus in CD susceptibility and highlight the role of the mucus layer in the development of CD.

OBJECTIVE

Global gene expression profiling has been widely used in lung cancer research to identify clinically relevant molecular subtypes as well as to predict prognosis and therapy response. So far, the value of these multigene signatures in clinical practice is unclear, and the biologic importance of individual genes is difficult to assess, as the published signatures virtually do not overlap.

METHODS

Here, we describe a novel single institute cohort, including 196 non-small lung cancers (NSCLC) with clinical information and long-term follow-up. Gene expression array data were used as a training set to screen for single genes with prognostic impact. The top 450 probe sets identified using a univariate Cox regression model (significance level P < 0.01) were tested in a meta-analysis including five publicly available independent lung cancer cohorts (n = 860).

RESULTS

The meta-analysis revealed 14 genes that were significantly associated with survival (P < 0.001) with a false discovery rate <1%. The prognostic impact of one of these genes, the cell adhesion molecule 1 (CADM1), was confirmed by use of immunohistochemistry on tissue microarrays from 2 independent NSCLC cohorts, altogether including 617 NSCLC samples. Low CADM1 protein expression was significantly associated with shorter survival, with particular influence in the adenocarcinoma patient subgroup.

CONCLUSIONS

Using a novel NSCLC cohort together with a meta-analysis validation approach, we have identified a set of single genes with independent prognostic impact. One of these genes, CADM1, was further established as an immunohistochemical marker with a potential application in clinical diagnostics.

OBJECTIVE

We wanted to identify the most promising methylation marker candidates for cervical cancer early detection.

METHODS

A systematic literature review was performed in Medline and weighted average frequencies for methylated genes stratified by tissue source and methods used were computed.

RESULTS

51 studies were identified analyzing 68 different genes for methylation in 4376 specimens across all stages of cervical carcinogenesis. 15 genes, DAPK1, RASSF1, CDH1, CDKN2A, MGMT, RARB, APC, FHIT, MLH1, TIMP3, GSTP1, CADM1, CDH13, HIC1, and TERT have been analyzed in 5 or more studies. The published data on these genes is highly heterogeneous; 7 genes (CDH1, FHIT, TERT, CDH13, MGMT, TIMP3, and HIC1) had a reported range of methylation frequencies in cervical cancers of greater than 60% between studies. Stratification by analysis method did not resolve the heterogeneity. Three markers, DAPK1, CADM1, and RARB, showed elevated methylation in cervical cancers consistently across studies.

CONCLUSIONS

There is currently no methylation marker that can be readily translated for use in cervical cancer screening or triage settings. Large, well-conducted methylation profiling studies of cervical carcinogenesis could yield new candidates that are more specific for HPV-related carcinogenesis. New candidate markers need to be thoroughly validated in highly standardized assays.

The mouse lymphoid organ-resident CD8alpha(+) dendritic cell (DC) subset is specialized in Ag presentation to CD8(+) T cells. Recent evidence shows that mouse nonlymphoid tissue CD103(+) DCs and human blood DC Ag 3(+) DCs share similarities with CD8alpha(+) DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26(+) skin lymph DC subset shares significant transcriptomic similarities with mouse CD8alpha(+) and human blood DC Ag 3(+) DCs. This allowed the identification of a common set of phenotypic characteristics for CD8alpha-like DCs in the three mammalian species (i.e., SIRP(lo), CADM1(hi), CLEC9A(hi), CD205(hi), XCR1(hi)). Compared to CD26(-) DCs, the sheep CD26(+) DCs show 1) potent stimulation of allogeneic naive CD8(+) T cells with high selective induction of the Ifngamma and Il22 genes; 2) dominant efficacy in activating specific CD8(+) T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8alpha(+)-like DCs across mammalian species and identify molecular candidates that could be used for the design of vaccines applying to mammals in general.

OBJECTIVE

Screening women for high-grade cervical intraepithelial neoplasia or cervical cancer (CIN3(+)) by high-risk human papillomavirus (hrHPV) testing has as side-effect the detection of hrHPV-positive women without clinically relevant lesions. Here, we developed an objective assay assessing the methylation status of the promoter regions of CADM1 and MAL to triage hrHPV-positive women for CIN3(+).

METHODS

In a training set (51 women with CIN3(+) and 224 without CIN2(+)), panels consisting of one to four quantitative methylation-specific PCR (qMSP) assays (CADM1-m12,CADM1-m18,MAL-m1,MAL-m2) were analyzed. Cross-validated receiver-operating characteristics (ROC) curves were constructed and the panel with highest partial cross-validated area under the curve (AUC) was used for validation in an independent set of 236 consecutive hrHPV-positive women from a screening cohort. In the validation set, the ROC curve of the panel was compared with CIN3(+) sensitivity and specificity of cytology and of cytology combined with HPV16/18 genotyping.

RESULTS

In the training set, CADM1-m18 combined with MAL-m1 was the best panel (cross-validated partial AUC = 0.719). In the validation set, this panel revealed CIN3(+) sensitivities ranging from 100% (95% CI: 92.4-100) to 60.5% (95% CI: 47.1-74.6), with corresponding specificities ranging from 22.7% (95% CI: 20.2-25.2) to 83.3% (95% CI: 78.4-87.4). For cytology these were 65.8% (95% CI: 52.3-79.0) and 78.8% (95% CI: 73.7-83.1) and for cytology/HPV16/18, these were 84.2% (95% CI: 72.0-92.7) and 54.0% (95% CI: 49.2-58.7), respectively. The point estimates of both cytology and cytology/HPV16/18 were equal to the values of the ROC curve of CADM1-m18/MAL-m1.

CONCLUSIONS

We developed an objective methylation marker panel that was equally discriminatory for CIN3(+) as cytology or cytology with HPV16/18 genotyping in hrHPV-positive women. This opens the possibility for complete cervical screening by objective, nonmorphological molecular methods.

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA. Whereas no methylation was evident in pre-immortal cells, progression to anchorage independence was associated with an accumulation of frequent methylation events involving five genes, all of which were also methylated in cervical carcinomas. TP73 and ESR1 methylation became manifest in early immortal cells followed by RARbeta and DAPK1 methylation in late immortal passages. Complementary methylation of MGMT was related to anchorage independence. Analysis of nine cervical cancer cell lines, representing the tumorigenic phenotype, revealed in addition to these five genes frequent methylation of CADM1, CDH13 and CHFR. In conclusion, eight recurrent methylation events in cervical carcinomas could be assigned to different stages of HPV-induced transformation. Hence, our in vitro model system provides a valuable tool to further functionally address the epigenetic alterations that are common in cervical carcinomas.

Given the lower specificity for high-grade cervical lesions of high-risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test-positive women is needed to detect high-grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation-specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma. When qMSPs were combined and positivity for at least one of the qMSPs in the combination was taken into account, the highest positivity rates for cervical intraepithelial neoplasia grade 3 (CIN3) lesions (97%) and squamous cell- and adeno-carcinomas (99%) were obtained by combining a single CADM1 marker with a single MAL marker. Subsequent qMSP analysis of 70 GP5+/6+-PCR hrHPV-positive scrapings revealed that a two-marker panel consisting of CADM1-M18 and MAL-M1 was most discriminative, detecting 90% of women with CIN3 (n = 30), whereas it showed a positive result in only 13.5% of women without cervical disease (n = 40). Finally, we applied hrHPV GP5+/6+-PCR testing followed by CADM1-M18/MAL-M1 methylation analysis to a cohort of 79 women visiting the outpatient colposcopy clinic. hrHPV testing revealed a sensitivity of 97% and a specificity of 33% for CIN3+. Additional CADM1-M18/MAL-M1 methylation analysis on the hrHPV-positive women increased the specificity to 78% with a sensitivity of 70%. In conclusion, the methylation marker panel CADM1-M18 and MAL-M1 may serve as an alternative molecular triage tool for hrHPV-positive women.

Pancreatic β cells adapt to compensate for increased metabolic demand during insulin resistance. Although the microRNA pathway has an essential role in β cell proliferation, the extent of its contribution is unclear. Here, we report that miR-184 is silenced in the pancreatic islets of insulin-resistant mouse models and type 2 diabetic human subjects. Reduction of miR-184 promotes the expression of its target Argonaute2 (Ago2), a component of the microRNA-induced silencing complex. Moreover, restoration of miR-184 in leptin-deficient ob/ob mice decreased Ago2 and prevented compensatory β cell expansion. Loss of Ago2 during insulin resistance blocked β cell growth and relieved the regulation of miR-375-targeted genes, including the growth suppressor Cadm1. Lastly, administration of a ketogenic diet to ob/ob mice rescued insulin sensitivity and miR-184 expression and restored Ago2 and β cell mass. This study identifies the targeting of Ago2 by miR-184 as an essential component of the compensatory response to regulate proliferation according to insulin sensitivity.

The microlocalization of mast cells within specific tissue compartments is thought to be critical for the pathophysiology of many diverse diseases. This is particularly evident in asthma where they localize to the airway smooth muscle (ASM) bundles. Mast cells are recruited to the ASM by numerous chemoattractants and adhere through CADM1, but the functional consequences of this are unknown. In this study, we show that human ASM maintains human lung mast cell (HLMC) survival in vitro and induces rapid HLMC proliferation. This required cell-cell contact and occurred through a cooperative interaction between membrane-bound stem cell factor (SCF) expressed on ASM, soluble IL-6, and CADM1 expressed on HLMC. There was a physical interaction in HLMC between CADM1 and the SCF receptor (CD117), suggesting that CADM1-dependent adhesion facilitates the interaction of membrane-bound SCF with its receptor. HLMC-ASM coculture also enhanced constitutive HLMC degranulation, revealing a novel smooth muscle-driven allergen-independent mechanism of chronic mast cell activation. Targeting these interactions in asthma might offer a new strategy for the treatment of this common disease.

We previously showed that silencing of TSLC1, recently renamed <em>CADM1</em>, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. <em>CADM1</em> silencing often results from promoter methylation. Here, we determined the extent of <em>CADM1</em> promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a <em>CADM1</em>-based methylation marker for identification of women at risk of cervical cancer. Methylation-specific PCRs targeting three regions within the <em>CADM1</em> promoter were performed on high-risk HPV-containing cell lines, PBMCs, normal cervical smears, and (pre)malignant lesions. <em>CADM1</em> protein expression in cervical tissues was analysed by immunohistochemistry. All statistical tests were two-sided. Density of methylation was associated with the degree of anchorage-independent growth and <em>CADM1</em> gene silencing in vitro. In cervical squamous lesions, methylation frequency and density increased with severity of disease. Dense methylation (defined as>>or= 2 methylated regions) increased from 5% in normal cervical samples to 30% in CIN3 lesions and 83% in squamous cell carcinomas (SCCs) and was significantly associated with decreased <em>CADM1</em> protein expression (p < 0.00005). The frequency of dense methylation was significantly higher in>>or= CIN3 compared with <or= CIN1 (p = 0.005), as well as in SCCs compared with adenocarcinomas (83% versus 23%; p = 0.002). Detection of dense <em>CADM1</em> promoter methylation will contribute to the assembly of a valuable marker panel for the triage of high-risk HPV-positive women at risk of>>or= CIN3.

MicroRNA-10b (miR-10b) was recently reported to be dysregulated in some types of cancer and to play a role in invasion and metastasis. However, effects and potential mechanisms of action of miR-10b in the metastasis of hepatocellular carcinoma (HCC) have not been explored. In this study, we confirmed that miR-10b is highly expressed in metastatic HCC tissues and in metastatic HCC cell lines by qRT-PCR. Moreover, patients with higher miR-10b expression had significantly poorer overall survival, and high miR-10b expression was an independent predictor of poor prognosis. Inhibition of miR-10b reduced cell migration and invasion in MHCC97H cells, whereas over-expression of miR-10b in HepG2 cells increased cell migration and invasion. Bioinformatics and luciferase reporter assays revealed that miR-10b binds the 3'-UTR of CADM1 mRNA and represses its translation. Western blot and qRT-PCR showed that CADM1 is inhibited by miR-10b over-expression. Silencing of CADM1 resulted in substantially increased cell motility and invasion similar to that observed with over-expression of miR-10b in HepG2 cells. These results suggest that miR-10b may positively regulate the invasion and metastasis of HCC through targeting CADM1.

The unified idea on the molecular pathogenesis of Autism Spectrum Disorder (ASD) is still unknown although mutations in genes encoding neuroligins and SHANK3 have been shown in a small part of the patients. RA175/SynCAM1/CADM1(CADM1), a member of immunoglobulin superfamily, is another synaptic cell adhesion molecule. To clarify the idea that impaired synaptogenesis underlies the pathogenesis of ASD, we examined the relationship between mutations in the CADM1 gene and ASD. We found two missense mutations, C739A(H246N) and A755C(Y251S), in the CADM1 gene of male Caucasian ASD patients and their family members. Both mutations were located in the third immunoglobulin domain, which is essential for trans-active interaction. The mutated CADM1 exhibited less amount of high molecular weight with the matured oligosaccharide, defective trafficking to the cell surface, and more susceptibility to the cleavage and or degradation. Our findings provide key support for the unified idea that impaired synaptogenesis underlies the pathogenesis of ASD.

OBJECTIVE

Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA-protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia.

METHODS

Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n=46), CIN2 (n=12), and CIN3+ (n=27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing.

RESULTS

Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, 2/16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+.

CONCLUSIONS

These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and, thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.

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.

Worldwide, one person dies every 40 seconds by suicide, a potentially preventable tragedy. A limiting step in our ability to intervene is the lack of objective, reliable predictors. We have previously provided proof of principle for the use of blood gene expression biomarkers to predict future hospitalizations due to suicidality, in male bipolar disorder participants. We now generalize the discovery, prioritization, validation, and testing of such markers across major psychiatric disorders (bipolar disorder, major depressive disorder, schizoaffective disorder, and schizophrenia) in male participants, to understand commonalities and differences. We used a powerful within-participant discovery approach to identify genes that change in expression between no suicidal ideation and high suicidal ideation states (n=37 participants out of a cohort of 217 psychiatric participants followed longitudinally). We then used a convergent functional genomics (CFG) approach with existing prior evidence in the field to prioritize the candidate biomarkers identified in the discovery step. Next, we validated the top biomarkers from the prioritization step for relevance to suicidal behavior, in a demographically matched cohort of suicide completers from the coroner's office (n=26). The biomarkers for suicidal ideation only are enriched for genes involved in neuronal connectivity and schizophrenia, the biomarkers also validated for suicidal behavior are enriched for genes involved in neuronal activity and mood. The 76 biomarkers that survived Bonferroni correction after validation for suicidal behavior map to biological pathways involved in immune and inflammatory response, mTOR signaling and growth factor regulation. mTOR signaling is necessary for the effects of the rapid-acting antidepressant agent ketamine, providing a novel biological rationale for its possible use in treating acute suicidality. Similarly, MAOB, a target of antidepressant inhibitors, was one of the increased biomarkers for suicidality. We also identified other potential therapeutic targets or biomarkers for drugs known to mitigate suicidality, such as omega-3 fatty acids, lithium and clozapine. Overall, 14% of the top candidate biomarkers also had evidence for involvement in psychological stress response, and 19% for involvement in programmed cell death/cellular suicide (apoptosis). It may be that in the face of adversity (stress), death mechanisms are turned on at a cellular (apoptosis) and organismal level. Finally, we tested the top increased and decreased biomarkers from the discovery for suicidal ideation (CADM1, CLIP4, DTNA, KIF2C), prioritization with CFG for prior evidence (SAT1, SKA2, SLC4A4), and validation for behavior in suicide completers (IL6, MBP, JUN, KLHDC3) steps in a completely independent test cohort of psychiatric participants for prediction of suicidal ideation (n=108), and in a future follow-up cohort of psychiatric participants (n=157) for prediction of psychiatric hospitalizations due to suicidality. The best individual biomarker across psychiatric diagnoses for predicting suicidal ideation was SLC4A4, with a receiver operating characteristic (ROC) area under the curve (AUC) of 72%. For bipolar disorder in particular, SLC4A4 predicted suicidal ideation with an AUC of 93%, and future hospitalizations with an AUC of 70%. SLC4A4 is involved in brain extracellular space pH regulation. Brain pH has been implicated in the pathophysiology of acute panic attacks. We also describe two new clinical information apps, one for affective state (simplified affective state scale, SASS) and one for suicide risk factors (Convergent Functional Information for Suicide, CFI-S), and how well they predict suicidal ideation across psychiatric diagnoses (AUC of 85% for SASS, AUC of 89% for CFI-S). We hypothesized a priori, based on our previous work, that the integration of the top biomarkers and the clinical information into a universal predictive measure (UP-Suicide) would show broad-spectrum predictive ability across psychiatric diagnoses. Indeed, the UP-Suicide was able to predict suicidal ideation across psychiatric diagnoses with an AUC of 92%. For bipolar disorder, it predicted suicidal ideation with an AUC of 98%, and future hospitalizations with an AUC of 94%. Of note, both types of tests we developed (blood biomarkers and clinical information apps) do not require asking the individual assessed if they have thoughts of suicide, as individuals who are truly suicidal often do not share that information with clinicians. We propose that the widespread use of such risk prediction tests as part of routine or targeted healthcare assessments will lead to early disease interception followed by preventive lifestyle modifications and proactive treatment.

BACKGROUND

The tumor suppressor genes CADM1/TSLC1 and DAL-1/4.1B are frequently inactivated by promoter methylation in non-small cell lung cancer. The proteins they encode, CADM1 and 4.1B, form a complex in human epithelial cells and are involved in cell-cell adhesion.

METHODS

Expression of CADM1 and 4.1B proteins was examined by immunohistochemistry in 67 primary breast cancer and adjacent noncancerous tissues. CADM1 and 4.1B messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction (RT-PCR). The methylation status of the CADM1 and 4.1B promoters was determined quantitatively by bisulfite treatment followed by pyrosequencing.

RESULTS

CADM1 and 4.1B protein signals were detected along the cell membrane in normal mammary epithelia. By contrast, 47 (70%) and 49 (73%) of 67 primary breast cancers showed aberrant CADM1 and 4.1B staining, respectively. Aberrant CADM1 staining was more frequently observed in pT2 and pT3 tumors and for stages II and III (P = 0.045 and P = 0.020, respectively), while aberrant 4.1B staining was more often observed in tumors with lymph node metastasis, for pT2 and pT3 tumors, and for stages II and III (P = 0.0058, P = 0.0098, and P = 0.0007, respectively). Furthermore, aberrant CADM1 and 4.1B expression was preferentially observed in invasive relative to noninvasive lesions from the same specimen (P = 0.036 and P = 0.0009, respectively). Finally, hypermethylation of CADM1 and 4.1B genes was detected in 46% and 42% of primary breast cancers, respectively.

CONCLUSIONS

Our findings suggest that aberrant CADM1 and 4.1B expression is involved in progression of breast cancer, especially in invasion into the stroma and metastasis.

Metastasis is a complex process utilizing both tumor-cell-autonomous properties and host-derived factors, including cellular immunity. We have previously shown that germline polymorphisms can modify tumor cell metastatic capabilities through cell-autonomous mechanisms. However, how metastasis susceptibility genes interact with the tumor stroma is incompletely understood. Here, we employ a complex genetic screen to identify Cadm1 as a novel modifier of metastasis. We demonstrate that Cadm1 can specifically suppress metastasis without affecting primary tumor growth. Unexpectedly, Cadm1 did not alter tumor-cell-autonomous properties such as proliferation or invasion, but required the host's adaptive immune system to affect metastasis. The metastasis-suppressing effect of Cadm1 was lost in mice lacking T cell-mediated immunity, which was partially phenocopied by depleting CD8(+) T cells in immune-competent mice. Our data show a novel function for Cadm1 in suppressing metastasis by sensitizing tumor cells to immune surveillance mechanisms, and this is the first report of a heritable metastasis susceptibility gene engaging tumor non-autonomous factors.

The porcine skin has striking similarities to the human skin in terms of general structure, thickness, hair follicle content, pigmentation, collagen and lipid composition. This has been the basis for numerous studies using the pig as a model for wound healing, transdermal delivery, dermal toxicology, radiation and UVB effects. Considering that the skin also represents an immune organ of utmost importance for health, immune cells present in the skin of the pig will be reviewed. The focus of this review is on dendritic cells, which play a central role in the skin immune system as they serve as sentinels in the skin, which offers a large surface area exposed to the environment. Based on a literature review and original data we propose a classification of porcine dendritic cell subsets in the skin corresponding to the subsets described in the human skin. The equivalent of the human CD141(+) DC subset is CD1a(-)CD4(-)CD172a(-)CADM1(high), that of the CD1c(+) subset is CD1a(+)CD4(-)CD172a(+)CADM1(+/low), and porcine plasmacytoid dendritic cells are CD1a(-)CD4(+)CD172a(+)CADM1(-). CD209 and CD14 could represent markers of inflammatory monocyte-derived cells, either dendritic cells or macrophages. Future studies for example using transriptomic analysis of sorted populations are required to confirm the identity of these cells.

Targeting antigens to cross-presenting dendritic cells (DCs) is a promising method for enhancing CD8(+) T-cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross-presenting murine CD8α(+) DCs, and that the expression is conserved on homologous DC subsets in humans (CD141(+) DCs), sheep (CD26(+) DCs), and macaques (CADM1(+) DCs). We therefore tested if targeting antigens to Xcr1 on cross-presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α(+) DCs and increased proliferation of antigen-specific T cells. DNA vaccines encoding dimeric Xcl1-hemagglutinin (HA) fusion proteins induced cytotoxic CD8(+) T-cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8(+) T-cell responses, targeting of antigen to Xcr1 induced CD4(+) Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α(+) DCs using Xcl1 represents a novel and promising method for induction of protective CD8(+) T-cell responses.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with an unknown molecular pathogenesis. A recent molecular focus has been the mutated neuroligin 3, neuroligin 3(R451C), in gain-of-function studies and for its role in induced impairment of synaptic function, but endoplasmic reticulum (ER) stress induced by mutated molecules also deserves investigation. We previously found two missense mutations, H246N and Y251S, in the gene-encoding synaptic cell adhesion molecule-1 (CADM1) in ASD patients, including cleavage of the mutated CADM1 and its intracellular accumulation. In this study, we found that the mutated CADM1 showed slightly reduced homophilic interactions in vitro but that most of its interactions persist. The mutated CADM1 also showed morphological abnormalities, including shorter dendrites, and impaired synaptogenesis in neurons. Wild-type CADM1 was partly localized to the ER of C2C5 cells, whereas mutated CADM1 mainly accumulated in the ER despite different sensitivities toward 4-phenyl butyric acid with chemical chaperone activity and rapamycin with promotion activity for degradation of the aggregated protein. Modeling analysis suggested a direct relationship between the mutations and the conformation alteration. Both mutated CADM1 and neuroligin 3(R451C) induced upregulation of C/EBP-homologous protein (CHOP), an ER stress marker, suggesting that in addition to the trafficking impairment, this CHOP upregulation may also be involved in ASD pathogenesis.

Combined detection of cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation-associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high-risk human papillomavirus (hrHPV)-positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV-positive women with no underlying high-grade disease, high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN-grade of the corresponding biopsy and second to CIN-grade stratified by the presence of 'normal' or 'abnormal' cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with ≤ CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV-positive scrapes of women with CIN2/3 (n = 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and ≥ 5 years). Methylation levels were determined by quantitative methylation-specific PCR and normal cytology scrapes of hrHPV-positive women with histologically ≤ CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3- and 6.2-fold in CIN2/3, respectively, and 143.5- and 454.9-fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5- and 13.6-fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.

The tumor suppressor, CADM1, is involved in cell adhesion and preferentially inactivated in invasive cancer. We have previously reported that CADM1 associates with an actin-binding protein, 4.1B/DAL-1, and a scaffold protein, membrane protein palmitoylated 3 (MPP3)/DLG3. However, underlying mechanism of tumor suppression by CADM1 is not clarified yet. Here, we demonstrate that MPP1/p55 and MPP2/DLG2, as well as MPP3, interact with both CADM1 and 4.1B, forming a tripartite complex. We then examined cell biological roles of CADM1 and its complex in epithelia using HEK293 cells. Among MPP1-3, MPP2 is recruited to the CADM1-4.1B complex in the early process of adhesion in HEK293 cells. By suppression of CADM1 expression using siRNA, HEK293 lose epithelia-like structure and show flat morphology with immature cell adhesion. 4.1B and MPP2, as well as E-cadherin and ZO-1, are mislocalized from the membrane by depletion of CADM1 in HEK293. Mislocalization of MPP2 is also observed in several cancer cells lacking CADM1 expression with the transformed morphology. These findings suggest that CADM1 is involved in the formation of epithelia-like cell structure with 4.1B and MPP2, while loss of its function could cause morphological transformation of cancer cells.

BACKGROUND

Undifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection.

METHODS

Nasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied.

RESULTS

NPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%.

CONCLUSIONS

Multiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.