Nitric oxide produces HLA-G nitration and induces metalloprotease-dependent shedding creating a tolerogenic milieu

Human leucocyte antigen G (HLA-G) is a tolerogenic molecule that protects the fetus from maternal immune attack, may favour tumoral immunoescape and is up-regulated in viral and inflammatory diseases. The aim of this work was to discover if nitric oxide (NO) could affect HLA-G expression or function because NO is an important modulator of innate and adaptive immunity. For this purpose HLA-G expression and function were analysed following treatment with a NO donor or a peroxynitrite donor in various cell lines expressing HLA-G either spontaneously or upon transfection. Results showed NO-dependent nitration of both cellular and soluble HLA-G protein, but not all HLA-G moieties underwent nitration. Endogenous biosynthesis of NO by both U-937-HLA-G1 and M8-HLA-G5 stable transfectants also caused HLA-G nitration. The NO decreased total HLA-G cellular protein content and expression on the cell surface, while increasing HLA-G shedding into the culture medium. This effect was post-transcriptional and the result of metalloprotease activity. By contrast, NO pretreatment did not affect HLA-G capability to suppress NK cytotoxicity and lymphocyte proliferation. Our studies show that NO regulates the availability of HLA-G molecules without modifying their biological activities

Genome-wide DNA methylation pattern in visceral adipose tissue differentiates insulin-resistant from insulin-sensitive obese subject

Elucidating the potential mechanisms involved in the detrimental effect of excess body weight on insulin action is an important priority in counteracting obesity-associated diseases. The present study aimed to disentangle the epigenetic basis of insulin resistance by performing a genome-wide epigenetic analysis in visceral adipose tissue (VAT) from morbidly obese patients depending on the insulin sensitivity evaluated by the clamp technique. The global human methylome screening performed in VAT from 7 insulin-resistant (IR) and 5 insulin-sensitive (IS) morbidly obese patients (discovery cohort) analyzed using the Infinium HumanMethylation450 BeadChip array identified 982 CpG sites able to perfectly separate the IR and IS samples. The identified sites represented 538 unique genes, 10% of which were diabetes-associated genes. The current work identified novel IR-related genes epigenetically regulated in VAT, such as COL9A1, COL11A2, CD44, MUC4, ADAM2, IGF2BP1, GATA4, TET1, ZNF714, ADCY9, TBX5, and HDACM. The gene with the largest methylation fold-change and mapped by 5 differentially methylated CpG sites located in island/shore and promoter region was ZNF714. This gene presented lower methylation levels in IR than in IS patients in association with increased transcription levels, as further reflected in a validation cohort (n = 24; 11 IR and 13 IS). This study reveals, for the first time, a potential epigenetic regulation involved in the dysregulation of VAT that could predispose patients to insulin resistance and future type 2 diabetes in morbid obesity, providing a potential therapeutic target and biomarkers for counteracting this process

MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b

Cancer stem cells (CSCs) are a small part of the heterogeneous tumor cell population possessing self-renewal and multilineage differentiation potential as well as a great ability to sustain tumorigenesis. The molecular pathways underlying CSC phenotype are not yet well characterized. MicroRNAs (miRs) are small noncoding RNAs that play a powerful role in biological processes. Early studies have linked miRs to the control of self-renewal and differentiation in normal and cancer stem cells. We aimed to study the functional role of miRs in human breast cancer stem cells (BCSCs), also named mammospheres. We found that miR-221 was upregulated in BCSCs compared to their differentiated counterpart. Similarly, mammospheres from T47D cells had an increased level of miR-221 compared to differentiated cells. Transfection of miR-221 in T47D cells increased the number of mammospheres and the expression of stem cell markers. Among miR-221's targets, we identified DNMT3b. Furthermore, in BCSCs we found that DNMT3b repressed the expression of various stemness genes, such as Nanog and Oct 3/4, acting on the methylation of their promoters, partially reverting the effect of miR-221 on stemness. We hypothesize that miR-221 contributes to breast cancer tumorigenicity by regulating stemness, at least in part through the control of DNMT3b expression

Genome-wide profiling of p53-regulated enhancer RNAs uncovers a subset of enhancers controlled by a lncRNA

p53 binds enhancers to regulate key target genes. Here, we globally mapped p53-regulated enhancers by looking at enhancer RNA (eRNA) production. Intriguingly, while many p53-induced enhancers contained p53-binding sites, most did not. As long non-coding RNAs(lncRNAs) are prominent regulators of chromatin dynamics, we hypothesized that p53-induced lncRNAs contribute to the activation of enhancers by p53. Among p53-induced lncRNAs, we identified LED and demonstrate that its suppression attenuates p53 function. Chromatin-binding and eRNA expression analyses show that LED associates with and activates strong enhancers. One prominent target of LED was located at an enhancer region within CDKN1A gene, a potent p53-responsive cell cycle inhibitor. LED knockdown reduces CDKN1A enhancer induction and activity, and cell cycle arrest following p53 activation. Finally, promoter-associated hypermethylation analysis shows silencing of LED in human tumours. Thus, our study identifies a new layer of complexity in the p53 pathway and suggests its dysregulation in cancer

The role of clonal communication and heterogeneity in breast cancer

Background: Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones. Methods: We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays. Results: Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration. Conclusions: These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression

Identification of novel synthetic lethal vulnerability in non small cell lung cancer by co targeting TMPRSS4 and DDR1

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4

DNA methylation map in circulating leukocytes mirrors subcutaneous adipose tissue methylation pattern: a genome-wide analysis from non-obese and obese patients

The characterization of the epigenetic changes within the obesity-related adipose tissue will provide new insights to understand this metabolic disorder, but adipose tissue is not easy to sample in population-based studies. We aimed to evaluate the capacity of circulating leukocytes to reflect the adipose tissue-specific DNA methylation status of obesity susceptibility. DNA samples isolated from subcutaneous adipose tissue and circulating leukocytes were hybridized in the Infinium HumanMethylation 450 BeadChip. Data were compared between samples from obese (n = 45) and non-obese (n = 8-10) patients by Wilcoxon-rank test, unadjusted for cell type distributions. A global hypomethylation of the differentially methylated CpG sites (DMCpGs) was observed in the obese subcutaneous adipose tissue and leukocytes. The overlap analysis yielded a number of genes mapped by the common DMCpGs that were identified to reflect the obesity state in the leukocytes. Specifically, the methylation levels of FGFRL1, NCAPH2, PNKD and SMAD3 exhibited excellent and statistically significant efficiencies in the discrimination of obesity from non-obesity status (AUC > 0.80; p < 0.05) and a great correlation between both tissues. Therefore, the current study provided new and valuable DNA methylation biomarkers of obesity-related adipose tissue pathogenesis through peripheral blood analysis, an easily accessible and minimally invasive biological material instead of adipose tissue

Barriers of mental health treatment utilization among first-year college students: First cross-national results from the WHO World Mental Health International College Student Initiative.

BACKGROUND: Although mental disorders and suicidal thoughts-behaviors (suicidal thoughts and behaviors) are common among university students, the majority of students with these problems remain untreated. It is unclear what the barriers are to these students seeking treatment. AIMS: The aim of this study is to examine the barriers to future help-seeking and the associations of clinical characteristics with these barriers in a cross-national sample of first-year college students. METHOD: As part of the World Mental Health International College Student (WMH-ICS) initiative, web-based self-report surveys were obtained from 13,984 first-year students in eight countries across the world. Clinical characteristics examined included screens for common mental disorders and reports about suicidal thoughts and behaviors. Multivariate regression models adjusted for socio-demographic, college-, and treatment-related variables were used to examine correlates of help-seeking intention and barriers to seeking treatment. RESULTS: Only 24.6% of students reported that they would definitely seek treatment if they had a future emotional problem. The most commonly reported reasons not to seek treatment among students who failed to report that they would definitely seek help were the preference to handle the problem alone (56.4%) and wanting to talk with friends or relatives instead (48.0%). Preference to handle the problem alone and feeling too embarrassed were also associated with significantly reduced odds of having at least some intention to seek help among students who failed to report that they would definitely seek help. Having 12-month major depression, alcohol use disorder, and suicidal thoughts and behaviors were also associated with significantly reduced reported odds of the latter outcome. CONCLUSIONS: The majority of first-year college students in the WMH-ICS surveys report that they would be hesitant to seek help in case of future emotional problems. Attitudinal barriers and not structural barriers were found to be the most important reported reasons for this hesitation. Experimental research is needed to determine whether intention to seek help and, more importantly, actual help-seeking behavior could be increased with the extent to which intervention strategies need to be tailored to particular student characteristics. Given that the preference to handle problems alone and stigma and appear to be critical, there could be value in determining if internet-based psychological treatments, which can be accessed privately and are often build as self-help approaches, would be more acceptable than other types of treatments to student who report hesitation about seeking treatment.status: publishe

WHO World Mental Health Surveys International College Student Project: Prevalence and Distribution of Mental Disorders

Increasingly, colleges across the world are contending with rising rates of mental disorders, and in many cases, the demand for services on campus far exceeds the available resources. The present study reports initial results from the first stage of the WHO World Mental Health International College Student project, in which a series of surveys in 19 colleges across 8 countries (Australia, Belgium, Germany, Mexico, Northern Ireland, South Africa, Spain, United States) were carried out with the aim of estimating prevalence and basic sociodemographic correlates of common mental disorders among first-year college students. Web-based self-report questionnaires administered to incoming first-year students (45.5% pooled response rate) screened for six common lifetime and 12-month DSM-IV mental disorders: major depression, mania/hypomania, generalized anxiety disorder, panic disorder, alcohol use disorder, and substance use disorder. We focus on the 13,984 respondents who were full-time students: 35% of whom screened positive for at least one of the common lifetime disorders assessed and 31% screened positive for at least one 12-month disorder. Syndromes typically had onsets in early to middle adolescence and persisted into the year of the survey. Although relatively modest, the strongest correlates of screening positive were older age, female sex, unmarried-deceased parents, no religious affiliation, nonheterosexual identification and behavior, low secondary school ranking, and extrinsic motivation for college enrollment. The weakness of these associations means that the syndromes considered are widely distributed with respect to these variables in the student population. Although the extent to which cost-effective treatment would reduce these risks is unclear, the high level of need for mental health services implied by these results represents a major challenge to institutions of higher education and governments. (PsycINFO Database Record (c) 2018 APA, all rights reserved).status: publishe

Characterizing the invasive tumor front of aggressive uterine adenocarcinoma and leiomyosarcoma

The invasive tumor front (the tumor-host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors