Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

Differential CpG DNA methylation in peripheral naïve CD4+ T-cells in early rheumatoid arthritis patients.

BACKGROUND: The genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation, one of the hallmarks of epigenetic re-programing, as well as many T-cell genes, with a strong MHC association, pointing to immunogenetic mechanisms as disease triggers leading to chronicity. The aim of our study was to explore DNA methylation in early, drug-naïve RA patients, towards a better understanding of early events in pathogenesis. RESULT: Monocytes, naïve and memory CD4+ T-cells were sorted from 6 healthy controls and 10 RA patients. DNA methylation was assessed using a genome-wide Illumina 450K CpG promoter array. Differential methylation was confirmed using bisulfite sequencing for a specific gene promoter, ELISA for several cytokines and flow cytometry for cell surface markers. Differentially methylated (DM) CpGs were observed in 1047 genes in naïve CD4+ T-cells, 913 in memory cells and was minimal in monocytes with only 177 genes. Naive CD4+ T-cells were further investigated as presenting differential methylation in the promoter of > 500 genes associated with several disease-relevant pathways, including many cytokines and their receptors. We confirmed hypomethylation of a region of the TNF-alpha gene in early RA and differential expression of 3 cytokines (IL21, IL34 and RANKL). Using a bioinformatics package (DMRcate) and an in-house analysis based on differences in ß values, we established lists of DM genes between health and RA. Publicly available gene expression data were interrogated to confirm differential expression of over 70 DM genes. The lists of DM genes were further investigated based on a functional relationship database analysis, which pointed to an IL6/JAK1/STAT3 node, related to TNF-signalling and engagement in Th17 cell differentiation amongst many pathways. Five DM genes for cell surface markers (CD4, IL6R, IL2RA/CD25, CD62L, CXCR4) were investigated towards identifying subpopulations of CD4+ T-cells undergoing these modifications and pointed to a subset of naïve T-cells, with high levels of CD4, IL2R, and CXCR4, but reduction and loss of IL6R and CD62L, respectively. CONCLUSION: Our data provided novel conceptual advances in the understanding of early RA pathogenesis, with implications for early treatment and prevention.

Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma.

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)-ß-mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single-cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation.

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.

[Increased frequency of icterus in parenterally fed patients after a change of lipid emulsion]. / Toegenomen frequentie van icterus bij parenteraal gevoede patiënten na verandering van vetemulsie.

UNLABELLED: Parenteral nutrition is associated with liver enzyme abnormalities. Until 1993 the incidence of icterus was low in both academic hospitals in Amsterdam, the Netherlands (Academic Medical Centre (AMC) and Academic Hospital of the Free University (AZVU)). In 1993 Intralipid in the nutrition was replaced by Endolipid in the home total parenteral nutrition programme (AMC) and by Lipofundin S in AZVU. Fifty per cent of the patients in the home programme developed severe fatigue, jaundice and thrombocytopenia. These signs and symptoms disappeared over months when parenteral nutrition without fat was given. After reintroduction of Intralipid these signs and symptoms never recurred. In AZVU the incidence of jaundice increased from 21% in 1992 to 79% in 1993 (p = 0.0002). After reintroduction of Intralipid in 1994 the incidence of jaundice decreased to 16%. CONCLUSION: Although the lipid emulsions are equivalent according to the product specification, the described observation suggests that Lipofundin S and Endolipid cause more icterus than Intralipid, possibly caused bij an impurity in the fat emulsion.

Visceral fat loss measured by magnetic resonance imaging in relation to changes in serum lipid levels of obese men and women.

The effect of weight reduction on serum lipids in relation to visceral fat accumulation was studied in 78 healthy obese subjects (40 premenopausal women and 38 men) aged 27-51 years and with an initial body mass index of 30.7 +/- 2.2 kg/m2 (mean +/- SD). The subjects received a 4.2 MJ/day energy-deficit diet for 13 weeks. Magnetic resonance imaging was used to assess abdominal fat areas before and after weight loss. Weight reductions of 12.6 +/- 3.2 kg in men and 11.7 +/- 3.8 kg in women resulted in larger reductions in the fasting serum levels of total cholesterol (p < 0.05), low density lipoprotein cholesterol (p = 0.06), and triglycerides (p < 0.01) and a larger increase in the high density lipoprotein cholesterol/low density lipoprotein cholesterol ratio (p = 0.05) in men compared with women. Men also lost more visceral fat (p < 0.0001), whereas the reductions in the total and subcutaneous abdominal fat depots were similar. In women, visceral fat loss was significantly related with an increase of the high density lipoprotein cholesterol level, independent of the degree of total fat loss. In men, however, no significant correlations were observed between changes in visceral fat and any of the serum lipids. Comparisons of average changes in obese men and women suggest that visceral fat loss is associated with an improvement of the serum lipid profile. However, correlation analysis does not support a critical role of visceral fat in determining serum lipid concentrations on an individual level, except for an improvement of the high density lipoprotein cholesterol level with visceral fat loss in obese women.

Waist-hip ratio is a poor predictor of changes in visceral fat.

Magnetic resonance imaging was used to study the effect of weight loss on three fat depots: the visceral and subcutaneous abdominal depots and the subcutaneous depot at trochanter level. Changes in fat depots were compared with changes in circumference measures and the waist-hip ratio (WHR) in obese men (n = 38) and women (n = 40). Mean weight loss was (mean +/- SD) 12.9 +/- 3.5 kg (P < 0.001). The proportional reduction of fat was largest in the visceral depot (men 40%, women 33%). Less fat was lost subcutaneously, especially at trochanter level (men 29%, women 26%). WHR decreased significantly in both sexes (P < 0.001). Change in WHR was not significantly related to the absolute reduction in visceral fat. Total body-fat loss showed a stronger association with subcutaneous fat loss than with visceral fat loss. The findings suggest that fat distribution may change with weight loss, particularly by the loss of visceral fat, but changes in WHR are not appropriate for evaluating changes in this fat depot.