Expression and regulation of mitogen-activated protein kinase (MAPK) phosphatases (MKPs) in adipogenesis

Obesity has reached epidemic proportions in western industrialized countries and can be linked to a number of health risks such as hypertension, type 2 diabetes, coronary heart disease, and certain types of cancer. Obesity is due to a positive energy balance in the body that results in expansion of the adipose tissue caused by both hyperplasia and hypertrophy of adipocytes. Previous studies have found that MAPK activity is required in early stages of adipogenesis. It has also been reported that two of the MKPs, phosphatases known to dephosphorylate MAPKs, have a role in adipocyte differentiation. Based on this knowledge we carried out a systematic analysis of the expression and possible regulation of members of the MKP family during differentiation of 3T3-L1 preadipocytes. Results showed that most members of the MKP family are down-regulated during adipogenesis. We hypothesized that ectopic expression of one of the MKPs that was significantly down-regulated at early stages of differentiation, MKP-5, would have an effect on adipogenesis. Retroviral expression of MKP-5 in 3T3-L1 cells had a positive effect on adipocyte differentiation. In rodent obesity models ob/ob and Ay, most of the MKPs showed up-regulation in white adipose tissue, including MKP-5, with especially dramatic differences in subcutaneous fat depots. Although further studies are needed to verify these data, the results suggest that MKP-5, and possibly other MKPs, may have distinct roles during adipogenesis

Plasma fatty acid levels and gene expression related to lipid metabolism in peripheral blood mononuclear cells: A cross-sectional study in healthy subjects

Background: Solid evidence indicates that intake of marine n-3 fatty acids lower serum triglycerides, and that replacing saturated fatty acids (SFA) with polyunsaturated fatty acids (PUFA) reduces plasma total cholesterol and LDL-cholesterol. The molecular mechanisms underlying these health beneficial effects are however not completely elucidated. The aim of this study was therefore to investigate the expression of genes related to lipid metabolism in peripheral blood mononuclear cells (PBMC) depending on the plasma levels of n-6 and n-3 39 fatty acids and the SFA to PUFA ratio. Methods: Fifty-four healthy subjects were grouped into tertiles (n=18) based on plasma levels of n-6 and n-3 fatty acids and the SFA to PUFA ratio. The PBMC gene expression levels among subjects in the highest versus the lowest tertiles were compared. In total, 285 genes related to cholesterol and triglyceride metabolism were selected for this explorative study. Results: Among the 285 selected genes, 161 were defined as expressed in the PBMCs. The 47 plasma SFA to PUFA ratio was associated with the highest number of significantly different expressed genes (25 gene transcripts), followed by plasma n-6 fatty acid level (15 gene transcripts) and plasma n-3 fatty acid level (8 gene transcripts). In particular, genes involved in cholesterol homeostasis were significantly different expressed among subjects with high compared to low plasma SFA to PUFA ratio. Conclusion: Genes involved in lipid metabolism were differentially expressed in PBMCs depending on the plasma fatty acid levels. This finding may increase our understanding of how fatty acids influence lipid metabolism at a molecular level in humans

Differences in peripheral blood mononuclear cell gene expression and triglyceride composition in lipoprotein subclasses in plasma triglyceride responders and non-responders to omega-3 supplementation

Background:Intake of the marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)reduces fasting triglyceride (TG) levels and may thereby lower cardiovascular disease risk. However, there are largeinter-individual differences in the TG-lowering effect of omega-3 supplementation. Genotype differences partly explainthis variation, but gene-environment interactions leading to gene expression differences may also be important. In thisstudy, we aimed to investigate baseline differences and differences in the change in peripheral blood mononuclear cell(PBMC) gene expression and lipoprotein subclass TG levels between TG responders and non-responders to omega-3fatty acid supplementation.Methods:In a previous randomized controlled trial, healthy normotriglyceridemic subjects (n= 35, 71% women)received 1.6 g EPA + DHA/day for 7 weeks. In this exploratory sub-study, we defined TG responders as subjects havinga TG reduction beyond the 20% day-to-day variation and non-responders as having a TG change between−20% and+ 20% after omega-3 supplementation. PBMC gene expression was measured using microarray, and lipoproteinsubclasses were measured using nuclear magnetic resonance spectroscopy.Results:Eight subjects were defined as responders with a median TG reduction of 37%, and 16 subjects were definedas non-responders with a median TG change of 0%. At baseline, responders had higher TG levels in two of four high-density lipoprotein (HDL) subclasses and 909 gene transcripts (p≤0.05) were differentially expressed compared to non-responders. During the intervention, the plasma TG reduction among responders was reflected in TG reductions in fourof six different very low-density lipoprotein subclasses and three of four different HDL subclasses. Compared to non-responders, the expression of 454 transcripts was differentially altered in responders (p≤0.05). Pathway analyses revealed that responders had altered signaling pathways related to development and immune function. In addition,two of the top 10 enriched pathways in responders compared to non-responders were related to lysophosphatidic acid signaling.Conclusion:TG responders and non-responders to omega-3 supplementation have different lipoprotein subclass andPBMC gene expression profiles at baseline and different lipoprotein subclass and PBMC gene expression responses toomega-3 supplementation. These gene expression differences may partially explain the variability in TG response observed after omega-3 supplementation

Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas

Background Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. Methods Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. Results The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. Conclusions ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors

Crosstalk between microRNA expression and DNA methylation drives the hormone-dependent phenotype of breast cancer

Background Abnormal DNA methylation is observed as an early event in breast carcinogenesis. However, how such alterations arise is still poorly understood. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play key roles in various biological processes. Here, we integrate miRNA expression and DNA methylation at CpGs to study how miRNAs may affect the breast cancer methylome and how DNA methylation may regulate miRNA expression. Methods miRNA expression and DNA methylation data from two breast cancer cohorts, Oslo2 (n = 297) and The Cancer Genome Atlas (n = 439), were integrated through a correlation approach that we term miRNA-methylation Quantitative Trait Loci (mimQTL) analysis. Hierarchical clustering was used to identify clusters of miRNAs and CpGs that were further characterized through analysis of mRNA/protein expression, clinicopathological features, in silico deconvolution, chromatin state and accessibility, transcription factor binding, and long-range interaction data. Results Clustering of the significant mimQTLs identified distinct groups of miRNAs and CpGs that reflect important biological processes associated with breast cancer pathogenesis. Notably, two major miRNA clusters were related to immune or fibroblast infiltration, hence identifying miRNAs associated with cells of the tumor microenvironment, while another large cluster was related to estrogen receptor (ER) signaling. Studying the chromatin landscape surrounding CpGs associated with the estrogen signaling cluster, we found that miRNAs from this cluster are likely to be regulated through DNA methylation of enhancers bound by FOXA1, GATA2, and ER-alpha. Further, at the hub of the estrogen cluster, we identified hsa-miR-29c-5p as negatively correlated with the mRNA and protein expression of DNA methyltransferase DNMT3A, a key enzyme regulating DNA methylation. We found deregulation of hsa-miR-29c-5p already present in pre-invasive breast lesions and postulate that hsa-miR-29c-5p may trigger early event abnormal DNA methylation in ER-positive breast cancer. Conclusions We describe how miRNA expression and DNA methylation interact and associate with distinct breast cancer phenotypes

Subtype and cell type specific expression of lncRNAs provide insight into breast cancer

Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer

Widespread alternative exon usage in clinically distinct subtypes of Invasive Ductal Carcinoma

Cancer cells can have different patterns of exon usage of individual genes when compared to normal tissue, suggesting that alternative splicing may play a role in shaping the tumor phenotype. The discovery and identification of gene variants has increased dramatically with the introduction of RNA-sequencing technology, which enables whole transcriptome analysis of known, as well as novel isoforms. Here we report alternative splicing and transcriptional events among subtypes of invasive ductal carcinoma in The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) cohort. Alternative exon usage was widespread, and although common events were shared among three subtypes, ER+ HER2−, ER− HER2−, and HER2+, many events on the exon level were subtype specific. Additional RNA-seq analysis was carried out in an independent cohort of 43 ER+ HER2− and ER− HER2− primary breast tumors, confirming many of the exon events identified in the TCGA cohort. Alternative splicing and transcriptional events detected in five genes, MYO6, EPB41L1, TPD52, IQCG, and ACOX2 were validated by qRT-PCR in a third cohort of 40 ER+ HER2− and ER− HER2− patients, showing that these events were truly subtype specific

The expression of the long NEAT1_2 isoform is associated with human epidermal growth factor receptor 2-positive breast cancers

The long non-coding RNA NEAT1 locus is transcribed into two overlapping isoforms, NEAT1_1 and NEAT1_2, of which the latter is essential for the assembly of nuclear paraspeckles. NEAT1 is abnormally expressed in a wide variety of human cancers. Emerging evidence suggests that the two isoforms have distinct functions in gene expression regulation, and recently it was shown that NEAT1_2, but not NEAT1_1, expression predicts poor clinical outcome in cancer. Here, we report that NEAT1_2 expression correlates with HER2-positive breast cancers and high-grade disease. We provide evidence that NEAT1_1 and NEAT1_2 have distinct expression pattern among different intrinsic breast cancer subtypes. Finally, we show that NEAT1_2 expression and paraspeckle formation increase upon lactation in humans, confirming what has previously been demonstrated in mice

DataSheet_1_Complex molecular profile of DNA repair genes in epithelial ovarian carcinoma patients with different sensitivity to platinum-based therapy.pdf

Epithelial ovarian carcinoma (EOC) is known for high mortality due to diagnosis at advanced stages and frequent therapy resistance. Previous findings suggested that the DNA repair system is involved in the therapeutic response of cancer patients and DNA repair genes are promising targets for novel therapies. This study aimed to address complex inter-relations among gene expression levels, methylation profiles, and somatic mutations in DNA repair genes and EOC prognosis and therapy resistance status. We found significant associations of DUT expression with the presence of peritoneal metastases in EOC patients. The high-grade serous EOC subtype was enriched with TP53 mutations compared to other subtypes. Furthermore, somatic mutations in XPC and PRKDC were significantly associated with worse overall survival of EOC patients, and higher FAAP20 expression in platinum-resistant than platinum-sensitive patients was observed. We found higher methylation of RAD50 in platinum-resistant than in platinum-sensitive patients. Somatic mutations in BRCA1 and RAD9A were significantly associated with higher RBBP8 methylation in platinum-sensitive compared to platinum-resistant EOC patients. In conclusion, we discovered associations of several candidate genes from the DNA repair pathway with the prognosis and platinum resistance status of EOC patients, which deserve further validation as potential predictive biomarkers.</p

Ubiquitination of Fibroblast Growth Factor Receptor 1 Is Required for Its Intracellular Sorting but Not for Its Endocytosis

Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation