The Oncogenic role of miR-155 in breast cancer

miR-155 is an oncogenic miRNA with well described roles in leukemia. However, additional roles of miR-155 in breast cancer progression have recently been described. A thorough literature search was conducted to review all published data to date, examining the role of miR-155 in breast cancer. Data on all validated miR-155 target genes was collated to identify biologic pathways relevant to miR-155 and breast cancer progression. Publications describing the clinical relevance, functional characterization, and regulation of expression of miR-155 in the context of breast cancer are reviewed. A total of 147 validated miR-155 target genes were identified from the literature. Pathway analysis of these genes identified likely roles in apoptosis, differentiation, angiogenesis, proliferation, and epithelial-mesenchymal transition. The large number of validated miR-155 targets presented here provide many avenues of interest as to the clinical potential of miR-155. Further investigation of these target genes will be required to elucidate the specific mechanisms and functions of miR-155 in breast cancer. This is the first review examining the role of miR-155 in breast cancer progression. The collated data of target genes and biologic pathways of miR-155 identified in this review suggest new avenues of research for this oncogenic miRNA.Sam Mattiske, Rachel J. Suetani, Paul M. Neilsen, and David F. Calle

Proteomic Analysis of Aortae from Human Lipoprotein(a) Transgenic Mice Shows an Early Metabolic Response Independent of Atherosclerosis

Background: Elevated low density lipoprotein (LDL) and lipoprotein(a) are independent risk factors for the development of atherosclerosis. Using a proteomic approach we aimed to determine early changes in arterial protein expression in transgenic mice containing both human LDL and lipoprotein(a) in circulation. Methods and Results: Plasma lipid analyses showed the lipoprotein(a) transgenic mice had significantly higher lipid levels than wildtype, including a much increased LDL and high density lipoprotein (HDL) cholesterol. Analysis of aortae from lipoprotein(a) mice showed lipoprotein(a) accumulation but no lipid accumulation or foam cells, leaving the arteries essentially atherosclerosis free. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 34 arterial proteins with significantly altered abundance (P,0.05) in lipoprotein(a) transgenic mice compared to wildtype including 17 that showed a $2 fold difference. Some proteins of interest showed a similarly altered abundance at the transcript level. These changes collectively indicated an initial metabolic response that included a down regulation in energy, redox and lipid metabolism proteins and changes in structural proteins at a stage when atherosclerosis had not yet developed. Conclusions: Our study shows that human LDL and lipoprotein(a) promote changes in the expression of a unique set o

Genetic variants impacting metabolic outcomes among people on clozapine: a systematic review and meta-analysis

Clozapine is the gold standard medication for treatment refractory schizophrenia, but unfortunately, its use is also associated with many adverse metabolic side effects. There may be a strong genetic component to the development of these adverse effects. We undertook a systematic review to examine the evidence for genetic variation being associated with secondary metabolic outcomes in patients with schizophrenia on clozapine, under both longitudinal and cross-sectional study designs. We limited studies to those examining patients definitely taking clozapine, unlike prior reviews that have examined metabolic effects of patients taking a range of antipsychotic medications. We found associations with outcomes such as increases in BMI and metabolic syndrome for variants in genes such as LEP and HTR2C. Meta-analysis of rs381328 in HTR2C revealed that the presence of the T allele led to a 0.63\ua0kg/m (95% CI −\ua01.06 to −\ua00.19; p\ua0=\ua00.005) decrease in BMI compared to the C allele. Study and population heterogeneity and lack of statistical power among reviewed articles mean that evidence is lacking to warrant prophylactic genotyping of patients commencing clozapine to predict those at increased risk of developing adverse metabolic effects. Further efforts to establish collaborative consortia, consensus around study design and replication studies in independent populations should be encouraged

Homology modeling and functional testing of an ABCA1 mutation causing Tangier disease

Objective: To investigate the impact of the p.R1068H mutation on the structure and function of the ATP-binding cassette A1 (ABCA1) protein. Methods: A homology model of the nucleotide binding domains of ABCA1 was constructed to identify the three-dimensional orientation of R1068. Cholesterol efflux assays were performed on fibroblasts obtained from members of a Tangier disease (TD) family carrying the p.R1068H mutation and in HEK293 cells transfected with a p.R1068H mutant cDNA vector. Confocal microscopy was used to investigate the localisation of the wildtype and mutant p.R1068H protein in HEK293 cells. Results: Sequence alignments and modeling indicated residue R1068 to be located in an α-helix downstream of the Walker B motif in the first nucleotide binding domain (NBD-1), in a position to form ionic interactions with D1092 and E1093. Cholesterol efflux studies showed the efflux from TD fibroblasts and HEK293 cells expressing the mutant p.R1068H protein to be markedly reduced compared to wildtype. Localisation of the mutant p.R1068H protein in HEK293 cells showed intracellular retention of the protein indicating a defect in trafficking to the plasma membrane. Conclusion: Homology modeling of the ABCA1 protein showed that the p.R1068H mutation would likely disrupt the conformation of NBD-1. Functional studies of p.R1068H showed a lack of cholesterol efflux function due to defective trafficking to the plasma membrane, most likely caused by impaired oligomerisation

Specific-site methylation of tumour suppressor ANKRD11 in breast cancer

ANKRD11 is a putative tumour suppressor gene in breast cancer, which has been shown in our laboratory to be a co-activator of p53. Our data suggest that down-regulation of ANKRD11 is associated with breast tumourigenesis. Breast cancer cell lines treated with DNA demethylating agents resulted in up-regulation of ANKRD11 expression suggesting that promoter DNA methylation may be responsible for its down-regulation. The transcriptional activity of a CpG-rich region 2 kb upstream of the transcription initiation site of ANKRD11 was investigated using dual-luciferase reporter assays. The constructs carrying -661 to -571 bp promoter sequence showed significant transcriptional activity. Using the SEQUENOM Epityper Platform, the region between -770 and +399 bp was analysed in 25 breast tumours, four normal breast tissues and five normal blood samples. The region between -770 and -323 bp was shown to be frequently methylated in breast tumours. The methylation patterns of all analysed CpGs in this region were identical in the normal and tumour samples, except for a 19 bp region containing three CpG sites. These sites had significantly higher levels of methylation in tumours (40%) compared to normal samples (6%). Our findings support the role of ANKRD11 as a tumour suppressor gene and suggest that aberrant DNA methylation of three CpGs in a 19 bp region within the ANKRD11 promoter may be responsible for its down-regulation in breast cancer

Comparison of aorta lipids in wildtype and Lp(a) mice.

<p>A, Total cholesterol (TC), triglyceride (TG) and phospholipids (PL) in homogenized aorta lipid extracts (n = 6). TC and TG concentrations in the aorta of Lp(a) mice were significantly reduced compared to wildtype mice. B, Lp(a) mice had a significantly elevated concentration of thiobarbituric acid-reactive substances (TBARS) in the aorta compared to wildtype suggesting an accumulation of aldehydes from lipid oxidation. Data represented as mean concentration ± SEM.^**<i>P</i><0.01 versus wildtype.</p

RT-PCR analysis of transcripts of interest.

<p>Quantitative RT-PCR was performed to investigate if proteins of interest showing a ≥2 fold change at the protein level were also regulated at the mRNA transcript level between the Lp(a) and wildtype mice. Total RNA was isolated from aorta samples (n = 6) of wildtype and Lp(a) mice. RNA was reverse transcribed to cDNA and quantitative PCR for transcripts of interest relative to 18S rRNA was performed. Glucose-6-phosphate dehydrogenase (G6pdx) and peroxiredoxin 4 (Prdx4) transcripts were increased in Lp(a) mice compared to wildtype. Dihydrolipopoyllysine succinyltransferase (Dlst), Glycerol-3-phosphate dehydrogenase (Gpd1) and fatty acid-binding protein 4 (Fabp4) transcripts were decreased in Lp(a) mice compared to wildtype. Isocitrate dehydrogenase (Idh3a) transcript showed no significant difference in Lp(a) mice compared to wildtype mice. Results are presented as relative levels of transcript normalized to wildtype. ^***<i>P</i><0.001 versus wildtype.</p

Representative histological analysis of aortae from wildtype and Lp(a) mice.

<p>Aortic arches and sinuses of wildtype and Lp(a) mice (n = 8) were stained with haematoxylin and eosin (A and B) or Verhoeff's elastic stain and Curtis' modified van Gieson stain (C and D), which stained the elastic laminar (black), smooth muscle (brown), and collagen-rich fibrous tissue (red/pink). There was no evidence of atherosclerosis in the arteries of Lp(a) mice or wildtype mice, including no evidence of foam cells in the aortic arch or aortic sinus. Aortic arches were also immunostained with an anti-human Lp(a) antibody (brown) and counterstained with haematoxylin (blue). E, The wildtype mice were negative for Lp(a). F, The Lp(a) mice showed staining with the Lp(a)-specific antibody, indicating retention of Lp(a) in the arterial wall. Scale bar represents 100 µm.</p