Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine.

BACKGROUND: Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.

An ABCA1 truncation shows no dominant negative effect in a familial hypoalphalipoproteinemia pedigree with three ABCA1 mutations.

The ATP binding cassette transporter (ABCA1) A1 is a key determinant of circulating high density lipoprotein cholesterol (HDL-C) levels. Mutations in ABCA1 are a major genetic contributor to low HDL-C levels within the general population. Following the finding of three different ABCA1 mutations, p.C978fsX988, p.T1512M and p.N1800H in a subject with hypoalphalipoproteinemia, we aimed to establish whether the p.C978fsX988 truncation exerted a dominant negative effect on the full-length ABCA1 alleles within family members as has been reported for other ABCA1 truncations. Characterisation of the p.C978fsX988 mutant in transfected HEK 293 cells showed it to be expressed as a GFP fusion protein but lacking in cholesterol efflux function. This was in keeping with results from cholesterol efflux assays in the fibroblasts of p.C978fsX988 carriers which also showed impaired efflux. Allele- specific quantification of p.C978fsX988 mRNA and analysis of ABCA1 protein levels in the fibroblasts of p.C978fsX988 heterozygotes showed negligible levels of mRNA and protein expression. There was no evidence of a dominant negative effect on wildtype or p.N1800H protein levels. We conclude that in the case of the p.C978fsX988 truncated mutant a lack of expression precludes it from having a dominant negative effect.

Association of Schizophrenia Risk With Disordered Niacin Metabolism in an Indian Genome-wide Association Study.

Importance: Genome-wide association studies (GWASs) in European populations have identified more than 100 schizophrenia-associated loci. A schizophrenia GWAS in a unique Indian population offers novel findings. Objective: To discover and functionally evaluate genetic loci for schizophrenia in a GWAS of a unique Indian population. Design, Setting, and Participants: This GWAS included a sample of affected individuals, family members, and unrelated cases and controls. Three thousand ninety-two individuals were recruited and diagnostically ascertained via medical records, hospitals, clinics, and clinical networks in Chennai and surrounding regions. Affected participants fulfilled DSM-IV diagnostic criteria for schizophrenia. Unrelated control participants had no personal or family history of psychotic disorder. Recruitment, genotyping, and analysis occurred in consecutive phases beginning January 1, 2001. Recruitment was completed on February 28, 2018, and genotyping and analysis are ongoing. Main Outcomes and Measures: Associations of single-nucleotide polymorphisms and gene expression with schizophrenia. Results: The study population included 1321 participants with schizophrenia, 885 family controls, and 886 unrelated controls. Among participants with schizophrenia, mean (SD) age was 39.1 (11.4) years, and 52.7% were male. This sample demonstrated uniform ethnicity, a degree of inbreeding, and negligible rates of substance abuse. A novel genome-wide significant association was observed between schizophrenia and a chromosome 8q24.3 locus (rs10866912, allele A; odds ratio [OR], 1.27 [95% CI, 1.17-1.38]; P = 4.35 × 10-8) that attracted support in the schizophrenia Psychiatric Genomics Consortium 2 data (rs10866912, allele A; OR, 1.04 [95% CI, 1.02-1.06]; P = 7.56 × 10-4). This locus has undergone natural selection, with the risk allele A declining in frequency from India (approximately 72%) to Europe (approximately 43%). rs10866912 directly modifies the abundance of the nicotinate phosphoribosyltransferase gene (NAPRT1) transcript in brain cortex (normalized effect size, 0.79; 95% CI, 0.6-1.0; P = 5.8 × 10-13). NAPRT1 encodes a key enzyme for niacin metabolism. In Indian lymphoblastoid cell lines, (risk) allele A of rs10866912 was associated with NAPRT1 downregulation (AA: 0.74, n = 21; CC: 1.56, n = 17; P = .004). Preliminary zebrafish data further suggest that partial loss of function of NAPRT1 leads to abnormal brain development. Conclusions and Relevance: Bioinformatic analyses and cellular and zebrafish gene expression studies implicate NAPRT1 as a novel susceptibility gene. Given this gene's role in niacin metabolism and the evidence for niacin deficiency provoking schizophrenialike symptoms in neuropsychiatric diseases such as pellagra and Hartnup disease, these results suggest that the rs10866912 genotype and niacin status may have implications for schizophrenia susceptibility and treatment.

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 kg/m2 (95% CI - 1.06 to - 0.19; p = 0.005) 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.

TAp63 regulates oncogenic miR-155 to mediate migration and tumour growth.

miR-155 is an oncogenic microRNA which is upregulated in many solid cancers. The targets of miR-155 are well established , with over 100 confirmed mRNA targets. However, the regulation of miR-155 and the basis of its upregulation in cancer is not well understood. We have previously shown that miR-155 is regulated by p63, and here we investigate the role of the major p63 isoforms TAp63 and ΔNp63 in this regulation. When the TAp63 isoform was knocked down, or exogenously overexpressed, miR-155 levels were elevated in response to TAp63 knockdown or reduced in response to TAp63 overexpression. The ΔNp63 isoform is shown to directly bind to the p63 response element on the miR-155 host gene, and this binding is enriched when TAp63 is knocked down. This could indicate that TAp63 prevents ΔNp63 from binding to the miR-155 host gene. The knockdown of TAp63, and the subsequent elevation of miR-155, enhances migration and tumour growth similar to that seen when directly overexpressing miR-155. The migratory phenotype is abrogated when miR-155 is inhibited, indicating that miR-155 is responsible for the phenotypic effect of TAp63 knockdown.

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.

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 of arterial proteins which may be early indicators of the metabolic disturbances preceding atherosclerosis.

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 2kb 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.

A comparison of vitamin D activity in paired non-malignant and malignant human breast tissues.

Links between a low vitamin D status and an increased risk of breast cancer have been observed in epidemiological studies. These links have been investigated in human tissue homogenates and cultured cell lines. We have used non-malignant, malignant and normal reduction mammoplasty breast tissues to investigate the biological and metabolic consequences of the application of vitamin D to intact ex vivo human breast tissue. Tissues were exposed to 1α,25(OH)(2)D(3) (1,25D; active metabolite) and 25(OH)D (25D; pre-metabolite). Changes in mRNA expression and protein expression after vitamin D exposure were analysed. Results indicate that while responses in normal and non-malignant breast tissues are similar between individuals, different tumour tissues are highly variable with regards to their gene expression and biological response. Collectively, malignant breast tissue responds well to active 1,25D, but not to the inactive pre-metabolite 25D. This may have consequences for the recommendation of vitamin D supplementation in breast cancer patients.

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.

Mutant p53 uses p63 as a molecular chaperone to alter gene expression and induce a pro-invasive secretome.

Mutations in the TP53 gene commonly result in the expression of a full-length protein that drives cancer cell invasion and metastasis. Herein, we have deciphered the global landscape of transcriptional regulation by mutant p53 through the application of a panel of isogenic H1299 derivatives with inducible expression of several common cancer-associated p53 mutants. We found that the ability of mutant p53 to alter the transcriptional profile of cancer cells is remarkably conserved across different p53 mutants. The mutant p53 transcriptional landscape was nested within a small subset of wild-type p53 responsive genes, suggesting that the oncogenic properties of mutant p53 are conferred by retaining its ability to regulate a defined set of p53 target genes. These mutant p53 target genes were shown to converge upon a p63 signalling axis. Both mutant p53 and wild-type p63 were co-recruited to the promoters of these target genes, thus providing a molecular basis for their selective regulation by mutant p53. We demonstrate that mutant p53 manipulates the gene expression pattern of cancer cells to facilitate invasion through the release of a pro-invasive secretome into the tumor microenvironment. Collectively, this study provides mechanistic insight into the complex nature of transcriptional regulation by mutant p53 and implicates a role for tumor-derived p53 mutations in the manipulation of the cancer cell secretome.