Citations

Abstract An ovarian cancer Genome Wide Association Study (GWAS) identified 9p22.2 as a novel susceptibility locus with the most statistically significant single nucleotide polymorphisms (SNPs) located in an intergenic region near the Basonuclin 2 (BNC2) gene, which codes for a putative transcription regulator containing three pairs of zinc finger (ZF) domains. The minor alleles are protective in terms of ovarian cancer susceptibility. However, the molecular mechanisms by which these SNPs modify susceptibility remain largely unknown. The significant SNPs in the 9p22.2 locus lie in non-coding regions and therefore are hypothesized to affect the activity of regulatory elements and modify the expression of a target gene(s). In order to test this hypothesis we conducted fine mapping for the locus which delimited a ∼74kb region containing SNPs with a p-value less than 10-8. FAIRE-Seq and ChIP-Seq experiments for histone markers conducted in immortalized ovarian surface epithelial cells (IOSE) and fallopian tube epithelial cells (IFTE) were used to prioritize functional SNP candidates which overlap with regulatory elements. Luciferase assays tested the ability of these regulatory elements to activate transcription. Chromosome conformation capture (3C) experiments demonstrate a physical interaction between the BNC2 promoter and candidate regulatory elements containing risk-associated SNPs. Methylation Quantification at Trait Loci (mQTL) revealed an association between decreased methylation at the BNC2 promoter and the protective minor alleles. Expression analysis shows decreased expression of BNC2 in cancer versus normal tissue implicating tumor suppressor function of BNC2. Therefore the protective minor allele likely increases expression of BNC2 which in turn contains tumor suppressing properties that decrease ovarian cancer risk. BNC2 protein is in complex with transcriptional regulatory proteins, in particular the NURD complex components. Moreover, BNC2 acts as a transcriptional repressor in in vitro transfection assays indicating that it functions in transcriptional repression. We then used protein binding microarrays and CHIP-Seq experiments to identify its putative DNA binding motifs and its downstream target genes. Analysis of this data suggests that BNC2 functions in a regulatory transcription network that impacts on genes implicated in ovarian cancer with enrichment for genes in the TGF-beta response pathway. Citation Format: Melissa A. Buckley, Howard C. Shen, Gustavo A. Mendoza-Fandino, Nicholas T. Woods, Anxhela Gjyshi, Juliet French, Kate Lawrenson, Honglin Song, Jonathan Tyrer, Renato S. Carvalho, Alexandra Valle, Ann Chen, Sean Yoder, Gregory Bloom, Ya-Yu Tsai, Ally Yang, Timothy R. Hughes, Xiaotao Qu, Mine Cicek, Melissa Larson, Ellen Goode, Brooke Fridley, Susan Ramus, Georgia Chenevix-Trench, Paul Pharoah, Thomas A. Sellers, Simon Gayther, Alvaro N.A. Monteiro, Ovarian Cancer Association Consortium. Functional analysis of the 9p22 locus implicates the transcriptional regulation of BNC2 as a mechanism in ovarian cancer predisposition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2014-3285

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.

The failing ventricular myocardium is characterized by reduction of high-energy phosphates and reduced activity of the phosphotransfer enzymes creatine kinase (CK) and adenylate kinase (AK), which are responsible for transfer of high-energy phosphoryls from sites of production to sites of utilization, thereby compromising excitation-contraction coupling. In humans with chronic atrial fibrillation (AF) unassociated with congestive heart failure (CHF), impairment of atrial myofibrillar energetics linked to oxidative modification of myofibrillar CK has been observed. However, the bioenergetic status of the failing atrial myocardium and its potential contribution to atrial electrical instability in CHF have not been determined. Dogs with ( n = 6) and without ( n = 6) rapid pacing-induced CHF underwent echocardiography (conscious) and electrophysiological (under anesthesia) studies. CHF dogs had more pronounced mitral regurgitation, higher atrial pressure, larger atrial area, and increased atrial fibrosis. An enhanced propensity to sustain AF was observed in CHF, despite significant increases in atrial effective refractory period and wavelength. Profound deficits in atrial bioenergetics were present with reduced activities of the phosphotransfer enzymes CK and AK, depletion of high-energy phosphates (ATP and creatine phosphate), and reduction of cellular energetic potential (ATP-to-ADP and creatine phosphate-to-Cr ratios). AF duration correlated with left atrial area ( r = 0.73, P = 0.01) and inversely with atrial ATP concentration ( r = −0.75, P = 0.005), CK activity ( r = −0.57, P = 0.054), and AK activity ( r = −0.64, P = 0.02). Atrial levels of malondialdehyde, a marker of oxidative stress, were significantly increased in CHF. Myocardial bioenergetic deficits are a conserved feature of dysfunctional atrial and ventricular myocardium in CHF and may constitute a component of the substrate for AF in CHF.

We used whole‐exome sequencing to identify variants other than APOE associated with the rate of hippocampal atrophy in amnestic mild cognitive impairment. An in‐silico predicted missense variant in REST (rs3796529) was found exclusively in subjects with slow hippocampal volume loss and validated using unbiased whole‐brain analysis and meta‐analysis across 5 independent cohorts. REST is a master regulator of neurogenesis and neuronal differentiation that has not been previously implicated in Alzheimer's disease. These findings nominate REST and its functional pathways as protective and illustrate the potential of combining next‐generation sequencing with neuroimaging to discover novel disease mechanisms and potential therapeutic targets. Ann Neurol 2015;77:547–552

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

Abstract This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM. Mol Cancer Ther; 13(12); 2919–29. ©2014 AACR.