👤 Sarah C Edwards

The purpose of these studies was to evaluate the effects of evacetrapib on male and female fertility and on embryo-fetal development (EFD). Evacetrapib, a potent and selective inhibitor of cholesteryl ester transfer protein (CETP), was administered daily by oral gavage starting 2 weeks (for female) or 4 weeks (for male) before mating, during cohabitation, and until necropsy in the male rat fertility study or through gestation day (GD) 17 in the female rat combined fertility/EFD study. For rabbit EFD studies, animals were dosed from GDs 7 to 19 or from 1 week before mating through GD 19. Dose levels of evacetrapib ranged from 60 to 600 mg/kg for rats and from 1 to 100 mg/kg/day for rabbits. Parental findings in rats included decreased body weight and food consumption and moribund euthanasia in animals given 600 mg/kg/day and decreased food consumption at 300 mg/kg/day. There were no adverse effects on estrus cycling, fertility indices, sperm parameters, maternal reproductive parameters, male reproductive tissue, or fetal viability, growth, or external/visceral morphology. An increase in the incidence of 14th rudimentary ribs, a minor, transient variation considered nonadverse, was the only significant developmental finding in rats given 600 mg/kg/day. Slight decreases in body weight and food consumption at 100 mg/kg/day were the only maternal effects observed in rabbits with no adverse developmental effects noted. No adverse effects on fertility or EFD were observed in rats at doses up to 600 mg/kg/day and no adverse effects on EFD were noted in rabbits at doses up to 100 mg/kg/day. Birth Defects Research 109:513-527, 2017. © 2017 Wiley Periodicals, Inc. Show less

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10-5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression. Show less

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer. Show less

White blood cells play diverse roles in innate and adaptive immunity. Genetic association analyses of phenotypic variation in circulating white blood cell (WBC) counts from large samples of otherwise healthy individuals can provide insights into genes and biologic pathways involved in production, differentiation, or clearance of particular WBC lineages (myeloid, lymphoid) and also potentially inform the genetic basis of autoimmune, allergic, and blood diseases. We performed an exome array-based meta-analysis of total WBC and subtype counts (neutrophils, monocytes, lymphocytes, basophils, and eosinophils) in a multi-ancestry discovery and replication sample of ∼157,622 individuals from 25 studies. We identified 16 common variants (8 of which were coding variants) associated with one or more WBC traits, the majority of which are pleiotropically associated with autoimmune diseases. Based on functional annotation, these loci included genes encoding surface markers of myeloid, lymphoid, or hematopoietic stem cell differentiation (CD69, CD33, CD87), transcription factors regulating lineage specification during hematopoiesis (ASXL1, IRF8, IKZF1, JMJD1C, ETS2-PSMG1), and molecules involved in neutrophil clearance/apoptosis (C10orf54, LTA), adhesion (TNXB), or centrosome and microtubule structure/function (KIF9, TUBD1). Together with recent reports of somatic ASXL1 mutations among individuals with idiopathic cytopenias or clonal hematopoiesis of undetermined significance, the identification of a common regulatory 3' UTR variant of ASXL1 suggests that both germline and somatic ASXL1 mutations contribute to lower blood counts in otherwise asymptomatic individuals. These association results shed light on genetic mechanisms that regulate circulating WBC counts and suggest a prominent shared genetic architecture with inflammatory and autoimmune diseases. Show less

The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC. Show less

Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve-Melchior-Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin-deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation. Show less

Prostate enlargement leading to clinical benign prostatic hyperplasia (BPH) is associated with metabolic dysregulation and obesity. The genetic basis of this association is unclear. Our objective was to evaluate whether single nucleotide polymorphisms (SNPs) previously associated with metabolic disorders are also associated with prostate volume (PV). Participants included 876 men referred for prostate biopsy and found to be prostate cancer free. PV was measured by transrectal ultrasound. Samples were genotyped using the Illumina Cardio-MetaboChip platform. Multivariable adjusted linear regression models were used to evaluate SNPs (additive coding) in relation to natural-log transformed (log) PV. We compared SNP-PV results from biopsy-negative men to 442 men with low-grade prostate cancer with similar levels of obesity and PV. Beta-coefficients from the discovery and replication samples were then aggregated with fixed effects inverse variance weighted meta-analysis. SNP rs11736129 (near the pseudo-gene LOC100131429) was significantly associated with log-PV (beta: 0.16, p-value 1.16x10(-8)) after adjusting for multiple testing. Other noteworthy SNPs that were nominally associated (p-value < 1x10(-4)) with log-PV included rs9583484 (intronic SNP in COL4A2), rs10146527 (intronic SNP in NRXN3), rs9909466 (SNP near RPL32P31), and rs2241606 (synonymous SNP in SLC12A7). We found several SNPs in metabolic loci associated with PV. Further studies are needed to confirm our results and elucidate the mechanism between these genetic loci, PV, and clinical BPH. Show less

Central nervous system hemangioblastomas are the most common manifestation of von Hippel-Lindau (VHL) disease, an autosomal dominant tumor suppressor syndrome that results in loss of VHL protein function and continuous upregulation of hypoxia-inducible factors. These tumors are composed of neoplastic stromal cells and abundant vasculature. Stromal cells express markers consistent with multipotent embryonically arrested hemangioblasts, which are precursors for hematopoietic and vascular lineages. Notch receptors are transmembrane signaling molecules that regulate multiple developmental processes including hematopoiesis and vasculogenesis. To investigate the importance of notch signaling in the development of VHL disease-associated CNS hemangioblastomas, the authors examined the presence of the four notch receptors and downstream notch effectors in this setting. The authors used surgical specimens obtained from confirmed VHL-associated hemangioblastomas. Immunohistochemical analysis for the four notch receptors and the downstream effectors was performed on formalin-fixed paraffin-embedded sections. Western blot analysis for HES1 was performed on frozen specimens. All four notch receptors are present in hemangioblastomas. NOTCH1 and NOTCH4 receptors were widely and prominently expressed in both the stromal and vascular cells, NOTCH2 receptor expression was limited to primarily stromal cells, and NOTCH3 receptor expression was limited to vascular cells. All 4 receptors displayed a nuclear presence. Immunohistochemical analysis also demonstrated that downstream notch effectors, HES1 and HES5, were uniformly expressed in tumor stromal and vascular cells, but HES3, HEY1, and HEY2 were not. Strong HES1 expression was confirmed by Western blot analysis. The presence of all four notch receptors and downstream effector molecules suggests that the notch signaling pathway plays a critical role in the maintenance of the undifferentiated pluripotent phenotype of these tumors and in the associated vascular response. Moreover, the prominent expression of notch receptors in VHL-associated CNS hemangioblastomas reveals a new and possibly potent therapeutic target. Show less

Aberrant mitogen/extracellular signal-regulated kinase 5 (MEK5)-extracellular signal-regulated protein kinase 5 (ERK5)-mediated signalling has been implicated in a number of tumour types including prostate cancer (PCa). The molecular basis of ERK5-driven carcinogenesis and its clinical relevance remain to be fully characterised. Modulation of ERK5 expression or function in human PCa PC3 and PC3-ERK5 (stably transfected with ERK5) cells was performed using siRNA-mediated knockdown or the MEK inhibitor PD18435 respectively. In vitro significance of ERK5 signalling was assessed by assays for proliferation, motility, invasion and invadopodia. Expression of matrix metalloproteinases/tissue inhibitors of metalloproteases was determined by Q-RT-PCR. Extracellular signal-regulated protein kinase 5 expression in primary and metastatic PCa was examined using immunohistochemistry. Reduction of ERK5 expression or signalling significantly inhibited the motility and invasive capability of PC3 cells. Extracellular signal-regulated protein kinase 5-mediated signalling significantly promoted formation of in vivo metastasis in an orthotopic PCa model (P<0.05). Invadopodia formation was also enhanced by forced ERK5 expression in PC3 cells. Furthermore, in metastatic PCa, nuclear ERK5 immunoreactivity was significantly upregulated when compared with benign prostatic hyperplasia and primary PCa (P=0.013 and P<0.0001, respectively). Our in vitro, in vivo and clinical data support an important role for the MEK5-ERK5 signalling pathway in invasive PCa, which represents a potential target for therapy in primary and metastatic PCa. Show less

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. Show less

Abnormal intracellular signaling contributes to carcinogenesis and may represent novel therapeutic targets. mitogen/extracellular signal-regulated kinase kinase-5 (MEK5) overexpression is associated with aggressive prostate cancer. In this study, we examined the role of extracellular signal-regulated kinase (ERK5, an MAPK and specific substrate for MEK5) in prostate cancer. ERK5 immunoreactivity was significantly upregulated in high-grade prostate cancer when compared to benign prostatic hyperplasia (P<0.0001). Increased ERK5 cytoplasmic signals correlated closely with Gleason sum score (P<0.0001), bony metastases (P=0.0044) and locally advanced disease at diagnosis (P=0.0023), with a weak association with shorter disease-specific survival (P=0.036). A subgroup of patients showed strong nuclear ERK5 localization, which correlated with poor disease-specific survival and, on multivariant analysis, was an independent prognostic factor (P<0.0001). Analysis of ERK5 expression in matched tumor pairs (before and after hormone relapse, n=26) revealed ERK5 nuclear expression was significantly associated with hormone-insensitive disease (P=0.0078). Similarly, ERK5 protein expression was increased in an androgen-independent LNCaP subline. We obtained the following in vitro and in vivo evidence to support the above expression data: (1) cotransfection of ERK5wt and MEK5D constructs in PC3 cells results in predominant ERK5 nuclear localization, similar to that observed in aggressive clinical disease; (2) ERK5-overexpressing PC3 cells have enhanced proliferative, migrative and invasive capabilities in vitro (P<0.0001), and were dramatically more efficient in forming tumors, with a shorter mean time for tumors to reach a critical volume of 1000 mm(3), in vivo (P<0.0001); (3) the MEK1 inhibitor, PD184352, blocking ERK1/2 activation at low dose, did not suppress proliferation but did significantly decrease proliferation at a higher dose required to inhibit ERK5 activation. Taken together, our results establish the potential importance of ERK5 in aggressive prostate cancer. Show less

The Liver X Receptor (LXR) alpha and beta isoforms are members of the type II nuclear receptor family which function as a heterodimer with the Retinoid X Receptor (RXR). Upon agonist binding, the formation of the LXR/RXR heterodimer takes place and ultimately the regulation of a number of genes begins. The LXR isoforms share 77% sequence homology, with LXRalpha having highest expression in liver, intestine, adipose tissue, and macrophages and LXRbeta being ubiquitously expressed. The aim of this article is to review the reported medicinal chemistry strategies towards the optimisation of novel non-steroidal chemotypes as LXR agonists. An analysis of the structural features important for LXR ligand binding will be given, utilising both structural activity relationship data obtained from LXR assays as well as X-ray co-crystallographic data obtained with LXR ligands and the LXR ligand binding domain (LBD). The X-ray co-crystallographic data analysis will detail the key structural interactions required for LXR binding/agonist activity and reveal the differences observed between chemotype classes. It has been postulated that a LXRbeta selective compound may have a beneficial outcome on the lipid profile for a ligand by dissociating the favourable and unfavourable effects of LXR agonists. Whilst there have been a few examples of compounds showing a modest level of LXRalpha selectivity, obtaining a potent LXRbeta selective compound has been more challenging. Analysis of the SAR and X-ray co-crystallographic data suggests that the rational design of a LXRbeta selective compound will not be trivial. Show less

A phase 1, dose-escalating trial was conducted in healthy adults to evaluate immunogenicity and reactogenicity of a type V group B streptococcal (GBS) capsular polysaccharide (CPS)-tetanus toxoid (TT) conjugate vaccine. Participants received one dose of unconjugated V CPS (37 microg), V-TT (2.4 microg CPS/1.1 microg TT), V-TT (9.6 microg CPS/4.3 microg TT) or V-TT (38.5 microg CPS/17.0 microg TT). Each vaccine and all doses of V-TT were well-tolerated. V CPS-specific antibodies reached a peak 4-8 weeks after immunization and were significantly higher through 52 weeks post-immunization in recipients of V-TT at each dose than in uncoupled CPS vaccinees. V-TT vaccine-induced antibodies promoted opsonophagocytic killing of type V GBS and avidity maturation of V CPS-specific IgG. Show less

The Liver X Receptor (LXR) alpha and beta isoforms are members of the type II nuclear receptor family which function as obligate heterodimers with the Retinoid X Receptor (RXR). Upon agonist binding, the DNA Binding Domain (DBD) of LXR interacts with LXR response elements on target genes to initiate transcription. A number of genes have been shown to be modulated by LXR function, including the ATP-binding cassette transporter A1 (ABCA1). ABCA1 is involved in the process of reverse cholesterol transport (RCT) from macrophages in atherosclerotic plaques to high-density lipoproteins (HDL) in the plasma. Both homozygous and heterozygous mutations in ABCA1 result in conditions characterised by decreased levels of HDL and an earlier onset of atherosclerosis. A number of other genes are upregulated by LXR activation which would be expected to have either pro- or anti-atherogenic effects. One such target gene is sterol regulatory element binding protein-1c (SREBP-1c), which is involved in the process of lipogenesis leading to increased levels of triglycerides which are pro-atherogenic. The complexity of LXR responses, however, makes it difficult to extrapolate the 'positive' or 'negative' effects of each target gene in isolation to a conclusion as to the outcome in humans when all target genes are being modulated in concert. This review will cover the structural features and associated biological data of non-steroidal LXR modulators claimed for the treatment of cardiovascular disease, as well as highlighting preferred compounds where this information can be discerned. In addition to this patent information a précis of literature data relevant to the utility of specific compounds in the treatment of cardiovascular disease will be given where available. Show less

A new apolipoprotein (apo) gene, APOA5, was recently identified on chromosome 11q23, and common variants in the gene have been associated with plasma triglyceride (TG) levels in several studies. The purpose of the present study was to examine the association of five single nucleotide polymorphisms (SNPs) and haplotypes in the APOA5 gene with low-density lipoprotein (LDL) particle size using a community-based sample of Japanese American families, including examining whether the associations with LDL size are independent of, or primarily reflecting, TG levels. Genetic association analyses were performed using 154 unrelated individuals, quantitative transmission disequilibrium tests (TDT) in 238 nuclear families, a sample of 24 hypertriglyceridemic subjects with matched, normotriglyceridemic controls, and using haplotype analyses. There was a high degree of allelic association between several of the SNPs, with complete linkage disequilibrium (LD) between -1131C>T and the -3A>G SNP which alters a potential Kozak sequence. All approaches demonstrated associations between the -3A>G APOA5 variant and both decreased LDL size and increased TG levels. The frequency of the rare allele was higher than reported for Caucasian, Hispanic, and African Americans, but similar to that in Japan and China. Therefore, the haplotype containing the -1131C and -3G variants, and possibly specifically the -3A>G SNP in APOA5, may be a major genetic determinant of LDL particle size and TG levels among ethnic Asians. Show less

The gene coding for human cyclin K was isolated as a CPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far- phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1, CLN2, and CLN3. The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II). Murine and Drosophila melanogaster homologs of cyclin K have also been identified. Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries. Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II. Thus, cyclin K represents a new member of the "transcription" cyclin family which may play a dual role in regulating Cdk and RNAP II activity. Show less

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G1 arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G1 arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles. Show less