👤 Debbie Hughes

We have screened small molecule libraries specifically for inhibitors that target WWP2, an E3 ubiquitin ligase associated with tumour outgrowth and spread. Selected hits demonstrated dose-dependent WWP2 inhibition, low micromolar IC50 values, and inhibition of PTEN substrate-specific ubiquitination. Binding to WWP2 was confirmed by ligand-based NMR spectroscopy. Furthermore, we used a combination of STD NMR, the recently developed DEEP-STD NMR approach, and docking calculations, to propose for the first time an NMR-validated 3D molecular model of a WWP2-inhibitor complex. These first generation WWP2 inhibitors provide a molecular framework for informing organic synthetic approaches to improve activity and selectivity. Show less

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis. Show less

Apolipoprotein C-III (APOC3) is a key regulator of plasma triglyceride levels. Elevated triglyceride levels are associated with a risk of adverse cardiovascular events and pancreatitis. ISIS 304801 is a second-generation antisense inhibitor of APOC3 synthesis. We conducted a randomized, double-blind, placebo-controlled, dose-ranging, phase 2 study to evaluate ISIS 304801 in untreated patients with fasting triglyceride levels between 350 mg per deciliter (4.0 mmol per liter) and 2000 mg per deciliter (22.6 mmol per liter) (ISIS 304801 monotherapy cohort), as well as in patients receiving stable fibrate therapy who had fasting triglyceride levels between 225 mg per deciliter (2.5 mmol per liter) and 2000 mg per deciliter (ISIS 304801-fibrate cohort). Eligible patients were randomly assigned to receive either ISIS 304801, at doses ranging from 100 to 300 mg, or placebo, once weekly for 13 weeks. The primary outcome was the percentage change in APOC3 level from baseline. A total of 57 patients were treated in the ISIS 304801 monotherapy cohort (41 received active agent, and 16 received placebo), and 28 patients were treated in the ISIS 304801-fibrate cohort (20 received active agent, and 8 received placebo). The mean (±SD) baseline triglyceride levels in the two cohorts were 581±291 mg per deciliter (6.6±3.3 mmol per liter) and 376±188 mg per deciliter (4.2±2.1 mmol per liter), respectively. Treatment with ISIS 304801 resulted in dose-dependent and prolonged decreases in plasma APOC3 levels when the drug was administered as a single agent (decreases of 40.0±32.0% in the 100-mg group, 63.8±22.3% in the 200-mg group, and 79.6±9.3% in the 300-mg group, vs. an increase of 4.2±41.7% in the placebo group) and when it was administered as an add-on to fibrates (decreases of 60.2±12.5% in the 200-mg group and 70.9±13.0% in the 300-mg group, vs. a decrease of 2.2±25.2% in the placebo group). Concordant reductions of 31.3 to 70.9% were observed in triglyceride levels. No safety concerns were identified in this short-term study. We found that treatment with ISIS 304801 was associated with significant lowering of triglyceride levels, among patients with a broad range of baseline levels, through selective antisense inhibition of APOC3 synthesis. (Funded by Isis Pharmaceuticals; ClinicalTrials.gov number, NCT01529424.). Show less

The familial chylomicronemia syndrome is a genetic disorder characterized by severe hypertriglyceridemia and recurrent pancreatitis due to a deficiency in lipoprotein lipase (LPL). Currently, there are no effective therapies except for extreme restriction in the consumption of dietary fat. Apolipoprotein C-III (APOC3) is known to inhibit LPL, although there is also evidence that APOC3 increases the level of plasma triglycerides through an LPL-independent mechanism. We administered an inhibitor of APOC3 messenger RNA (mRNA), called ISIS 304801, to treat three patients with the familial chylomicronemia syndrome and triglyceride levels ranging from 1406 to 2083 mg per deciliter (15.9 to 23.5 mmol per liter). After 13 weeks of study-drug administration, plasma APOC3 levels were reduced by 71 to 90% and triglyceride levels by 56 to 86%. During the study, all patients had a triglyceride level of less than 500 mg per deciliter (5.7 mmol per liter) with treatment. These data support the role of APOC3 as a key regulator of LPL-independent pathways of triglyceride metabolism. Show less

The development of male internal and external genitalia in an XY fetus requires a complex interplay of many critical genes, enzymes, and cofactors. The enzyme 17β-hydroxysteroid-dehydrogenase type 3 (17βHSD3) is present almost exclusively in the testicles and converts Delta 4-androstenodione (Δ4) to testosterone. A deficiency in this enzyme is rare and is a frequently misdiagnosed autosomal recessive cause of 46,XY, disorder of sex development. The case report is of a 15-year-old adolescent, who was raised according to female gender. At puberty, the adolescent had a severe virilization and primary amenorrhea. The physical examination showed a male phenotype with micropenis and blind vagina. The Tanner stage was A3B1P4, nonpalpable gonads. The karyotype revealed 46,XY. The endocrinology study revealed: testosterone=2.38 ng/ml, Δ4>10.00 ng/ml, and low testosterone/Δ4 ratio=0.23. Magnetic resonance imaging of the abdominal-pelvic showed the presence of testicles in inguinal canal, seminal vesicle, prostate, micropenis, and absence of uterus and vagina. The genetic study confirmed the mutation p.Glu215Asp on HSD17B3 gene in homozygosity. The dilemma of sex reassignment was seriously considered when the diagnosis was made. During all procedures the patient was accompanied by a child psychiatrist/psychologist. The teenager desired to continue being a female, so gonadectomy was performed. Estrogen therapy and surgical procedure to change external genitalia was carried out. In this case, there was a severe virilization at puberty. It is speculated to be due to a partial activity of 17βHSD3 in the testicles and/or extratesticular ability to convert Δ4 to testosterone by 17βHSD5. Prenatal exposure of the brain to androgens has increasingly been put forward as a critical factor in gender identity development, but in this case the social factor was more important for the gender assignment. In this case, we highlight the late diagnosis, probably because the patient belongs to a poor family without proper primary medical care.We emphasize the psychological and social aspects in the sex assignment decision. Show less

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ~12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH. Show less

Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs. Show less

The aim of the present study was to test the hypotheses that exercise is associated with generation of peroxisome proliferator-activated receptor-γ (PPARγ) ligands in the plasma and that this may activate PPARγ signaling within circulating monocytes, thus providing a mechanism to underpin the exercise-induced antiatherogenic benefits observed in previous studies. A cohort of healthy individuals undertook an 8-wk exercise-training program; samples were obtained before (Pre) and after (Post) standardized submaximal exercise bouts (45 min of cycling at 70% of maximal O(2) uptake, determined at baseline) at weeks 0, 4, and 8. Addition of plasma samples to PPARγ response element (PPRE)-luciferase reporter gene assays showed increased PPARγ activity following standardized exercise bouts (Post/Pre = 1.23 ± 0.10 at week 0, P < 0.05), suggesting that PPARγ ligands were generated during exercise. However, increases in PPARγ/PPRE-luciferase activity in response to the same standardized exercise bout were blunted during the training program (Post/Pre = 1.18 ± 0.14 and 1.10 ± 0.10 at weeks 4 and 8, respectively, P > 0.05 for both), suggesting that the relative intensity of the exercise may affect PPARγ ligand generation. In untrained individuals, specific transient increases in monocyte expression of PPARγ-regulated genes were observed within 1.5-3 h of exercise (1.7 ± 0.4, 2.6 ± 0.4, and 1.4 ± 0.1 fold for CD36, liver X receptor-α, and ATP-binding cassette subfamily A member 1, respectively, P < 0.05), with expression returning to basal levels within 24 h. In contrast, by the end of the exercise program, expression at the protein level of PPARγ target genes had undergone sustained increases that were not associated with an individual exercise bout (e.g., week 8 Pre/week 0 Pre = 2.79 ± 0.61 for CD36, P < 0.05). Exercise is known to upregulate PPARγ-controlled genes to induce beneficial effects in skeletal muscle (e.g., mitochondrial biogenesis and aerobic respiration). We suggest that parallel exercise-induced benefits may occur in monocytes, as monocyte PPARγ activation has been linked to beneficial antidiabetic effects (e.g., exercise-induced upregulation of monocytic PPARγ-controlled genes is associated with reverse cholesterol transport and anti-inflammatory effects). Thus, exercise-triggered monocyte PPARγ activation may constitute an additional rationale for prescribing exercise to type 2 diabetes patients. Show less

Twenty human proteins encode Phox/Bem1p (PB1) domains, which are involved in forming protein heterodimers. MEKK2, MEKK3, and MEK5 are 3 serine-threonine protein kinases that have PB1 domains. MEKK2, MEKK3, and MEK5 are the MAP3Ks and the MAP2K in the ERK5 mitogen-activated protein kinase (MAPK) signaling module. ERK5 is a critical MAPK for both development of the vasculature and vascular homeostasis in the adult, but no other MAPK has been shown to be critical in vascular maintenance in the adult animal. MEKK2 and MEKK3 are the only MAP3Ks shown to physically interact with and activate the MEK5-ERK5 signaling module. Interaction of MEKK2 or MEKK3 with MEK5 is mediated by heterodimerization of the MEKK2 (or MEKK3) PB1 and MEK5 PB1 domains. The authors have developed a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor PB1-PB1 domain heterodimerization. The assay uses a europium-chelate conjugated GST-MEK5 PB1 domain chimera, biotinylated MEKK2 PB1 domain, and streptavidin-Cy5. Interaction of the MEKK2 and MEK5 PB1 domains gives a robust FRET signal (Z' factor = 0.93), which is completely abrogated by mutation of 2 acidic residues (64D65E-->AA) within the MEK5 PB1 domain that causes loss of stable PB1-PB1 domain interaction. This assay can be used to study the specificity of PB1-PB1 domain interactions and to screen for molecules that can regulate MEKK2/MEKK3-MEK5 interactions. Disruption of PB1 domain interactions represents a novel approach for selectively regulating the ERK5 signaling pathway independent of kinase active site-directed adenosine triphosphate competitive inhibitors. Show less

17beta-hydroxysteroid dehydrogenase type 3 isoenzyme (17beta-HSD3) is required to produce testosterone for male sex differentiation. Mutations in the HSD17B3 gene cause 17betaHSD3 deficiency and result in XY sex reversal of varying degree. We report the phenotypes of 14 subjects with 17betaHSD3 deficiency in relation to sex of rearing, androgen production, and HSD17B3 mutations. Cases were identified through the Cambridge Disorders of Sex Development Database where detailed clinical information was recorded, results of hCG stimulation tests were available, and HSD17B3 mutation was identified. Fourteen subjects from seven pedigrees (four consanguineous) had the following seven mutations: A56T, N130S, E215D, S232L, C268Y, V205E, and a novel mutation M197K. XY sex reversal was classified as complete in 10 infants at birth. Inguinal masses suggestive of androgen insensitivity syndrome (AIS) occurred in five infants. Contrasexual virilization reminiscent of 5alpha-reductase deficiency occurred in four subjects at puberty. The median (range) testosterone : androstenedione (T/A) ratio after a short hCG stimulation test was 0.32 (0.12-3.4). The S232L mutation identified in three affected family members caused isolated, severe hypospadias in one member who was raised male; virilization occurred despite in vitro studies showing an inactive mutant enzyme. Ratios of T/A in this pedigree were more than 0.8. XY sex reversal is sufficiently variable in 17betaHSD3 deficiency to cause problems in accurate diagnosis, particularly in distinguishing it from AIS. It should be considered in undervirilized male infants with normal Wolffian duct structures, absent Müllerian ducts, and normal adrenal steroid biosynthesis; or when an assigned female subject virilizes at puberty. Elevated hCG-stimulated T/A ratio may occur, and sex of rearing may not be concordant within affected families with the same HSD17B3 mutation. The T/A ratio, mutation analysis and functional analysis of the mutant enzyme taken in isolation, respectively, may not conclusively establish a diagnosis of 17betaHSD3 deficiency in undervirilized male subjects; the reasons for these discrepancies remain unknown. Show less