The present study was performed to clarify the significance of DNA methylation alterations during endometrial carcinogenesis. Genome-wide DNA methylation analysis and targeted sequencing of tumor-related genes were performed using the Infinium MethylationEPIC BeadChip and the Ion AmpliSeq Cancer Hotspot Panel v2, respectively, for 31 samples of normal control endometrial tissue from patients without endometrial cancer and 81 samples of endometrial cancer tissue. Principal component analysis revealed that tumor samples had a DNA methylation profile distinct from that of control samples. Gene Ontology enrichment analysis revealed significant differences of DNA methylation at 1034 CpG sites between early-onset endometrioid endometrial cancer (EE) tissue (patients aged ≤40 years) and late-onset endometrioid endometrial cancer (LE) tissue, which were accumulated among 'transcriptional factors'. Mutations of the CTNNB1 gene or DNA methylation alterations of genes participating in <em>Wnt</em> signaling were frequent in EEs, whereas genetic and epigenetic alterations of fibroblast growth factor signaling genes were observed in LEs. Unsupervised hierarchical clustering grouped EE samples in Cluster EA (n = 22) and samples in Cluster EB (n = <em>12</em>). Clinicopathologically less aggressive tumors tended to be accumulated in Cluster EB, and DNA methylation levels of 18 genes including HOXA9, HOXD10 and SOX11 were associated with differences in such aggressiveness between the two clusters. We identified 11 marker CpG sites that discriminated EB samples from EA samples with 100% sensitivity and specificity. These data indicate that genetically and epigenetically different pathways may participate in the development of EEs and LEs, and that DNA methylation profiling may help predict tumors that are less aggressive and amenable to fertility preservation treatment.

Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1β as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and <em>12</em>. Despite the contribution of both mediators to the stimulation of Runx2 and <em>Wnt</em>/β-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1β played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-<em>12</em> were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints.

The aim of the present study was to identify common microRNAs (miRNAs) in ovarian cancer (OC) cells and their exosomes using microarray data (accession number GSE76449) available from the Gene Expression Omnibus database, including exosomal samples from 3 OC cell lines, 1 normal ovarian surface epithelial cell line and their original cell samples. Differentially-expressed miRNAs (DE-miRNAs) were identified using the Linear Models for Microarray data method, and mRNA targets of DE-miRNAs were predicted using the miRWalk2 database. The potential functions of the target genes of the DE-miRNAs were analyzed using the Database for Annotation, Visualization and Integrated Discovery tool. The association between crucial miRNAs and target genes, and their clinical associations, were validated using The Cancer Genome Atlas data. As a result, <em>12</em> upregulated and <em>12</em> downregulated DE-miRNAs were shared by the 3 OC cell lines compared with normal controls in the exosomal samples, while 5 upregulated and 65 downregulated DE-miRNAs were shared between the original cells. Among them, 9 downregulated DE-miRNAs were shared between exosomal and original cells. The target genes of 4 common DE-miRNAs between exosomal and original cells (miR-<em>12</em>7-3p, miR-339-5p, miR-409-3p and miR-654-3p) were predicted. Functional enrichment analysis indicated that these target genes may be involved in the <em>Wnt</em> signaling pathway (miR-409-3p-CTBP1 and miR-339-5p-CHD8) and Proteoglycans in cancer (miR-<em>12</em>7-3p-PPP1CA). The negative associations between these 3 miRNAs and target genes were confirmed by a Pearson's correlation analysis. miR-<em>12</em>7 was negatively associated with tumor grade. In conclusion, our results describe a set of miRNAs involved in OC development, in exosomal and non-exosomal manners, by regulating their target genes. They may be potential targets for treatment of OC.

<AbstractText>This study aimed to describe the use of a novel 4-lncRNA signature to predict prognosis in patients with laryngeal cancer and to explore its possible mechanisms.</AbstractText><AbstractText>We identified lncRNAs that were differentially expressed between 111 tumor tissue samples and <em>12</em> matched normal tissue samples from The Cancer Genome Atlas Database (TCGA). We used Cox regression analysis to identify lncRNAs that were correlated with prognosis. A 4-lncRNA signature was developed to predict the prognosis of patients with laryngeal cancer. The receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to verify the validity of this Cox regression model, and an independent prognosis analysis was used to confirm that the 4-lncRNA signature was an independent prognostic factor. Furthermore, the function of these lncRNAs was inferred using related gene prediction and Gene ontology (GO) enrichment analysis in order to clarify the possible mechanisms underlying their predictive ability.</AbstractText><AbstractText>In total, 214 differentially expressed lncRNAs were identified, and a 4-lncRNA signature was constructed using Cox survival analysis. The risk coefficients in the multivariate Cox analysis revealed that LINC02154 and MNX1-AS1 are risk factors for laryngeal cancer, whereas MYHAS and LINC0<em>12</em>81 appear to be protective factors. The results of a functional annotation analysis suggested that the mechanisms by which these lncRNAs influence prognosis in laryngeal cancer may involve the extracellular exosome, the Notch signaling pathway, voltage-gated calcium channels, and the <em>Wnt</em> signaling pathway.</AbstractText><AbstractText>We identified a novel 4-lncRNA signature that can predict the prognosis of patients with laryngeal cancer and that may influence the prognosis of laryngeal cancer by regulating immunity, tumor apoptosis, metastasis, invasion, and other characteristics through the Notch signaling pathway, voltage-gated calcium channels, and the <em>Wnt</em> signaling pathway.</AbstractText>

Alcohol-use disorders encompass a range of drinking levels and behaviors, including low, binge, and heavy drinking. In this regard, investigating the neural state of individuals who chronically self-administer lower doses of alcohol may provide insight into mechanisms that prevent the escalation of alcohol use. DNA methylation is one of the epigenetic mechanisms that stabilizes adaptations in gene expression and has been associated with alcohol use. Thus, we investigated DNA methylation, gene expression, and the predicted neural effects in the nucleus accumbens core (NAcc) of male rhesus macaques categorized as "low" or "binge" drinkers, compared to "alcohol-naïve" and "heavy" drinkers based on drinking patterns during a <em>12</em>-month alcohol self-administration protocol. Using genome-wide CpG-rich region enrichment and bisulfite sequencing, the methylation levels of 2.6 million CpGs were compared between alcohol-naïve (AN), low/binge (L/BD), and heavy/very heavy (H/VHD) drinking subjects (n = 24). Through regional clustering analysis, we identified nine significant differential methylation regions (DMRs) that specifically distinguished ANs and L/BDs, and then compared those DMRs among H/VHDs. The DMRs mapped to genes encoding ion channels, receptors, cell adhesion molecules, and cAMP, NF-κβ and <em>Wnt</em> signaling pathway proteins. Two of the DMRs, linked to PDE10A and PKD2L2, were also differentially methylated in H/VHDs, suggesting an alcohol-dose independent effect. However, two other DMRs, linked to the CCBE1 and FZD5 genes, had L/BD methylation levels that significantly differed from both ANs and H/VHDs. The remaining five DMRs also differentiated L/BDs and ANs. However, H/VHDs methylation levels were not distinguishable from either of the two groups. Functional validation of two DMRs, linked to FZD5 and PDE10A, support their role in regulating gene expression and exon usage, respectively. In summary, the findings demonstrate that L/BD is associated with unique DNA methylation signatures in the primate NAcc, and that the methylation signatures identify synaptic genes that may play a role in preventing the escalation of alcohol use.

Sertoli cells (Sc) are unique somatic cells of testis that are the target of both FSH and testosterone (T) and regulate spermatogenesis. Although Sc of neonatal rat testes are exposed to high levels of FSH and T, robust differentiation of spermatogonial cells becomes conspicuous only after 11-days of postnatal age. We have demonstrated earlier that a developmental switch in terms of hormonal responsiveness occurs in rat Sc at around <em>12</em> days of postnatal age during the rapid transition of spermatogonia A to B. Therefore, such "functional maturation" of Sc, during pubertal development becomes prerequisite for the onset of spermatogenesis. However, a conspicuous difference in robust hormone (both T and FSH) induced gene expression during the different phases of Sc maturation restricts our understanding about molecular events necessary for the spermatogenic onset and maintenance. Here, using microarray technology, we for the first time have compared the differential transcriptional profile of Sc isolated and cultured from immature (5 days old), maturing (<em>12</em> days old) and mature (60 days old) rat testes. Our data revealed that immature Sc express genes involved in cellular growth, metabolism, chemokines, cell division, MAPK and <em>Wnt</em> pathways, while mature Sc are more specialized expressing genes involved in glucose metabolism, phagocytosis, insulin signaling and cytoskeleton structuring. Taken together, this differential transcriptome data provide an important resource to reveal the molecular network of Sc maturation which is necessary to govern male germ cell differentiation, hence, will improve our current understanding of the etiology of some forms of idiopathic male infertility.

The RET receptor tyrosine kinase is a member of the cadherin superfamily and plays a pivotal role in cell survival, differentiation and proliferation. Currently, <em>12</em> ret/ptc chimeric oncogenes, characterized by the fusion between the intracellular domain of RET and different activating genes, which can cause ligand-independent dimerization and constitutive activation, have been described. β-catenin is usually involved in the maintenance of cell-to-cell adhesion and mediates the <em>Wnt</em>/β-catenin pathway important during embryogenesis and in cellular malignant transformation. Recently, a novel mechanism of RET-mediated function through the β-catenin pathway has been reported in multiple endocrine neoplasia type 2 and in sporadic thyroid carcinomas. Here, we investigated the effects of the ZD6474, a small molecule RET-inhibitor, on RET/β-catenin interaction. We confirmed the ZD6474 mediated-inhibition of recombinant RET kinase and of growth of cells expressing RET/PTC. Interestingly, we firstly observed reduced cellular mobility and changed morphology of TPC1 treated cells suggesting that RET-inhibitor could affect β-catenin cellular distribution as resulted in its co-immunoprecipitation with E-cadherin. We further investigated this hypothesis showing that TPC1 treated cells displayed predominantly β-catenin cytosolic localization. Surprisingly, RET and β-catenin co-immunoprecipitated in both ZD6474-treated and untreated TPC1 cells, suggesting that RET/β-catenin interaction might not be affected by RET kinase inactivation. All together these results suggest that RET kinase activation is crucial for β-catenin stabilization (pY654), localization and its signaling pathway activation but not for β-catenin/RET physical interactions, in human papillary thyroid carcinomas. In conclusion, ZD6474, by inhibiting RET kinase, down-modulates β-catenin pathway leading its recruitment to the membrane by E-cadherin.

UNASSIGNED

Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known.

UNASSIGNED

In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at <em>12</em> weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher's exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC.

UNASSIGNED

At <em>12</em> weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/β-catenin pathway were more frequently mutated and negative regulators of Wnt/β-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01).

UNASSIGNED

Wnt/β-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and <em>12</em> plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4(-/-) (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with <em>Wnt</em>2b and <em>Wnt</em>5a further validated the interplay between the <em>Wnt</em>/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si-β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc(Min/+) mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.

OBJECTIVE

To reveal the mechanism of Zuogui Pill (see text) in treatment of glucocorticoid-induced osteoporosis from the angle of the Wnt signal transduction pathway and to provide further experimental evidence for expounding the scientific connotation of "the kidney dominating the bones" in TCM.

METHODS

Forty-two male Wistar rats were selected and randomly divided into three groups, control group (n = 12), model group (n = 15) and Zuogui Pill group (n = 15). Form the beginning, The rats were injected dexamethasone for eight weeks to make the model of osteoporosis, and the Zuogui Pill were administered intragastrically to the rats of Zuogui Pill group for eight weeks. The relative morphological parameters were measured in the undecalcified tibial slices. And the protein expression levels of <em>Wnt</em>1, LRP-5 and beta-catenin in rat tibial osteoblasts (OB) and bone marrow stromal cells (BMC) were detected by immunohistochemistry.

RESULTS

Compared with the control group, TBV% and TFS% decreased significantly, while TRS% increased significantly, and the protein expression of <em>Wnt</em>1, LRP-5 and beta-catenin in OB and BMC decreased significantly in the model group. And compared with the model group, TBV% and TFS% increased significantly, and expression levels of <em>Wnt</em>1, LRP-5 and beta-catenin proteins increased significantly in the Zuogui pill group.

CONCLUSIONS

Zuogui Pill can prevent and treat glucocorticoid-induced osteoporosis in rats by up-regulating the expression of the key signal molecules WntWnt signal transduction pathway.

Osteoarthritis (OA) significantly influences the quality life of people around the world. It is urgent to find an effective way to understand the genetic etiology of OA. We used weighted gene coexpression network analysis (WGCNA) to explore the key genes involved in the subchondral bone pathological process of OA. Fifty gene expression profiles of GSE51588 were downloaded from the Gene Expression Omnibus database. The OA-associated genes and gene ontologies were acquired from JuniorDoc. Weighted gene coexpression network analysis was used to find disease-related networks based on 21756 gene expression correlation coefficients, hub-genes with the highest connectivity in each module were selected, and the correlation between module eigengene and clinical traits was calculated. The genes in the traits-related gene coexpression modules were subject to functional annotation and pathway enrichment analysis using ClusterProfiler. A total of 73 gene modules were identified, of which, <em>12</em> modules were found with high connectivity with clinical traits. Five modules were found with enriched OA-associated genes. Moreover, 310 OA-associated genes were found, and 34 of them were among hub-genes in each module. Consequently, enrichment results indicated some key metabolic pathways, such as extracellular matrix (ECM)-receptor interaction (hsa045<em>12</em>), focal adhesion (hsa04510), the phosphatidylinositol 3'-kinase (PI3K)-Akt signaling pathway (PI3K-AKT) (hsa04151), transforming growth factor beta pathway, and <em>Wnt</em> pathway. We intended to identify some core genes, collagen (COL)6A3, COL6A1, ITGA11, BAMBI, and HCK, which could influence downstream signaling pathways once they were activated. In this study, we identified important genes within key coexpression modules, which associate with a pathological process of subchondral bone in OA. Functional analysis results could provide important information to understand the mechanism of OA.

OBJECTIVE

Alopecia is one of the most common dermatological problems in the elderly; however, current therapies for it are limited by low efficacy and undesirable side effects. Although clinical reports on fractional laser treatment for various alopecia types are increasing, the exact mechanism remains to be clarified. The purposes of this study were to demonstrate the effect of ablative fractional laser treatment on hair follicle regrowth in vivo and investigate the molecular mechanism after laser treatment.

METHODS

Ablative CO2 fractional laser was applied to the shaved dorsal skin of 7-week-old C57BL/6 mice whose hair was in the telogen stage. After <em>12</em> mice were treated at various energy (10-40 mJ/spot) and density (100-400 spots/cm(2) ) settings to determine the proper dosage for maximal effect. Six mice were then treated at the decided dosage and skin specimens were sequentially obtained by excision biopsy from the dorsal aspect of each mouse. Tissue samples were used for the immunohistochemistry and reverse transcription polymerase chain reaction assays to examine hair follicle status and their related molecules.

RESULTS

The most effective dosage was the 10 mJ/spot and 300 spots/cm(2) setting. The anagen conversion of hair was observed in the histopathological examination, while Wnt/β-catenin expression was associated with hair regrowth in the immunohistochemistry and molecular studies.

CONCLUSIONS

Ablative fractional lasers appear to be effective for inducing hair regrowth via activation of the Wnt/β-catenin pathway in vivo. Our findings indicate that fractional laser treatment can potentially be developed as new treatment options for stimulating hair regrowth.

The canonical <em>Wnt</em> pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for <em>Wnt</em> pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the <em>12</em> Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand-receptor pairs likely facilitated the expansion of this pathway's capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFbeta signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand-receptor interactions as seen in the <em>Wnt</em> pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFbeta pathway. From a practical perspective, the former mechanism limits the investigator's ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer.

BACKGROUND

Growth hormone (GH) substitution of adult-onset growth hormone deficiency (aoGHD) patients partially reverses unfavorable body composition profile. Wnt signaling pathway has being acknowledged as an important modulator of bone mass and of energy metabolism in adipose tissue and in β-cells.

OBJECTIVE

To assess the role of selected Wnt antagonists in bone and glucose metabolism before and after GH replacement in aoGHD.

METHODS

Patients from two randomized placebo-controlled studies of GH replacement in aoGHD were used. In study 1, 39 patients received GH or placebo for 9 months with 4 months wash-out. In study 2, iliac bone biopsies were obtained before and after GH or placebo (n = 10 each) for 12 months. Body composition and serum (study 1) and bone matrix (study 2) levels of Wnt antagonists (DKK-1, sFRP-3, WIF-1, and SOST) were quantified before and after GH. In vitro effect of GH and IGF-1 on DKK-1 secretion and expression of Wnt signaling modulators was assessed in human osteoblasts and mature adipocytes.

RESULTS

GH replacement increased circulating and bone matrix levels of DKK-1, but not sFRP-3, WIF-1, and SOST. Furthermore, DKK-1 secretion increased in human osteoblasts stimulated by GH in vitro, with no effects on other cells. At baseline and after treatment, circulating DKK-1 was negatively associated with bone mass, but not fat mass or measures of insulin resistance, in aoGHD patients.

CONCLUSIONS

An increase in DKK-1 may limit the effects of GH on bone mass, but does not seem to impact the increase in insulin resistance following GH substitution.

Fascin-1, an actin-bundling protein, plays an important role in cancer cell migration and invasion; however, the underlying mechanism remains unclear. On the basis of a <em>12</em>-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell migration model, it was shown that TPA increased fascin-1 mRNA and protein expression and fascin-1-dependent cell migration. TPA dose- and time-dependently increased PKCδ and STAT3α activation and GSK3β phosphorylation; up-regulated <em>Wnt</em>-1, β-catenin, and STAT3α expression; and increased the nuclear translocation of β-catenin and STAT3α. Rottlerin, a PKCδ inhibitor, abrogated the increases in STAT3α activation and β-catenin and fascin-1 expression. WP1066, a STAT3 inhibitor, suppressed TPA-induced STAT3α DNA binding activity and β-catenin expression. Knockdown of β-catenin attenuated TPA-induced fascin-1 and STAT3α expression as well as cell migration. In addition to MCF-7, migration of Hs578T breast cancer cells was inhibited by silencing fascin-1, β-catenin, and STAT3α expression as well. TPA also induced <em>Wnt</em>-1 expression and secretion, and blocking <em>Wnt</em>-1 signaling abrogated β-catenin induction. DHA pretreatment attenuated TPA-induced cell migration, PKCδ and STAT3α activation, GSK3β phosphorylation, and <em>Wnt</em>-1, β-catenin, STAT3α, and fascin-1 expression. Our results demonstrated that TPA-induced migration is likely associated with the PKCδ and <em>Wnt</em>-1 pathways, which lead to STAT3α activation, GSK3β inactivation, and β-catenin increase and up-regulation of fascin-1 expression. Moreover, the anti-metastatic potential of DHA is partly attributed to its suppression of TPA-activated PKCδ and <em>Wnt</em>-1 signaling.

Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, lacks effective medical therapy. Large subsets of HCC demonstrate <em>Wnt</em>/β-catenin activation, making this an attractive therapeutic target. We report strategy and characterization of a novel small-molecule inhibitor, ICG-001, known to affect <em>Wnt</em> signaling by disrupting β-catenin-CREB binding protein interactions. We queried the ZINC online database for structural similarity to ICG-001 and identified PMED-1 as the lead compound, with ≥70% similarity to ICG-001. PMED-1 significantly reduced β-catenin activity in hepatoblastoma and several HCC cells, as determined by TOPflash reporter assay, with an IC50 ranging from 4.87 to 32 μmol/L. Although no toxicity was observed in primary human hepatocytes, PMED-1 inhibited <em>Wnt</em> target expression in HCC cells, including those with CTNNB1 mutations, and impaired cell proliferation and viability. PMED-1 treatment decreased β-catenin-CREB binding protein interactions without affecting total β-catenin levels or activity of other common kinases. PMED-1 treatment of Tg(OTM:d2EGFP) zebrafish expressing GFP under the β-catenin/Tcf reporter led to a notable decrease in β-catenin activity. The PMED effect on β-catenin signaling lasted from <em>12</em> to 24 hours in vitro and 6 to 15 hours in vivo. Thus, using a rapid and cost-effective computational methodology, we have identified a novel and specific small-molecule inhibitor of <em>Wnt</em> signaling that may have implications for HCC treatment.

BACKGROUND

Prickly pear cactus grown in Korea (Opuntia ficus-indica Mill, KC) and Buchema (Dioscorea nipponica Makino, B) have been traditionally used in East Asia and South America to treat various metabolic diseases. The aim of the present study was to determine whether the extracts of KC, B, and KC+B can prevent the impairments of energy, glucose, lipid and bone homeostasis in estrogen-deficient ovariectomized (OVX) rats and to explore their mechanisms.

METHODS

OVX rats were divided into 4 groups and fed high fat diets supplemented with either 3% dextrin (control), 3% KC, 3% B or 1.5% KC+1.5% B. Sham rats were fed 3% dextrin. After <em>12</em> weeks of diet consumption, energy, lipid, glucose and bone metabolisms were analyzed and <em>Wnt</em> signaling in the femur and hepatic signaling were determined.

RESULTS

OVX impaired energy, glucose and lipid metabolism and decreased uterine and bone masses. B and KC+B prevented the decrease in energy expenditure, especially from fat oxidation, in OVX rats, but did not affect food intake. KC+B and B reduced body weight and visceral fat levels, as compared to the OVX-control, by decreasing fat synthesis and inhibiting FAS and SREBP-1c expression. KC+B and B prevented the increases in serum lipid levels and insulin resistance by improving hepatic insulin signaling (pIRS→pAkt→pGSK-3β). KC and KC+B also prevented decreases in bone mineral density (BMD) in the femur and lumbar spine in OVX rats. This was related to decreased expressions of bone turnover markers such as serum osteocalcin, alkaline phosphatase (ALP) and bone-specific ALP levels, and increased serum P levels. KC and KC+B upregulated low-density lipoprotein receptor-related protein 5 and β-catenin in OVX rats, but suppressed the expression of dickkopf-related protein 1. B alone improved energy, lipid and glucose homeostasis, but not bone loss, whereas KC alone enhanced BMD, but not energy, lipid or glucose homeostasis.

CONCLUSIONS

KC+B synergistically attenuated impairments of bone, energy, lipid and glucose metabolism by OVX, suggesting potential efficacy of the combination for alleviating menopausal symptoms.

OBJECTIVE

<em>WNT</em>7A gene mutations were evaluated as a potential cause for Müllerian duct derivative abnormalities in human females. The <em>WNT</em> gene family encodes glycoproteins that serve as signaling molecules during early development. The <em>WNT</em>7A gene has been previously identified as necessary for normal murine Müllerian duct development. <em>WNT</em>7A mutant mice display several Müllerian duct derivative abnormalities.

METHODS

Molecular genetic analysis of female patients with Müllerian duct derivative abnormalities.

METHODS

Medical center-based academic research institution.

METHODS

40 women with developmental abnormalities of the uterus and vagina and 12 normal controls.

METHODS

Polymerase chain reaction DNA amplification from human genomic DNA and denaturing gradient gel electrophoresis analysis of amplified DNA fragments.

METHODS

Presence or absence of <em>WNT</em>7A gene mutations in analyzed DNA fragments.

RESULTS

No mutations were found in the <em>WNT</em>7A gene in any patient or control tested.

CONCLUSIONS

<em>WNT</em>7A mutations are an unlikely cause of Müllerian duct derivative abnormalities in humans.

Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the <em>Wnt</em>, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3β. GSK-3β is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for <em>12</em> days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/-) of TWS119, a GSK-3β inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (-). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3β on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (-) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/-) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (-). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/-), and platelet production was markedly decreased in TPO (-). This indicated that the GSK-3β inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3β inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (-) cells cultured in TWS119 (+/-) for 3 days. The results of the analysis indicated that GSK-3β inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the <em>Wnt</em> signaling and transforming growth factor-β signaling pathways. These observations suggest that GSK-3β inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.

BACKGROUND

Children presenting with medulloblastoma have a wide range of initial presenting symptoms. However, the influence of underlying tumor biology on the initial presentation of medulloblastoma is currently unknown. In light of the recent discovery of distinct medulloblastoma subgroups, we sought to define the initial presentation of childhood medulloblastoma in a subgroup specific manner.

METHODS

We assembled a cohort of <em>12</em>6 medulloblastoma cases at the Hospital for Sick Children between 1994 and 20<em>12</em> and determined subgroup affiliation using nanoString. Clinical details pertaining to the initial presentation were determined through a retrospective chart review.

RESULTS

The median pre-diagnostic interval across all medulloblastoma cases was 4 weeks (IQR: 4-<em>12</em> weeks). Strikingly, when the pre-diagnostic interval was then determined in a subgroup specific manner, cases with <em>WNT</em> and Group 4 tumors showed significantly longer median pre-diagnostic intervals of 8 weeks compared to 2 weeks for SHH and 4 weeks for Group 3 (P = 0.0001). Younger age was significantly associated with a prolonged pre-diagnostic interval (P = 0.02 for all). When stratifying by subgroup the association with age was only significant in Group 4 (P = 0.04 for Group 4). Improved survival was significantly associated with a longer pre-diagnostic interval (P = 0.02), however is no longer significant when controlling for subgroup (P = 0.07).

CONCLUSIONS

The duration of the pre-diagnostic interval in childhood medulloblastoma is highly subgroup dependent, further highlighting the clinical heterogeneity and biological relevance of the four principle subgroups of medulloblastoma.

OBJECTIVE

The adenomatous polyposis coli (APC) and beta-catenin (CTNNB1) genes are the two major components of the Wnt signaling pathway that has been shown to play an important role in the formation of certain cancers. The overactivation of the pathway, which results in abnormal accumulation of beta-catenin protein in nuclei, contributes to most colorectal cancers (CRCs), both sporadic and hereditary, as well as sporadic endometrial cancers (ECs). Here, we studied the involvement of APC and beta-catenin in hereditary nonpolyposis colorectal cancer (HNPCC)-related ECs, and compared the expression patterns to those in HNPCC-related CRCs.

METHODS

Nineteen ECs and 31 CRCs derived from HNPCC patients were immunohistochemically stained with anti-APC- and anti-beta-catenin-antibodies.

RESULTS

Tumor-specific loss of APC was observed in 16 of endometrial cancers (3 of 19) and in 39 of colorectal cancers (12 of 31). Consistently, the loss of APC expression was associated with nuclear beta-catenin staining. Altogether, aberrant beta-catenin localization was observed in 53 of ECs (10 of 19) as compared to 84 of CRCs (26 of 31) (P=0.02).

CONCLUSIONS

Our results suggest a frequent overactivation of the Wnt signaling pathway in hereditary endometrial cancer. In accordance with studies on sporadic cancers, abnormal accumulation of beta-catenin protein in nuclei occurred much less frequently in HNPCC-related ECs than CRCs, which may reflect organ-specific differences in their pathogenesis.

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical <em>Wnt</em> activation in a panel of <em>12</em> canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical <em>Wnt</em> activity that is dependent on <em>Wnt</em> ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical <em>Wnt</em> activity that is not responsive to inhibitors of <em>Wnt</em> ligand secretion. Tumor cell lines with highly active canonical <em>Wnt</em> signaling often carry mutations in key members of the <em>Wnt</em> signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular <em>Wnt</em> pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical <em>Wnt</em> activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical <em>Wnt</em> signaling in canine mammary tumors. We conclude that canonical <em>Wnt</em> activation may be a frequent event in canine mammary tumors both through <em>Wnt</em> ligand-dependent and novel ligand-independent mechanisms.

There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework project "PREDICTOMICS" was established in order to contribute to the further development of in vitro toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray technology is being used more frequently in the in vitro field, however, only very few studies have assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised conditions, each laboratory treated HK-2 cells with 5microM CsA for <em>12</em> and 48h. RNA was isolated and hybridised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes responsible for this separate clustering were removed all laboratories showed similar expression profiles. There was a major impact of time since feed, due to medium exhaustion in the 48h arrays compared to the <em>12</em>h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, amino acid metabolism, amino acid transport and amino acid biosynthesis were over-represented due to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-represented. CsA responsive genes were involved in cell cycle, the p53 pathway and <em>Wnt</em> signaling. Additionally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as medium exhaustion must also be considered in such analyses.

Renal cell carcinoma (RCC) is a seventh ranked malignancy with poor prognosis. RCC is lethal at metastatic stage as it does not respond to conventional systemic treatments, and there is an urgent need to find out promising novel biomarkers for effective treatment. The goal of this study was to evaluate the biomarkers that can be potential therapeutic target and predict effective inhibitors to treat the metastatic stage of RCC.

We conducted transcriptomic profiling to identify differentially expressed genes associated with RCC. Molecular pathway analysis was done to identify the canonical pathways and their role in RCC. Tissue microarrays (TMA) based immunohistochemical stains were used to validate the protein expression of cyclinD1 (CCND1) and were scored semi-quantitatively from 0 to 3+ on the basis of absence or presence of staining intensity in the tumor cell. Statistical analysis determined the association of CCND1 expression with RCC. Molecular docking analyses were performed to check the potential of two natural inhibitors, rutin and curcumin to bind CCND1.

We detected 1490 significantly expressed genes (1034, upregulated and 456, downregulated) in RCC using cutoff fold change 2 and p value < 0.05. Hes-related family bHLH transcription factor with YRPW motif 1 (HEY1), neuropilin 2 (NRP2), lymphoid enhancer-binding factor 1 (LEF1), and histone cluster 1 H3h (HIST1H3H) were most upregulated while aldolase B, fructose-bisphosphate (ALDOB), solute carrier family <em>12</em> (SLC<em>12</em>A1), calbindin 1 (CALB1) were the most down regulated genes in our dataset. Functional analysis revealed <em>Wnt</em>/β-catenin signaling as the significantly activated canonical pathway (z score = 2.53) involving cyclin D1 (CCND1). CCND1 was overexpressed in transcriptomic studies (FC = 2.26, p value = 0.0047) and TMA results also showed the positive expression of CCND1 in 53 % (73/139) of RCC cases. The ligands - rutin and curcumin bounded with CCND1 with good affinity.

CCND1 was one of the important upregulated gene identified in microarray and validated by TMA. Docking study showed that CCND1 may act as a potential therapeutic target and its inhibition could focus on the migratory, invasive, and metastatic potential of RCC. Further in vivo and in vitro molecular studies are needed to investigate the therapeutic target potential of CCND1 for RCC treatment.