Wingless-type MMTV Integration Site Research Articles

Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway.

Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/β-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/β-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/β-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of β-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced β-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of β-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation.

Reduced Wnt3a expression correlates with poor development of the hindgut in rats with anorectal malformations

Embryogenesis is orchestrated by the wingless-type MMTV integration site family (WNT) signaling pathways, including Wnt3a. This study was performed to investigate the expression of Wnt3a in the terminal hindgut in ethylenethiourea (ETU)-exposed rat embryos with anorectal malformations (ARMs) and its potential association between Wnt3a and the maldevelopment of the terminal hindgut in ARMs. ARM rat embryos were induced by ethylenethiourea on embryonic day 10 (E10). The expression levels of protein and mRNA of Wnt3a were confirmed using immunohistochemistry staining, Western blotting analyses, and quantitative real-time PCR (qRT-PCR) in normal rat and ARM embryos. Immunostaining revealed a variation in the expression of Wnt3a in the developing terminal hindgut of ARM embryos. The expression of Wnt3a in the terminal hindgut of ARM rat embryos decreased at both the mRNA level and protein level (P<0.05) compared with normal tissues. This study demonstrated that the expression of Wnt3a in the ARMs of ETU-exposed rat embryos was remarkably reduced, which indicated its potential role in the pathogenesis of the terminal hindgut maldevelopment in ARMs.

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor.

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.

Novel missense mutation in WNT6 in 100 couples with unexplained recurrent miscarriage.

Do mutations and/or polymorphisms in coding sequences in Wingless-Type MMTV Integration Site Family, Member 6 (WNT6) play a role in unexplained recurrent miscarriage (unexplained RM) in Chinese couples? We found four mutations in the coding sequences of WNT6 which appear to exist in a small proportion of Chinese women with unexplained RM. WNT6 has been proved to be essential for stromal cell proliferation during decidualization in mice, but in humans WNT6 has not been studied in recurrent miscarriage populations. For this study, 100 couples with unexplained RM (at least three or more unexplained spontaneous miscarriages), and 100 ethnically matched fertile couples (at least one live birth and no history of pregnancy pathologies) were recruited. All the participants were chosen over a 7-year period from the National Research Center for Assisted Reproductive Technology and Reproductive Genetics at Shandong University, Jinan, China. Patients were recruited following extensive clinical studies. Genomic DNA was isolated from peripheral blood. Mutation analysis in the coding regions of WNT6 was performed by PCR amplification and DNA sequences testing in all participants. Functional effects of missense variants were predicted using Polyphen-2 and sorting intolerant from tolerant (SIFT). Four rare novel mutations, including one missense mutation, were found in intron 1, exon 3 and the 3' untranslated region of WNT6 in four women with unexplained RM. Gene software predictions showed that the missense mutation in exon 3 could alter the function of WNT6. No mutations or polymorphisms were detected in the male partners of the unexplained RM patients or in the fertile controls. To further validate the findings, we continued to screen this missense mutation site in another 100 peripheral blood samples of normal fertile females, and there was still no positive result. There is no direct evidence to validate whether these novel mutations discovered in the present research are related to unexplained RM. Further studies are warranted to investigate the role of WNT6 in unexplained RM, including larger studies in an independent group. These results provide evidence to suggest the importance of WNT6 in reproductive failure and may support the hypothesis that WNT6 is essential for stromal cell proliferation during decidualization. This work was supported by Science and Technology Development Planning of Shandong (2013GGE27001), the National Natural Science Foundation of China (81300459), the Science Projection of Bureau of Public Health in Weifang (2012044) and the Science Research Foundation Item of No-earnings Health Vocation. The authors have no competing interests to declare.

Upper-body resistance exercise augments vastus lateralis androgen receptor–DNA binding and canonical Wnt/β-catenin signaling compared to lower-body resistance exercise in resistance-trained men without an acute increase in serum testosterone

The purpose of the study was to determine the effect of single bouts of lower-body (LB) and upper- and lower-body (ULB) resistance exercise on serum testosterone concentrations and the effects on muscle testosterone, dihydrotestosterone (DHT), androgen receptor (AR) protein content, and AR–DNA binding. A secondary purpose was to determine the effects on serum wingless-type MMTV integration site (Wnt4) levels and skeletal muscle β-catenin content. In a randomized cross-over design, exercise bouts consisted of a LB and ULB protocol, and each bout was separated by 1week. Blood and muscle samples were obtained before exercise and 3 and 24h post-exercise; blood samples were also obtained at 0.5, 1, and 2h post-exercise. Statistical analyses were performed by separate two-way factorial analyses of variance (ANOVA) with repeated measures. No significant differences from baseline were observed in serum total and free testosterone and skeletal muscle testosterone and DHT with either protocol (p>0.05). AR protein was significantly increased at 3h post-exercise and decreased at 24h post-exercise for ULB, whereas AR–DNA binding was significantly increased at 3 and 24h post-exercise (p<0.05). In response to ULB, serum Wnt4 was significantly increased at 0.5, 1, and 2h post-exercise (p<0.05) and β-catenin was significantly increased at 3 and 24h post-exercise (p<0.05). It was concluded that, despite a lack of increase in serum testosterone and muscle androgen concentrations from either mode of resistance exercise, ULB resistance exercise increased Wnt4/β-catenin signaling and AR–DNA binding.

Effects of Rosmarinus officinalis extract on human primary omental preadipocytes and adipocytes.

The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this invitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase 4, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, wingless-type MMTV integration site family, member 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-143. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement.

Psoriasis: in between the skin and the fat.

Substantial epidemiological evidence indicates that psoriasis associates with a predisposition to develop metabolic dysregulation leading to obesity and insulin resistance. However, the nature of this association and the potential underlying mechanisms remain unclear. In a recent report, Gerdes et al. explored the hypothesis that wingless-type MMTV integration site, Wnt5a, which has been linked to aberrant fat cell metabolism, may be driving this process. In this study, the authors compare circulating serum levels of Wnt5a in individuals with psoriasis and compare with healthy controls matched for age, gender and BMI. The bottom-line results show higher levels of Wnt5a in psoriasis patients irrespective of BMI compared to the matched non-psoriatic controls, indicating that psoriasis per se may result in increased secretion of Wnt5a into the circulation. In addition, there was a significant difference among patients with higher levels of Wnt5a in the obese psoriasis population. The study, even though being purely descriptive, may serve to inspire a more mechanistic approach exploring not only Wnt5a, but other inflammatory pathways in between the skin and the fat.

Melatonin improves experimental colitis with sleep deprivation.

Sleep deprivation (SD) is an epidemic phenomenon in modern countries, and its harmful effects are well known. SD acts as an aggravating factor in inflammatory bowel disease. Melatonin is a sleep-related neurohormone, also known to have antioxidant and anti-inflammatory effects in the gastrointestinal tract; however, the effects of melatonin on colitis have been poorly characterized. Thus, in this study, we assessed the measurable effects of SD on experimental colitis and the protective effects of melatonin. For this purpose, male imprinting control region (ICR) mice (n=24) were used; the mice were divided into 4 experimental groups as follows: the control, colitis, colitis with SD and colitis with SD and melatonin groups. Colitis was induced by the administration of 5% dextran sulfate sodium (DSS) in the drinking water for 6 days. The mice were sleep-deprived for 3 days. Changes in body weight, histological analyses of colon tissues and the expression levels of pro-inflammatory cytokines and genes were evaluated. SD aggravated inflammation and these effects were reversed by melatonin in the mice with colitis. In addition, weight loss in the mice with colitis with SD was significantly reduced by the injection of melatonin. Treatment with melatonin led to high survival rates in the mice, in spite of colitis with SD. The levels of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-17, interferon-γ and tumor necrosis factor-α, in the serum of mice were significantly increased by SD and reduced by melatonin treatment. The melatonin-treated group showed a histological improvement of inflammation. Upon gene analysis, the expression of the inflammatory genes, protein kinase Cζ (PKCζ) and calmodulin 3 (CALM3), was increased by SD, and the levels decreased following treatment with melatonin. The expression levels of the apoptosis-related inducible nitric oxide synthase (iNOS) and wingless-type MMTV integration site family, member 5A (Wnt5a) genes was decreased by SD, but increased following treatment with melatonin. Treatment with melatonin reduced weight loss and prolonged survival in mice with colitis with SD. Melatonin exerted systemic anti-inflammatory effects. Gene analysis revealed a possible mechanism of action of melatonin in inflammation and sleep disturbance. Thus, melatonin may be clinically applicable for patients with inflammatory bowel disease, particulary those suffering from sleep disturbances.

Disruption of insulin signaling in Myf5-expressing progenitors leads to marked paucity of brown fat but normal muscle development.

Insulin exerts pleiotropic effects on cell growth, survival, and metabolism, and its role in multiple tissues has been dissected using conditional knockout mice; however, its role in development has not been studied. Lineage tracing experiments have demonstrated that interscapular brown adipose tissue (BAT) arises from a Myf5-positive lineage shared with skeletal muscle and distinct from the majority of white adipose tissue (WAT) precursors. In this study, we sought to investigate the effects of impaired insulin signaling in the Myf5-expressing precursor cells by deleting the insulin receptor gene. Mice lacking insulin receptor in the Myf5 lineage (Myf5IRKO) have a decrease of interscapular BAT mass; however, muscle development appeared normal. Histologically, the residual BAT had decreased cell size but appeared mature and potentially functional. Expression of adipogenic inhibitors preadipocyte factor-1, Necdin, and wingless-type MMTV integration site member 10a in the residual BAT tissue was nonetheless increased compared with controls, and there was an enrichment of progenitor cells with impaired adipogenic differentiation capacity, suggesting a suppression of adipogenesis in BAT. Surprisingly, when cold challenged, Myf5IRKO mice did not show impaired thermogenesis. This resistance to cold could be attributed to an increased presence of uncoupling protein 1-positive brown adipocytes in sc WAT as well as increased expression of lipolytic activity in BAT. These data suggest a critical role of insulin signaling in the development of interscapular BAT from Myf5-positive progenitor cells, but it appears to be dispensable for muscle development. They also underscore the importance of compensatory browning of sc WAT in the absence of BAT for thermoregulation.

Wingless-type MMTV integration site family member 2 (WNT2) gene is associated with resistance to MAP in faecal culture and antibody response in Holstein cattle.

Mycobacterium avium subspecies paratuberculosis (MAP) is a pathogenic bacterium responsible for the lethal Johne's disease in cattle. So far, several genome-wide association studies (GWAS) have been carried out to identify chromosomal regions highly associated with Johne's disease. The aim of this study was to investigate the genetic variability within a pool of seven genes (LAMB1, DLD, WNT2, PRDM1, SOCS5, PTGER4 and IL10) indicated by former GWAS/RNA-Seq studies as putatively associated with MAP infections and to achieve a confirmation study of association with paratuberculosis susceptibility in a population of 324 German Holstein cattle (162 cases MAP positive and 162 controls MAP negative) using ELISA and fecal cultural tests. SNP validation and genotyping information are provided, quick methods for allelic discrimination were set up and transcription factor binding analyses were performed. The rs43390642:G>TSNP in the WNT2 promoter region is associated with paratuberculosis susceptibility (P = 0.013), suggesting a protective role of the T allele (P = 0.043; odds ratio 0.50 [0.25-0.97]). The linkage disequilibrium with the DLD rs134692583:A>T might suggest a combined mechanism of action of these neighboring genes in resistance to MAP infection, which is also supported by a significant effect shown by the haplotype DLD(T) /WNT2(T) (P = 0.047). In silico analysis predicted rs43390642:G>T and rs134692583:A>T as essential parts of binding sites for the transcription factors GR, C/EBPβ and GATA-1, hence suggesting a potential influence on WNT2 and DLD gene expression. This study confirmed the region on BTA 4 (UMD 3.1: 50639460-51397892) as involved in tolerance/resistance to Johne's disease. In addition, this study clarifies the involvement of the investigated genes in MAP infection and contributes to the understanding of genetic variability involved in Johne's disease susceptibility.

Computational Gene Expression Modeling Identifies Salivary Biomarker Analysis that Predict Oral Feeding Readiness in the Newborn

To combine mathematical modeling of salivary gene expression microarray data and systems biology annotation with reverse-transcription quantitative polymerase chain reaction amplification to identify (phase I) and validate (phase II) salivary biomarker analysis for the prediction of oral feeding readiness in preterm infants. Comparative whole-transcriptome microarray analysis from 12 preterm newborns pre- and postoral feeding success was used for computational modeling and systems biology analysis to identify potential salivary transcripts associated with oral feeding success (phase I). Selected gene expression biomarkers (15 from computational modeling; 6 evidence-based; and 3 reference) were evaluated by reverse-transcription quantitative polymerase chain reaction amplification on 400 salivary samples from successful (n = 200) and unsuccessful (n = 200) oral feeders (phase II). Genes, alone and in combination, were evaluated by a multivariate analysis controlling for sex and postconceptional age (PCA) to determine the probability that newborns achieved successful oral feeding. Advancing PCA (P < .001) and female sex (P = .05) positively predicted an infant's ability to feed orally. A combination of 5 genes, neuropeptide Y2 receptor (hunger signaling), adneosine-monophosphate-activated protein kinase (energy homeostasis), plexin A1 (olfactory neurogenesis), nephronophthisis 4 (visual behavior), and wingless-type MMTV integration site family, member 3 (facial development), in addition to PCA and sex, demonstrated good accuracy for determining feeding success (area under the receiver operator characteristic curve = 0.78). We have identified objective and biologically relevant salivary biomarkers that noninvasively assess a newborn's developing brain, sensory, and facial development as they relate to oral feeding success. Understanding the mechanisms that underlie the development of oral feeding readiness through translational and computational methods may improve clinical decision making while decreasing morbidities and health care costs.

Wnt4 in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides): identification and expression.

Wnt4 (Wingless-type MMTV integration site family member 4) has been demonstrated to play critical roles in ovarian development in mammals, but its function in fish reproduction is still unclear. In the present study, two full-length wnt4 cDNA sequences (named wnt4a and wnt4b) were cloned from the orange-spotted grouper (Epinephelus coioides). Amino acid alignment analysis showed that both orange-spotted grouper Wnt4s proteins had the typical characteristics of the Wnt family. RT-PCR revealed that both wnt4a and wnt4b were highly expressed in the ovaries of the orange-spotted grouper. Temporal expression profiles of both wnt4 genes during embryonic and ovarian development were examined. The expressions of wnt4a and wnt4b genes were first detected at the embryonic morula stage, but the gens showed different expression patterns. During ovarian development, high expression of wnt4a was observed in the ovarian lumen formation and gonium proliferation stage, while wnt4b exhibited strong expression in the early developmental stage of oocytes. Taken together, the present study indicates that the two wnt4 genes are involved in the regulation of ovarian development in the orange-spotted grouper.

Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer.

Despite recent advances in cancer management, colorectal cancer (CRC) remains the third most common cancer and a major health-care problem worldwide. MicroRNAs have recently emerged as key regulators of cancer development and progression by targeting multiple cancer-related genes; however, such regulatory networks are not well characterized in CRC. Thus, the aim of this study was to perform global messenger RNA (mRNA) and microRNA expression profiling in the same CRC samples and adjacent normal tissues and to identify potential miRNA-mRNA regulatory networks. Our data revealed 1273 significantly upregulated and 1902 downregulated genes in CRC. Pathway analysis revealed significant enrichment in cell cycle, integrated cancer, Wnt (wingless-type MMTV integration site family member), matrix metalloproteinase, and TGF-β pathways in CRC. Pharmacological inhibition of Wnt (using XAV939 or IWP-2) or TGF-β (using SB-431542) pathways led to dose- and time-dependent inhibition of CRC cell growth. Similarly, our data revealed up- (42) and downregulated (61) microRNAs in the same matched samples. Using target prediction and bioinformatics, ~77% of the upregulated genes were predicted to be targeted by microRNAs found to be downregulated in CRC. We subsequently focused on EZH2 (enhancer of zeste homolog 2 ), which was found to be regulated by hsa-miR-26a-5p and several members of the let-7 (lethal-7) family in CRC. Significant inverse correlation between EZH2 and hsa-miR-26a-5p (R2=0.56, P=0.0001) and hsa-let-7b-5p (R2=0.19, P=0.02) expression was observed in the same samples, corroborating the belief of EZH2 being a bona fide target for these two miRNAs in CRC. Pharmacological inhibition of EZH2 led to significant reduction in trimethylated histone H3 on lysine 27 (H3K27) methylation, marked reduction in cell proliferation, and migration in vitro. Concordantly, small interfering RNA-mediated knockdown of EZH2 led to similar effects on CRC cell growth in vitro. Therefore, our data have revealed several hundred potential miRNA-mRNA regulatory networks in CRC and suggest targeting relevant networks as potential therapeutic strategy for CRC.

WNT10A coding variants and maxillary lateral incisor agenesis with associated dental anomalies.

Congenital maxillary lateral incisor agenesis (MLIA) is one of the most common subtypes of dental agenesis. Because little is known with regard to the aetiology of this anomaly, the aim of the study was to determine the contribution of nucleotide variants in wingless-type MMTV integration site family, member 10A (WNT10A), msh homeobox 1 (MSX1), and paired box 9 (PAX9) to the risk of MLIA in a Polish population. Coding regions of the selected genes were analysed by direct sequencing in a group of 20 individuals with unilateral and bilateral MLIA, associated or not with other dental anomalies. The frequencies of the identified nucleotide variants were assessed in an additional cohort of patients with isolated dental agenesis (n = 147) and in 178 controls. Mutation screening showed four non-synonymous substitutions located in the highly conserved coding sequence of WNT10A in five (25%) of the 20 patients. Analysis of genotyping results revealed that three of these variants--p.Arg113Cys, p.Phe228Ile, and the newly identified p.Arg171Leu--may represent aetiological mutations underlying MLIA with associated dental anomalies. No mutations that were potentially aetiologic were identified in MSX1 and PAX9. In conclusion, this is the first report implicating coding variants in the WNT10A gene in the aetiology of MLIA. These results will require further confirmation using larger-scale studies.

Recent proteomic advances in developmental, regeneration, and cancer governing signaling pathways.

Embryonic development, adult tissue repair, and cancer share a number of common regulating pathways. The basic processes that govern the events that induce mesenchymal properties in epithelial cells-a process known as epithelial-mesenchymal transition-are central for embryonic development, and can be resumed in adults either during wound healing or tissue regeneration. A misregulation of these pathways is involved in pathological situations, such as tissue fibrosis and cancer. Proteomic approaches have emerged as promising tools to better understand the signaling pathways that govern these complex biological processes. This review focuses on the recent proteomic-based contributions to better understand the modulation of transforming growth factor-beta (TGF-β), wingless-type MMTV integration site family (Wnt), Notch and Receptor tyrosine kinase (RTK) signaling pathways. New advances include the description of new protein interactions, the formation of new protein complexes or the description on how some PTMs are regulating these pathways. Understanding protein interactions and the tempo-spatial modulation of these pathways might lead us to interesting research quests in cancer, embryonic development or even on improving adult tissue regeneration capabilities.

Insights into Molecular Features of Venerupis decussata Oocytes: A Microarray-Based Study

The production of Venerupis decussata relies on wild seed collection, which has been recently compromised due to recruitment failure and severe mortalities. To address this issue and provide an alternative source of seed, artificial spawning and larval rearing programs were developed. However, hatchery-based seed production is a relatively new industry and it is still underdeveloped. A major hurdle in the European clam seed production is the control of spawning and reproduction, which is further hindered by the impossibility of obtaining fertile gametes by gonadal “stripping”, as meiosis re-initiation is constrained to a maturation process along the genital ducts. In the present study, oocytes were collected from 15 females and microarray analyses was performed to investigate gene expression profiles characterizing released and stripped ovarian oocytes. A total of 198 differentially expressed transcripts between stripped and spawned oocytes were detected. Functional analysis carried out on these transcripts highlighted the importance of a few biological processes, which are most probably implicated in the control of oocyte competence. Significant differences were observed for transcripts encoding proteins involved in meiosis progression (e.g. dual specificity phosphatase CDC25), WNT signalling (e.g. frizzled class receptor 8, wingless-type MMTV integration site family member 4), steroid synthesis (e.g. progestin and adipoQ receptor family member 3, cytochrome P450-C17), mRNA processing (e.g. zinc finger protein XlCOF28), calcium regulation (e.g. regucalcin, calmodulin) and ceramide metabolism (ceramidase B, sphingomyelinase). This study provides new information on transcriptional profiles putatively associated with ovarian egg infertility, and suggests potential mechanisms regulating early oocyte development in clams. Genes which were differentially expressed between stripped and spawned oocytes might have a pivotal role during maturation process in the gonadal duct and could be interesting targets for further functional studies aiming to make ovarian oocytes fertilizable.

Suppression of wingless-type MMTV integration site family, member 1 expression by small interfering RNA inhibits U251 glioma cell growth in vitro.

A Wingless-type MMTV integration site family, member 1 (Wnt-1) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pGPU6/green fluorescent protein (GFP)/Neo and the sequence was confirmed by DNA sequencing. The pGPU6/GFP/Neo-short hairpin RNA (shRNA)-Wnt-1 vector was subsequently transfected into U251 cells, and reverse transcription polymerase chain reaction and western blot analysis were used to evaluate the Wnt-1 gene silencing effect on U251 cell growth by MTT assay and flow cytometry. The Wnt-1 protein expression was significantly reduced following transfection with the recombinant plasmid, as determined by western blot analysis of the transfected U251 cells. This transfection exhibited a significantly higher death rate, as shown by MTT. Thus, the present study demonstrated that the pGPU6/GFP/Neo-shRNA-Wnt-1 vector inhibited Wnt-1 protein expression. However, further investigations regarding the Wnt signaling pathway in glioma pathogenesis are required.

Autophagy regulator BECN1 suppresses mammary tumorigenesis driven by WNT1 activation and following parity

Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1+/−;MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).

Wingless-type MMTV integration site family member 16B (WNT16B)

Wingless-type MMTV integration site family member 16B (WNT16B)

Abstract 4067: TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways

Abstract A small fraction of cancer stem cells (CSC) initiate and maintain tumors thus driving glioma tumorigenesis and are responsible for resistance to classical chemo- and radio-therapies. It is desirable to identify signaling pathways related to CSC to develop novel therapies to selectively target them. TRPM7 is a ubiquitous Ca2+ and Mg2+ permeable ion channel which contains a serine/threonine kinase. Recent studies have demonstrated that TRPM7 plays an important role in the growth and proliferation of various tumor cells. To determine the possibility of an interaction among TRPM7, Notch, STAT3 and the ALDH1 signaling in the context of brain tumors, we investigated the effects of TRPM7 pathway activation on glioma cells and stem-like cells derived from them. Methods: 1) We analyzed human stem cell signaling pathways using real time RT2 Profile PCR arrays in the A172 glioma cells upon TRPM7 gene silencing. 2) We then examined Notch and STAT3 pathway activation, Aldehyde dehydrogenase 2 family, mitochondrial (ALDH1) and CD133 expressions in parental and glioma stem cell (GSC) populations using quantitative real time-PCR, Western blot, and immunofluorence images. 3) We conducted a ChIP analysis to examine the effects of STAT3 on ALDH1 promoter by introducing constitutive STAT3 (caSTAT3) and dominant STAT3 (dnSTAT3) into A172 cells. 4) Finally, we examined the role of TRPM7 in glioma cell migration and invasion using transwell invasion assay and wound healing assay. Results: 1) RT2 Profile PCR arrays showed that TRPM7 gene silencing down-regulated genes (≥2 fold-change) involved in Notch pathway [Notch1, Jagged 1(JAG1) and Delta-like 1, Drosophila (DLL1)], Wnt pathway, Wingless-type MMTV integration site family, member 1(WNT1) and ALDH1. 2) STAT3 activation and Notch1 expression were decreased in response to TRPM7 silencing. 3) Notch-regulated genes including Notch1, Hey2, JAG1, and Survivin as well as ALDH1 were significantly expressed higher in spheroid glioma CSCs when compared with monolayer cultures. 4) ALDH1 promoter activity was activated by STAT3 in A172 cells. 5) Knockdown of TRPM7 inhibited migration and invasion of A172 cells. Conclusions: TRPM7 activates JAK2/STAT3 and/or Notch signaling pathways and leads to increased cell proliferation and migration. These findings for the first time demonstrate that TRPM7 (1) activates a previously unrecognized STAT3→ALDH1 pathway, and (2) promotes the induction of ALDH1 activity in glioma cells. (This work was supported by the National Institutes of Health grant numbers NIH R01NS047506, R01NS066027, UL1 RR025008, U54 RR026137, AHA 0840132N, and ALZ IIRG-10-173350.) Citation Format: Mingli Liu, Koichi Inoue, Tiandong Leng, Shanchun Guo, Zhigang Xiong. TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways. [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 4067. doi:10.1158/1538-7445.AM2014-4067