Wnt Signaling Members Research Articles

Ellagic acid coordinately attenuates Wnt/β-catenin and NF-κB signaling pathways to induce intrinsic apoptosis in an animal model of oral oncogenesis

Constitutive activation of the Wnt signaling pathway and its downstream effectors plays a key role in neoplastic transformation. The objective of this study was to investigate the effect of ellagic acid, a plant-derived polyphenol on Wnt/β-catenin signaling and its downstream circuits- NF-κB and mitochondrial apoptosis in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model. Hamsters were divided into six groups. The right buccal pouches of animals in groups 1-4 were painted with 0.5% DMBA three times a week for 14 weeks. Animals in groups 2-4 received in addition basal diet containing ellagic acid at a concentration of 0.1, 0.2, and 0.4% in the diet. Group 5 animals were given 0.4% ellagic acid alone. Group 6 animals served as control. The expression of the members of Wnt and NF-κB signaling and intrinsic apoptosis was evaluated by western blot analysis. Dietary supplementation of 0.4% ellagic acid suppressed the development of HBP carcinomas by preventing the constitutive activation of Wnt pathway through the downregulation of Fz, Dvl-2, GSK-3β and nuclear translocation of β-catenin. Abrogation of Wnt signaling by ellagic acid was also associated with inactivation of NF-κB and modulation of key components of the mitochondrial apoptotic network. Our findings suggest a functional crosstalk between Wnt and NF-κB signaling pathways in HBP carcinomas that is blocked by ellagic acid supplementation. Dietary ellagic acid that targets the Wnt/β-catenin pathway as well as its downstream signaling mediators is a unique candidate for cancer chemoprevention.

Aberrant activation of Wnt/β-catenin signaling pathway contributes to the sequential progression of DMBA-induced HBP carcinomas

Wnt signaling pathway mediated via interactions between β-catenin and members of the TCF/LEF-1 family of transcription factors plays a central role in the regulation of epithelial cell proliferation, apoptosis, differentiation, adhesion, epithelial-mesenchymal transition, and invasion. Aberrant activation of the Wnt/β-catenin signaling pathway with overexpression of Wnt and Fz, mutations of APC, β-catenin, and axin 1, and cytoplasmic accumulation of β-catenin have been frequently reported in a broad spectrum of human malignancies including oral squamous cell carcinomas (OSCCs). However, changes in the components of the Wnt signaling pathway have not been documented during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis, a paradigm for oral oncogenesis and chemointervention. In this study, we evaluated the role of β-catenin accumulation and Wnt ligands, Wnt signaling members (Fz, Dvl, APC, GSK-3β, axin, and WIF) and the downstream targets of Wnt (cyclin D1, MMP-2, and MMP-9) during the sequential progression of DMBA-induced HBP carcinomas by semi-quantitative RT-PCR and western blot analyses. Our data reveal a correlation between β-catenin accumulation and activation of Wnt signaling, and its downstream effector molecules during the sequential development of HBP carcinomas from hyperplasia to invasive carcinoma through dysplasia. Our data also support a pivotal role for β-catenin in the malignant transition of the HBP. Aberrant Wnt signaling may be a hallmark of progression to malignancy during DMBA-induced HBP carcinogenesis and could be a potential preventive and therapeutic target for suppression of OSCC.

Wnt Signaling Is Required for Early Development of Zebrafish Swimbladder

BackgroundWnt signaling plays critical roles in mammalian lung development. However, Wnt signaling in the development of the zebrafish swimbladder, which is considered as a counterpart of mammalian lungs, have not been explored. To investigate the potential conservation of signaling events in early development of the lung and swimbladder, we wish to address the question whether Wnt signaling plays a role in swimbladder development.Methodology/Principal FindingsFor analysis of zebrafish swimbladder development, we first identified, by whole-mount in situ hybridization (WISH), has2 as a mesenchymal marker, sox2 as the earliest epithelial marker, as well as hprt1l and elovl1a as the earliest mesothelial markers. We also demonstrated that genes encoding Wnt signaling members Wnt5b, Fz2, Fz7b, Lef1, Tcf3 were expressed in different layers of swimbladder. Then we utilized the heat-shock inducible transgenic lines hs:Dkk1-GFP and hs:ΔTcf-GFP to temporarily block canonical Wnt signaling. Inhibition of canonical Wnt signaling at various time points disturbed precursor cells specification, organization, anterioposterior patterning, and smooth muscle differentiation in all three tissue layers of swimbladder. These observations were also confirmed by using a chemical inhibitor (IWR-1) of Wnt signaling. In addition, we found that Hedgehog (Hh) signaling was activated by canonical Wnt signaling and imposed a negative feedback on the latter.Significance/ConclusionWe first provided a new set of gene markers for the three tissue layers of swimbladder in zebrafish and demonstrated the expression of several key genes of Wnt signaling pathway in developing swimbladder. Our functional analysis data indicated that Wnt/β-catenin signaling is required for swimbladder early development and we also provided evidence for the crosstalk between Wnt and Hh signaling in early swimbladder development.

LEF-1 is a prosurvival factor in chronic lymphocytic leukemia and is expressed in the preleukemic state of monoclonal B-cell lymphocytosis

AbstractThe canonical Wnt signaling pathway is pathogenic in a variety of cancers. We previously identified aberrant expression of the Wnt pathway transcription factor and target gene lymphoid enhancer binding factor-1 (LEF1) in chronic lymphocytic leukemia (CLL). This suggested that the Wnt signaling pathway has a role in the biology of CLL. In this study, we performed a Wnt pathway analysis using gene expression profiling and identified aberrant regulation of Wnt pathway target genes, ligands, and signaling members in CLL cells. Furthermore, we identified aberrant protein expression of LEF-1 specifically in CLL but not in normal mature B-cell subsets or after B-cell activation. Using the T cell–specific transcription factor/LEF (TCF/LEF) dual luciferase reporter assay, we demonstrated constitutive Wnt pathway activation in CLL, although the pathway was inactive in normal peripheral B cells. Importantly, LEF-1 knockdown decreased CLL B-cell survival. We also identified LEF-1 expression in CD19+/CD5+ cells obtained from patients with monoclonal B-cell lymphocytosis, suggesting a role for LEF-1 early in CLL leukemogenesis. This study has identified the constitutive activation and prosurvival function of LEF-1 and the Wnt pathway in CLL and uncovered a possible role for these factors in the preleukemic state of monoclonal B-cell lymphocytosis.

Abstract 4680: Correlation between Wnt pathway and colorectal cancer recurrence using protein, mRNA and microRNA analysis

Abstract BACKGROUND: Wnt signaling pathway has a fundamental role in colorectal carcinogenesis. However, few studies have focused in the association of this pathway and the risk of postoperative recurrence. Besides microRNAs repress protein translation through binding to targets mRNAs and have shown association with specific targets. The main goal of this study was to evaluate several members of Wnt signaling pathway (Wnt protein family, proteins from β-catenin/APC complex, transcription factors and target proteins) and CRC recurrence. We also would like to assess association of microRNAs with specific Wnt targets or the direct implication of those small non coding RNAs with CRC recurrence. METHODS: The expression of Wnt proteins and mRNAs were assessed using immunohistochemistry and Real Time-PCR techniques, respectively. We also studied 381 microRNAs using Low Density TaqMan Array. This study was performed in samples from patients presenting stages I and II CRC and comparing those who had recurrence to those who did not. All patients undergone surgery with curative intention from 2000 to 2006 in A. C. Camargo Hospital. Statistical analyses used in this study were Chi-square, exact Fisher's test and T Paired. RESULTS: Regarding the protein expression and gene expression of Wnt signaling members only c-Myc showed statistically significant association with recurrence (p=0,001 and p=0,048, respectively). Regarding the analysis of 381 microRNAs, a statistically significant association was observed for miR-197 (p=0,03), where the group of patients who did not had recurrence demonstrated higher expression levels of this microRNA when compared to the group of patients who developed tumor recurrence. CONLUSION: Increased levels of c-Myc expression and low levels of miR-197 in the tumor may be used at the time of primary removal for defining patients at risk for tumor recurrence. miR-197 may regulate indirectly the expression of c-Myc. However, further experiments will be required to better evaluate this hypothesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4680.

Wnt and Notch signaling pathways selectively regulating hematopoiesis

Hematopoietic stem and progenitor cells (HSPCs) are the source of all blood cells in the adult body. The pool of HSPCs is formed during embryogenesis process through a well-characterized succession of intra-embryonic regions and organs. The spatial and temporal restrictions in definitive hematopoietic development and the signaling molecules involved are of great interest as these may prove useful for generating and expanding these clinically important cell populations ex vivo. To elucidate the mechanism by which definitive HSPCs expand during this limited developmental time frame, we analyzed the spatial and temporal programmed gene expression patterns of Wnt and Notch signaling members during hematopoietic development. Genes related to the Wnt signaling pathway were up-regulated in E10.5 aorta-gonad-mesonephros (AGM) and E14.5 fetal liver corresponding to the inherent proliferation potential of hematopoietic progenitors, whereas genes related to the Notch signaling pathway were identified as up-regulated in E10.5 AGM, and bone marrow coincides with the maintenance of undifferentiation state of hematopoietic progenitors. Our findings suggest that Wnt and Notch signalings are integrated and are selectively regulating hematopoiesis. The spatial and temporal balance between Wnt and Notch signaling orchestrates the precise progression of hematopoietic progenitors.

Retinoic acid induces discrete Wnt-signaling-dependent differentiation in F9 cells

Retinoic acid (RA) induces F9 cells, the mouse teratocarcinoma cells, to differentiate into primitive endoderm and further into visceral and parietal endoderm depending on the culture conditions. To elucidate the instructive mechanisms involved in the differentiation steps we investigated the effects of Wnt-signaling members, Wnt3a and β-catenin, on the differentiation of F9 cells and β-catenin-deficient F9 cells (βT cells). RA up-regulated the expression of differentiation markers for primitive, visceral and parietal endoderm in F9 cells but not for visceral endoderm in βT cells. Wnt3a or leukemia inhibitory factor (LIF) inhibited the RA-induced differentiation in F9 cells. LIF but not Wnt3a could inhibit differentiation in βT cells. RA evoked ZO-1α+ signals at cell-to-cell contacts in F9 cells in a Wnt3a sensitive manner. The results suggest that Wnt3a inhibits differentiation into endoderm through a pathway involving β-catenin, and β-catenin might be necessary in the process leading from primitive to visceral endoderm in F9 cells.

Mutational analysis of Wnt signaling molecules in ameloblastoma with aberrant nuclear expression of β‐catenin

To clarify the genetic background of ameloblastoma, expression of beta-catenin, and mutational status of genes involved in Wnt signaling pathway were investigated. We analyzed beta-catenin and cyclin D1 in 18 cases of ameloblastoma by immunohistochemical staining, and searched for mutations in CTNNB1 (gene for beta-catenin), APC, AXIN1, and AXIN2 by polymerase chain reaction (PCR) and direct sequencing method. We detected membranous and occasionally cytoplasmic expression of beta-catenin in 16 of 18 cases (89%), and nuclear expression of beta-catenin principally in the peripheral columnar cells in 11 of 18 cases (61%). In nine of the 18 cases (50%), we detected the expression of cyclin D1 principally in the peripheral columnar cells. However, there was no correlation between nuclear expressions of beta-catenin and cyclin D1. No missense mutations were found in CTNNB1, APC, AXIN1, and AXIN2 in all cases except for silent mutation and already-known single nucleotide polymorphism. Mutations in CTNNB1, APC, AXIN1, and AXIN2 are not implicated in nuclear accumulation of beta-catenin, and that the expression of cyclin D1 is accelerated independently of beta-catenin in ameloblastomas. Other Wnt signaling members or alternative pathways involved in the degradation of beta-catenin should be subject of further investigation.

Preliminary Protein Profile Analysis of the Late Embryonic B-Cell Stages in the Chicken Bursa of Fabricius

The bursa of Fabricius serves as a primary lymphoid organ for the development of a diverse repertoire of B-cells. The embryonic bursa is colonized during embryonic days (ED) 8-14 by stem cells expressing the sialyl Lewis carbohydrate (SLEX). At ED13-15, cells with the SLEX phenotype initiate X proliferation leading to the development of the bursal follicle. By ED15-17, a key differentiation event occurs resulting in the onset of repertoire development by immunoglobulin gene-conversion. This differentiation event is defined by a phenotypic transition in cell surface glycosylation from SLEX to a related carbohydrate structure termed Lewis (LEX). The goal of this study was to identify functional groups of genes in the two X stages that might be involved in critical biological processes of proliferation, differentiation, apoptosis and cell adhesion, and explain the observed functional differences. We found that prior to the onset of immunoglobulin gene conversion B-cells express genes related to ephrin receptor signaling, epidermal growth factor receptor (EGFR) signaling and Wnt signaling. B-cells undergoing immunoglobulin gene conversion express genes from TNFR signaling and both stages were found to express members of Wnt signaling, integrin signaling and EGFR signaling pathways. The differentially expressed pathways agree with previous observations, offering explanation to signals leading to proliferation, differentiation and apoptosis in the two B-cell stages.

Wnt signaling activation during bone regeneration and the role of Dishevelled in chondrocyte proliferation and differentiation

Wnt signaling is intrinsically involved in diverse cellular activities during cell differentiation, early embryonic development and organogenesis. Although much is known regarding the effects of Wnt signaling in the developing skeletal system, its role during regeneration remains unclear. Herein, we show transcriptional activation of specific members and target genes of the Wnt signaling pathway. Specifically, all of the Wnt signaling members and target genes analyzed were found to be upregulated during the early stages of fracture repair, with the exception of LEF1 whose expression was downregulated. In addition, spatial expression analysis of Dishevelled (Dvl) and β-catenin in the fracture callus revealed an identical pattern of expression with both proteins localizing in osteoprogenitor cells of the periosteum, osteoblasts and proliferating/pre-hypertrophic chondrocytes. Further, in vitro knockdown of all three Dvl isoforms in chondrocytes using small interfering RNAs (siRNA) leads to partial inhibition of cell proliferation and differentiation, decreased expression of chondrogenic markers (ColII, ColX, Sox9) and suppressed nuclear accumulation of unphosphorylated β-catenin. Taken together, these data verify our previous finding that the Wnt signaling pathway is activated during bone regeneration, by characterizing the temporal and spatial expression of a broad spectrum of Wnt-signaling molecules. Our data also suggest that all three Dvl isoforms, acting through the Wnt canonical pathway, are critical regulatory molecules for chondrocyte proliferation and differentiation.

Negative regulation of retinal-neurite extension by β-catenin signaling pathway

Although there have been many studies on the regulation of neurite extension in mouse brain, such a mechanism in neural retina has remained to be clarified. To delineate the role of Wnt signaling in retinal development, we used a retrovirus-vector-mediated expression system to express various mutants forms of Wnt signaling members in E17.5 mouse retinal explant cultures, which are an excellent system to examine retinal development in vitro. Expression of constitutively active beta-catenin or Lef-1 in the retinal cells resulted in failure of neurite extension, suggesting that beta-catenin negatively regulates neurite extension in the retina through Lef-1 transcriptional activity. However, proliferation and differentiation of retinal cells into mature retinal cells such as rod-photoreceptor cells and Muller glia cells were not affected by perturbation of the Wnt-Lef-1 pathway. As in retinal cells, activation of beta-catenin-Lef-1 signaling inhibited NGF-induced neurite extension in PC12 cells without affecting their proliferation. Interestingly, the Wnt-Lef-1 signaling pathway suppressed neurite extension without affecting Mek-1 signal activity, which is known to promote neurite extension. We found that MAPK was activated in retinal explant cultures, but that perturbation of MAPK signals did not affect neurite extension. Taken together, our data suggest that the Wnt pathway functions in proper neurite extension by opposing positive signals for promotion of neurite extension that are distinct from those of the MAPK pathway.