1.
Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures.
Abstract
ABSTRACT:
BACKGROUND:
Three dimensional (3D) growths of cancer cells in vitro are more reflective of in situ cancer cell growth than growth in monolayer (2D). The present study is designed to determine changes in protein and phosphoprotein that reflect adaptation of tumor cells to 3D as compared to 2D. Since relative hypoxia is a common feature of most solid tumors, the present study also aims to look at the impact of transition from normoxia to hypoxia in these two growth conditions.
RESULTS:
Using reverse-phase protein arrays, we compared levels of 121 different phosphorylated and non-phosphorylated proteins in 5 glioma and 6 adenocarcinoma lines under conditions of 3D and monolayer culture in normoxia and hypoxia. A three-way analysis of variance showed levels of 82 antibodies differed between media (2D vs. 3D) and 49 differed between treatments (hypoxia vs. normoxia). Comparing 2D to 3D growth, 7 proteins were commonly (i.e., >50% of tumors) elevated in 3D: FAK, AKT, Src, GSK3alphabeta, TSC2, p38, and NFkappabetap65. Conversely, 7 other proteins are commonly decreased: ATRIP, ATR, beta-catenin, BCL-X, cyclin B1, Egr-1, and HIF-1alpha. Comparing normoxia to hypoxia, only NCKIPSD was commonly elevated in hypoxia; 6 proteins were decreased: cyclin B1, 4EBP1(Ser65), c-Myc, SMAD3(Ser423), S6(Ser235), and S6(Ser240). Hypoxia affected glioma cell lines differently from adenocarcinoma cell lines: 8 proteins were increased in gliomas (BAX, caspase 7, HIF-1alpha, c-JUN, MEK1, PARP 1 cleaved, Src, and VEGFR2) and none in adenocarcinomas.
CONCLUSIONS:
We identified subsets of proteins with clearly concordant/discordant behavior between gliomas and adenocarcinomas. In general, monolayer to 3D culture differences are clearer than normoxia to hypoxia differences, with anti-apoptotic, cytoskeletal rearrangement and cell survival pathways emphasized in the former and mTOR pathway, transcription, cell-cycle arrest modulation, and increased cell motility in the latter.
- PMID:
- 22276931
- [PubMed - as supplied by publisher]
Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling.
Source
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Hematology, the Affiliated Hospital of Medical College Qingdao University, Qingdao, China.
Abstract
Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML-CP samples, 70% CML-AP samples and more than 80% CML-BP samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p<0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p<0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, besides, CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p<0.05), implying cell-autonomous activation of Shh signaling play a predominantly role in progenitor cells. Co-activation of Shh and β-cateninsignaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh neutralizing antibody or Wnt3a neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis, however, Wnt3a peptide could salvage anti-Shh-induced cell apoptosis while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G2/M cell cycle arrest of progenitor cells. Our results demonstrate that auto-activated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, thus suggesting a novel therapeutic approach in CML.
Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.
Frizzled7 as an emerging target for cancer therapy.
Source
Department of Biochemistry and Molecular Biology, Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA.
Abstract
Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Recent studies indicated that, among the Fzd family, Fzd7 is the Wnt receptor most commonly upregulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma and triple negative breast cancer. Fzd7 plays an important role in stem cell biology and cancer development and progression. In addition, it has been demonstrated that siRNA knockdown of Fzd7, the anti-Fzd7 antibody or the extracellular peptide of Fzd7 (soluble Fzd7 peptide) displayed anti-cancer activity in vitro and in vivo mainly due to the inhibition of the canonical Wnt signaling pathway. Furthermore, pharmacological inhibition of Fzd7 by small interfering peptides or a small molecule inhibitor suppressed β-catenin-dependent tumor cell growth. Therefore, targeted inhibition of Fzd7 represents a rational and promising new approach for cancer therapy.
Copyright © 2011 Elsevier Inc. All rights reserved.
Activation of voltage gated K(+) channel Kv1.5 by β-catenin.
Source
Department of Physiology, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.
Abstract
Voltage-gated Kv1.5 channels are expressed in a wide variety of tissues including cardiac myocytes, smooth muscle and tumor cells. Kv1.5 channel activity is modified by N-cadherin, which in turn binds the multifunctional oncogenic protein β-catenin. The present experiments explored the effect of β-catenin on Kv1.5 channel activity. To this end, Kv1.5 was expressed in Xenopus oocytes with or without β-catenin and the voltage-gated Kv current determined by dual electrode voltage clamp. As a result, expression of β-catenin significantly increased the voltage-gated Kv current at positive potentials. The stimulating effect of β-catenin on Kv1.5 was not dependent on the stimulation of transcription since it was observed even in the presence of the transcription inhibitor actinomycin D. Specific antibody binding to surface Kv1.5 in Xenopus oocytes revealed that β-catenin enhances the membrane abundance of Kv1.5. Further experiments with brefeldin A showed that β-catenin fosters the insertion of Kv1.5 into rather than delaying the retrieval from the plasma membrane. According to electrophysiological recordings with mutant β-catenin, the effect on Kv1.5 requires the same protein domains that are required for association of β-catenin with cadherin. The experiments disclose a completely novel function of β-catenin, i.e. the regulation of Kv1.5 channel activity.
Copyright © 2011 Elsevier Inc. All rights reserved.
An Anti-Wnt5a Antibody Suppresses Metastasis of Gastric Cancer Cells in vivo by Inhibiting Receptor-Mediated Endocytosis.
Source
1Molecular Biology and Biochemistry, Osaka University.
Abstract
Wnt5a is a representative ligand that activates the β-catenin-independent pathway in Wnt signaling. It was reported that the expression of Wnt5a in human gastric cancer is associated with aggressiveness and poor prognosis, and that knockdown of Wnt5a reduces the ability of gastric cancer cells to metastasize in nude mice. Therefore, Wnt5a and its signaling pathway might be important targets for the therapy of gastric cancer. The aim of this study was to examine whether an anti-Wnt5a antibody affects metastasis of gastric cancer cells. One anti-Wnt5a polyclonal antibody (pAb5a-5) inhibited the migration and invasion activities in vitro of gastric cancer cells with a high expression level of Wnt5a. Previously it was demonstrated that Wnt5a induces the internalization of receptors, which is required for Wnt5a-dependent activation of Rac1. pAb5a-5 inhibited Wnt5a-dependent internalization of receptors, thereby suppressed Wnt5a-dependent activation of Rac1. Laminin γ2 is one of target genes of Wnt5a signaling and Rac1 was involved in its expression. pAb5a-5 also inhibited Wnt5a-dependent expression of laminin γ2. In an experimental liver metastasis assay, gastric cancer cells were introduced into the spleens of nude mice. Laminin γ2 was required for liver metastatic ability of gastric cancer cells in vivo. Furthermore, intraperitoneal injection of pAb5a-5 inhibited the metastatic ability of gastric cancer cells. These results suggest that an anti-Wnt5a antibody was capable of suppressing Wnt5a-dependent internalization of receptors, resulting in the prevention of metastasis of gastric cancer cells probably through the inhibition of the activation of Rac1 and the expression of laminin γ2.
β-catenin/TCF4 complex induces the epithelial-to-mesenchymal transition (EMT)-activator ZEB1 to regulate tumor invasiveness.
Source
Group of Transcriptional Regulation of Gene Expression, Department of Oncology and Hematology, Institut d'Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain.
Abstract
In most carcinomas, invasion of malignant cells into surrounding tissues involves their molecular reprogramming as part of an epithelial-to-mesenchymal transition (EMT). Mutation of the APC gene in most colorectal carcinomas (CRCs) contributes to the nuclear translocation of the oncoprotein β-catenin that upon binding to T-cell and lymphoid enhancer (TCF-LEF) factors triggers an EMT and a proinvasive gene expression profile. A key inducer of EMT is the ZEB1 transcription factor whose expression promotes tumorigenesis and metastasis in carcinomas. As inhibitor of the epithelial phenotype, ZEB1 is never present in the epithelium of normal colon or the tumor center of CRCs where β-catenin remains membranous. We show here that ZEB1 is expressed by epithelial cells in intestinal tumors from human patients (familial adenomatous polyposis) and mouse models (APC(Min/+)) with germline mutations of APC that result in nuclear accumulation of β-catenin. However, ZEB1 is not expressed in the epithelium of hereditary forms of CRCs that carry wild-type APC and where β-catenin is excluded from the nucleus (Lynch syndrome). We found that β-catenin/TCF4 binds directly to the ZEB1 promoter and activates its transcription. Knockdown of β-catenin and TCF4 in APC-mutated CRC cells inhibited endogenous ZEB1, whereas forced translocation of β-catenin to the nucleus in APC-wild-type CRC cells induced de novo expression of ZEB1. Upregulation of MT1-MMP and LAMC2 by β-catenin/TCF4 has been linked to invasiveness in CRCs, and we show here that both proteins are activated by ZEB1 coexpressing with it in primary colorectal tumors with mutated APC. These results set ZEB1 as an effector of β-catenin/TCF4 signaling in EMT and tumor progression.
- PMID:
- 22080605
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3228467
- [Available on 2012/5/29]
The blood-testis barrier and its implications for male contraception.
Source
The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10065, USA. y-cheng@popcbr.rockefeller.edu
Abstract
The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. It divides the seminiferous epithelium into the basal and the apical (adluminal) compartments. Meiosis I and II, spermiogenesis, and spermiation all take place in a specialized microenvironment behind the BTB in the apical compartment, but spermatogonial renewal and differentiation and cell cycle progression up to the preleptotene spermatocyte stage take place outside of the BTB in the basal compartment of the epithelium. However, the BTB is not a static ultrastructure. Instead, it undergoes extensive restructuring during the seminiferous epithelial cycle of spermatogenesis at stage VIII to allow the transit of preleptotene spermatocytes at the BTB. Yet the immunological barrier conferred by the BTB cannot be compromised, even transiently, during the epithelial cycle to avoid the production of antibodies against meiotic and postmeiotic germ cells. Studies have demonstrated that some unlikely partners, namely adhesion protein complexes (e.g., occludin-ZO-1, N-cadherin-β-catenin, claudin-5-ZO-1), steroids (e.g., testosterone, estradiol-17β), nonreceptor protein kinases (e.g., focal adhesion kinase, c-Src, c-Yes), polarity proteins (e.g., PAR6, Cdc42, 14-3-3), endocytic vesicle proteins (e.g., clathrin, caveolin, dynamin 2), and actin regulatory proteins (e.g., Eps8, Arp2/3 complex), are working together, apparently under the overall influence of cytokines (e.g., transforming growth factor-β3, tumor necrosis factor-α, interleukin-1α). In short, a "new" BTB is created behind spermatocytes in transit while the "old" BTB above transiting cells undergoes timely degeneration, so that the immunological barrier can be maintained while spermatocytes are traversing the BTB. We also discuss recent findings regarding the molecular mechanisms by which environmental toxicants (e.g., cadmium, bisphenol A) induce testicular injury via their initial actions at the BTB to elicit subsequent damage to germ-cell adhesion, thereby leading to germ-cell loss, reduced sperm count, and male infertility or subfertility. Moreover, we also critically evaluate findings in the field regarding studies on drug transporters in the testis and discuss how these influx and efflux pumps regulate the entry of potential nonhormonal male contraceptives to the apical compartment to exert their effects. Collectively, these findings illustrate multiple potential targets are present at the BTB for innovative contraceptive development and for better delivery of drugs to alleviate toxicant-induced reproductive dysfunction in men.
Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy.
Source
Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, People's Republic of China.
Abstract
AIMS/HYPOTHESIS:
The wingless-type MMTV integration site (WNT) pathway mediates multiple physiological and pathological processes, such as inflammation, angiogenesis and fibrosis. The aim of this study was to investigate whether canonical WNT signalling plays a role in the pathogenesis of diabetic nephropathy.
METHODS:
Expression of WNT ligands and frizzled receptors in the canonical WNT pathway in the kidney was compared at the mRNA level using real-time RT-PCR between Akita mice, streptozotocin-induced diabetic rats and db/db mice and their respective non-diabetic controls. Renal function was evaluated by measuring the urine albumin excretion. Human renal proximal tubular epithelial cells were treated with high-glucose medium and 4-hydroxynonenal (HNE). Levels of β-catenin, connective tissue growth factor and fibronectin were determined by western blot analysis.
RESULTS:
Some of the WNT ligands and frizzled receptors showed increased mRNA levels in the kidneys of Akita mice, streptozotocin-induced diabetic rats and db/db mice compared with their non-diabetic controls. Renal levels of β-catenin and WNT proteins were upregulated in these diabetic models. Lowering the blood glucose levels by insulin attenuated the activation of WNT signalling in the kidneys of Akita mice. In cultured human renal proximal tubular epithelial cells, both high glucose and HNE activated WNT signalling. Inhibition of WNT signalling with a monoclonalantibody blocking LDL-receptor-related protein 6 ameliorated renal inflammation and fibrosis and reduced proteinuria in Akita mice.
CONCLUSIONS/INTERPRETATION:
The WNT pathway is activated in the kidneys of models of both type 1 and 2 diabetes. Dysregulation of the WNT pathway in diabetes represents a new pathogenic mechanism of diabetic nephropathy and renders a new therapeutic target.
The C-terminus of Apc does not influence intestinal adenoma development or progression.
Source
Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. lewisa@well.ox.ac.uk
Abstract
Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression.
Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
HGF/c-Met related activation of β-catenin in hepatoblastoma.
Source
Children's Cancer Research Group, University of Otago, Christchurch, Christchurch, New Zealand. rachel.purcell@otago.ac.nz
Abstract
BACKGROUND:
Activation of beta-catenin is a hallmark of hepatoblastoma (HB) and appears to play a crucial role in its pathogenesis. While aberrant accumulation of the beta-catenin is a common event in HB, mutations or deletions in CTNNB1 (beta-catenin gene) do not always account for the high frequency of protein expression. In this study we have investigated alternative activation of beta-catenin by HGF/c-Met signaling in a large cohort of 98 HB patients enrolled in the SIOPEL-3 clinical trial.
METHODS:
We performed immunohistochemistry, using antibodies to total beta-catenin and tyrosine654-phosphorylated beta-catenin, which is a good surrogate marker of HGF/c-Met activation. CTNNB1 mutation analysis was also carried out on all samples. We also investigated beta-catenin pathway activation in two liver cancer cell lines, HuH-6 and HuH-7.
RESULTS:
Aberrant beta-catenin expression was seen in the cytoplasm and/or nucleus of 87% of tumour samples. Our results also revealed a large subset of HB, 83%, with cytoplasmic expression of tyrosine654-phosphorylated beta-catenin and 30% showing additional nuclear accumulation. Sequence analysis revealed mutations in 15% of our cohort. Statistical analysis showed an association between nuclear expression of c-Met-activated beta-catenin and wild type CTNNB1 (P-value = 0.015). Analysis of total beta-catenin and Y654-beta-catenin in response to HGF activation in the cell lines, mirrors that observed in our HB tumour cohort.
RESULTS:
We identified a significant subset of hepatoblastoma patients for whom targeting of the c-Met pathway may be a treatment option and also demonstrate distinct mechanisms of beta-catenin activation in HB.
N-cadherin adherens junctions mediate osteogenesis through PI3K signaling.
Source
Department of Biochemistry University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA; Clinical and Translational Research, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA.
Abstract
During endochondral ossification, the cartilage is surrounded by a layer of cells that constitute the perichondrium. Communication between osteoblasts in the perichondrium via N-cadherin adherens junctions is essential for endochondral bone growth. We observed that adherens junction molecule N-cadherin and its interacting partners p120, β-catenin and PTEN are expressed by cells present in the perichondrium. To study if N-cadherin mediated adherens junctions play a role in mediating signal transduction events during bone development, we utilized MC3T3E1 preosteoblasts plated at sub confluent (low) and confluent (high) densities to mimic adherens junction formation. When MC3T3E1 cells were plated at high density we observed an increase in phosphorylation of AKTSer473 and its downstream target GSK3Ser9, which coincided with an increase in Osterix, Osteomodulin and Osteoglycin gene expression. Using immunofluorescence, we identified N-cadherin, p120 and β-catenin localized at the membrane of MC3T3E1 cells. Treatment of confluent MC3T3E1cells with an N-cadherin junction inhibitor-EGTA and a PI3K inhibitor LY294002 resulted in reduction of phosphorylation levels of AKT and GSK3 and expression of Osterix, Osteomodulin and Osteoglycin. Furthermore, utilizing an N-cadherin blocking antibody resulted in reduced AKT signaling and Osterix gene expression, suggesting that osteoblast junction formation is linked to activation of PI3K signaling, which leads to osteoblast differentiation. To further explore the strength of this linkage, we utilized a conditional knockout approach using Dermo1cre to delete β-catenin and PTEN, two important proteins known to be essential for adherens junctions and PI3K signaling, respectively. In the absence of β-catenin, we observed a decrease in adherens junctions and AKT signaling in the perichondrium. PTEN deletion, on the other hand, increased the number of cells expressing N-cadherin in the perichondrium. These observations show that N-cadherin mediated junctions between osteoblasts are needed for osteoblast gene transcription.
Copyright © 2011 Elsevier Inc. All rights reserved.
[Present and future of the treatment of osteoporosis with monoclonalantibodies].
Source
Unidad de Reumatología, Hospital Universitari Son Espases, Palma de Mallorca, España. 26824jfa@comb.es
Abstract
An improved knowledge of bone physiopathology has led to new therapeutic targets in osteoporosis, blocking some of them with monoclonal antibodies. The RANK-RANKL-OPG system is considered the final effector pathway of bone resorption regulating factors. Denosumab is a humanized monoclonal antibody (IgG2) with a high affinity for RANKL. Upon binding RANKL it simulates the action of OPG, impedes the interaction between RANK-RANKL, blocks osteoclast activation and inhibits bone resorption. Denosumab has shown, in several phase III trials, to be a rapid, potent and safe antiresorptive agent. When administered subcutaneously every 6 months, it increases bone mineral density and is accompanied by a fast reduction in bone remodeling markers. According to the FREEDOM trial, in postmenopausal women with osteoporosis, treatment with 60 mg/sc of denosumab every 6 months for 3 years is accompanied by a reduction in the relative risk of fracture of 68% (incidence 2,3% in patients treated with denosumab and 7,2% in the placebo group), 20% in the case of non vertebral fractures (incidence 6,5% with denosumab vs. 8% with placebo) and 40% in hip fractures (incidence 0,7% with denosumab vs. 1,2% with placebo). It is a safe drug, with a frequency of adverse events similar to placebo, although an increased risk for skin reactions. Research is being done into blocking the Wnt/β-catenin pathway with monoclonal antibodies, specifically antisclerostin antibodies and anti-Dkk antibodies. This block of the Wnt/β-catenin pathway would have an anabolic action on bone remodeling.
Copyright © 2011 Elsevier España, S.L. All rights reserved.
- PMID:
- 21924213
- [PubMed - in process]
Β-catenin expression in in situ and infiltrative ductal carcinomas of the breast.
Source
Department of Pathology, Mersin University, Faculty of Medicine, Mersin, Turkey. karabacaktuba@hotmail.com
Abstract
OBJECTIVE:
Cascades that include β-catenin that has a function in adhesion and interaction with tumor suppressor genes such as APC have important roles in many neoplasms. The aim of the current study was to confirm the effect of the β-catenin pathway in breast tumor carcinogenesis and invasion.
MATERIAL AND METHOD:
Polyclonal rabbit β-catenin antibody was applied to 52 cases of infiltrative ductal carcinoma and 28 cases of ductal carcinoma in situ using the Avidin Biotin complex immune peroxidase method. The intensity and cellular localization of immunostaining were evaluated and compared.
RESULTS:
β-catenin immunoreactivity similar to that of normal epithelium was observed in 7 (8.75%) cases and weak or absent β-catenin expression was noted in 45 (56.25%) infiltrative ductal carcinoma cases. β-catenin expression was strong in 5 (6.25%) cases of ductal carcinoma in situ but weak or absent immunostaining was observed in 23 (28.75%) cases. Membranous β-catenin immunoreactivity was observed in 18 (22.5%) cases of infiltrative and 14 (%17.5) cases of ductal carcinoma in situ. Cytoplasmic immunostaining or complete absence of staining was noted in 34 (42.5%) cases of infiltrative and 14 (17.5%) cases of ductal carcinoma in situ.
CONCLUSION:
Similar quantitative and qualitative changes in β-catenin expression were detected in a considerable proportion of in situ and infiltrative ductal carcinomas in the current study. These findings suggest that β-catenin plays a role in the carcinogenesis of infiltrative ductal carcinoma but similar expression patterns of β-catenin in infiltrative and in situ ductal carcinomas indicates that changes in β-catenin expression occur early in carcinogenesis.
Wnt5a skews dendritic cell differentiation to an unconventional phenotype with tolerogenic features.
Source
Department of Cell Biology, Faculty of Medicine, Complutense University, 28040 Madrid, Spain.
Abstract
Dendritic cells (DCs) are critical regulators of immune responses that integrate signals from the innate and adaptive immune system and orchestrate T cell responses toward either immunity or tolerance. Growing evidence points to the Wnt signaling pathway as a pivotal piece in the immune balance and focuses on DCs as a direct target for their immunoregulatory role. Our results show that the increase in Wnt5a signaling during the differentiation of human DCs from monocytes alters their phenotype and compromises their subsequent capacity to mature in response to TLR-dependent stimuli. These Wnt5a-DCs produce scant amounts of IL-12p70 and TNF-α but increased levels of IL-10. Consequently, these Wnt5a-DCs have a reduced capacity to induce Th1 responses that promote IL-10 secretion by CD4 T cells. Changes in the transcriptional profile of Wnt5a-DCs correlate with their unconventional phenotype caused presumably by increased IL-6/IL-10 signaling during the process of DC differentiation. The effect of Wnt5a is not a consequence of β-catenin accumulation but is dependent on noncanonical Ca(2+)/calmodulin-dependent protein kinase II/NF-κB signaling. Our results therefore suggest that under high levels of Wnt5a, typical of the inflammatory state and sepsis, monocytes could differentiate into unconventional DCs with tolerogenic features.
Biphasic and dosage-dependent regulation of osteoclastogenesis by β-catenin.
Source
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA.
Abstract
Wnt/β-catenin signaling is a critical regulator of skeletal physiology. However, previous studies have mainly focused on its roles in osteoblasts, while its specific function in osteoclasts is unknown. This is a clinically important question because neutralizing antibodies against Wnt antagonists are promising new drugs for bone diseases. Here, we show that in osteoclastogenesis, β-catenin is induced during the macrophage colony-stimulating factor (M-CSF)-mediated quiescence-to-proliferation switch but suppressed during the RANKL-mediated proliferation-to-differentiation switch. Genetically, β-catenin deletion blocks osteoclast precursor proliferation, while β-catenin constitutive activation sustains proliferation but prevents osteoclast differentiation, both causing osteopetrosis. In contrast, β-catenin heterozygosity enhances osteoclast differentiation, causing osteoporosis. Biochemically, Wnt activation attenuates whereas Wnt inhibition stimulates osteoclastogenesis. Mechanistically, β-catenin activation increases GATA2/Evi1 expression but abolishes RANKL-induced c-Jun phosphorylation. Therefore, β-catenin exerts a pivotal biphasic and dosage-dependent regulation of osteoclastogenesis. Importantly, these findings suggest that Wnt activation is a more effective treatment for skeletal fragility than previously recognized that confers dual anabolic and anti-catabolic benefits.
- PMID:
- 21876000
- [PubMed - indexed for MEDLINE]
- PMCID: PMC3232928
- [Available on 2012/6/1]
Protein kinase C ζ is a positive modulator of canonical Wnt signaling pathway in tumoral colon cell lines.
Source
Department of Biochemistry, Faculty of Medicine, Universidad Nacional Autónoma de México, Av. Universidad 3000, Mexico, 04510, Mexico.
Abstract
The colonic epithelium is a continuously renewing tissue with a dynamic turnover of cells. Wnt pathway is a key regulator of its homeostasis and is altered in a large proportion of colon cancers. Protein kinase C (PKC) family of serine/threonine kinases are also involved in colon tumor formation and progression; however, the molecular role played by them in the Wnt pathway, is poorly understood. Reciprocal coimmunoprecipitation and immunofluorescence studies revealed that PKCζ interacts with β-catenin mainly in tumoral colon cells, which overexpressed this PKC isoform. The pharmacological inhibition, the small interference RNA-mediated knockdown of PKCζ or the expression of a dominant-negative form of it in tumoral SW480 cells, blocked in a dose-dependent manner the constitutive transcriptional activity mediated by β-catenin, the cell proliferation and the expression of the Wnt target gene c-myc. Remarkably, the PKCζ stably depleted cells exhibited diminished tumorigenic activity in grafted mice. We show that PKCζ functions in a mechanism that does not involve β-catenin degradation since the effects produced by PKCζ inhibition were also obtained in the presence of proteosome inhibitor and in cells expressing a β-catenin degradation-resistant mutant. It was found that PKCζ activity regulates the nuclear localization of β-catenin since PKCζ inhibition induces a rapid export of β-catenin from the nucleus to the cytoplasm in a Leptomycin B sensitive manner. Taken together, our results indicate that the atypical PKCζ plays an important role in the positive regulation of canonical Wnt pathway.
Platelet-derived growth factor regulates breast cancer progression via β-catenin expression.
Source
Department of Molecular Pathology, Osaka University Graduate School of Medicine and Health Science, Osaka, Japan.
Abstract
OBJECTIVE:
The knowledge on the association between platelet-derived growth factor (PDGF) signaling and epithelial cancers is scarce, although overexpression of PDGF and PDGF receptors has been reported in some human mesenchymal tumors. Thus, we studied the effect of PDGF on breast cancer cells in vitro and the distribution of PDGF in breast cancer tissues.
METHODS:
The effect of PDGF-BB on breast cancer cells was assessed by Western blotting, immunofluorescence, WST and 5-bromo-2-deoxyuridine incorporation experiments. PDGF-B and β-catenin expression was investigated in breast cancer tissues by immunohistochemistry.
RESULTS:
PDGF-BB induces β-catenin expression in breast cancer cells, and immunohistochemically the distribution of PDGF-B was similar to β-catenin in breast cancer cells. PDGF-B-positive cancer cells were more frequent in cases of ductal carcinoma in situ (87.5%) than invasive carcinoma (61.2%). In addition, PDGF-B staining was stronger in intraductal than invasive cancer cells. PDGF-BB tended to induce nuclear translocation of β-catenin, cell proliferation and DNA incorporation in MDA-MB231 cells, while these results were not found in MCF-7 cells.
CONCLUSION:
Our results suggest that PDGF-BB regulates protein expression of β-catenin and is associated with cancer cell behavior.
Copyright © 2011 S. Karger AG, Basel.
Clinical and histopathological features and immunoreactivity of human choroidal and ciliary melanomas as prognostic factors for metastasis and death.
Source
Department of Ophthalmology, University of Cincinnati College of Medicine, Stetson Building - Suite 5300, 260 Stetson Street, Cincinnati, OH 45267-0527, USA.
Abstract
PURPOSES:
To determine the relationship between immunohistochemical reactivity to osteopontin, vimentin, keratin 8/18, LZTS1, and beta-catenin and clinical and histopathological prognostic factors for metastasis and death in archival specimens of primary uveal melanomas, and the prognostic value of the evaluated study variables for death from metastasis.
METHODS:
Retrospective analysis of clinical records and formalin-fixed, paraffin-embedded slides of primary uveal melanomas treated by enucleation during May 1 1999, through June 30 2009. Immunofluorescent staining of each tumor was assessed on newly prepared histologic slides after the application of antibodies directed against five biomarkers associated with unfavorable prognosis in uveal melanoma.
RESULTS:
After exclusions, our study group consisted of 82 cases. Immunofluorescence was observed in 40.2% of specimens evaluated for keratin, 50.0% evaluated for osteopontin, 26.8% evaluated for β-catenin, 65.9% evaluated for vimentin, and 70.7% evaluated for LZTS1. Through available follow-up, 27 patients (32.9%) were dead of confirmed or suspected metastatic uveal melanoma. None of the patients whose tumor exhibited strong immunoreactivity to β-catenindied of metastasis. In contrast, patients whose tumor exhibited immunoreactivity of any intensity to LZTS1 were more likely to develop metastasis. In multivariate Cox proportional hazards modeling, a composite variable that took into account the immunostaining for both β-catenin and LZTS1 had a statistically significant relationship with patient's survival time.
CONCLUSIONS:
Our study suggests that conventional clinical and histopathological prognostic factors, and immunoexpression of β-catenin and LZTS1 combined may allow better prognostication of metastasis than clinical and histomorphological factors alone.
Tumor endothelial marker 8 amplifies canonical Wnt signaling in blood vessels.
Source
Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
Abstract
Tumor Endothelial Marker 8/Anthrax Toxin Receptor 1 (TEM8/ANTXR1) expression is induced in the vascular compartment of multiple tumors and therefore, is a candidate molecule to target tumor therapies. This cell surface molecule mediates anthrax toxin internalization, however, its physiological function in blood vessels remains largely unknown. We identified the chicken chorioallantoic membrane (CAM) as a model system to study the endogenous function of TEM8 in blood vessels as we found that TEM8 expression was induced transiently between day 10 and 12 of embryonic development, when the vascular tree is undergoing final development and growth. We used the cell-binding component of anthrax toxin, Protective Antigen (PA), to engage endogenous TEM8 receptors and evaluate the effects of PA-TEM8 complexes on vascular development. PA applied at the time of highest TEM8 expression reduced vascular density and disrupted hierarchical branching as revealed by quantitative morphometric analysis of the vascular tree after 48h. PA-dependent reduced branching phenotype was partially mimicked by Wnt3a application and ameliorated by the Wnt antagonist, Dikkopf-1. These results implicate TEM8 expression in endothelial cells in regulating the canonical Wnt signaling pathway at this day of CAM development. Consistent with this model, PA increased beta catenin levels acutely in CAM blood vessels in vivo and in TEM8 transfected primary human endothelial cells in vitro. TEM8 expression in Hek293 cells, which neither express endogenous PA-binding receptors nor Wnt ligands, stabilized beta catenin levels and amplified beta catenin-dependent transcriptional activity induced by Wnt3a. This agonistic function is supported by findings in the CAM, where the increase in TEM8 expression from day 10 to day 12 and PA application correlated with Axin 2 induction, a universal reporter gene for canonical Wnt signaling. We postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical Wnt signaling to regulate vessel patterning and density.
Silymarin targets β-catenin signaling in blocking migration/invasion of human melanoma cells.
Source
Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Abstract
Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i) the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser(45), Ser(33/37) and Thr(41), and (ii) the binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241) and β-catenin-inactivated (Mel 1011) melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway.
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