E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120(ctn), also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

The orphan nuclear receptor LRH-1 promotes breast cancer motility and invasion

The orphan nuclear receptor liver receptor homologue-1 (LRH-1) has roles in the development, cholesterol and bile acid homeostasis, and steroidogenesis. It also enhances proliferation and cell cycle progression of cancer cells. In breast cancer, LRH-1 expression is associated with invasive breast cancer; positively correlates with ERα status and aromatase activity; and promotes oestrogen-dependent cell proliferation. However, the mechanism of action of LRH-1 in breast cancer epithelial cells is still not clear. By silencing or over-expressing LRH-1 in ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells, we have demonstrated that LRH-1 promotes motility and cell invasiveness. Similar effects were observed in the non-tumourigenic mammary epithelial cell line, MCF-10A. Remodelling of the actin cytoskeleton and E-cadherin cleavage was observed with LRH-1 over-expression, contributing to increased migratory and invasive properties. Additionally, in LRH-1 over-expressing cells, the truncation of the 120 kDa E-cadherin to the inactive 97 kDa form was observed. These post-translational modifications in E-cadherin may be associated with LRH-1-dependent changes to matrix metalloproteinase 9 expression. These findings suggest a new role of LRH-1 in promoting migration and invasion in breast cancer, independent of oestrogen sensitivity. Therefore, LRH-1 may represent a new target for breast cancer therapeutics.

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α-Mediated Nuclear Factor-κB Activation

Objective: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process.

Approach and Results: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α–mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation.

Conclusions: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α–mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Morphological and functional characterization of placenta during gestation in bovine clones derived by somatic nuclear transfer

La technique de clonage par transfert nucléaire de cellules somatiques (SCNT) présente une page importante dans les annales scientifiques, mais son application pratique demeure incertaine dû à son faible taux de succès. Les anomalies placentaires et de développement fœtal se traduisent par des pertes importantes de gestation et des mortalités néonatales. Dans un premier temps, la présente étude a caractérisé les changements morphologiques des membranes fœtales durant la gestation clonée en les comparant à des gestations contrôles obtenues à partir de l’insémination artificielle. Les différentes anomalies morphologiques des placentomes telles que l’œdème chorioallantoique, la présence de zones hyperéchoiques et irrégulières dans la membrane amniotique et la présence de cellules inflammatoires dégénérées compromettent le développement fœtal normal de la gestation clonée. L’examen ultrasonographique représente une technique diagnostique importante pour faire le suivi d’une gestation et de caractériser les changements placentaires dans le cadre d’évaluation globale du bien-être fœtal. Le profil hormonal de trois stéroïdes (progestérone (P4), estrone sulfate (E1S), et œstradiol (E2)) et de la protéine B spécifique de gestation (PSPB) dans le sérum des vaches porteuses de clones SCNT a été déterminé et associé aux anomalies de gestations clonées. Une diminution de la P4 sérique au jour 80, une élévation du niveau de la concentration de la PSPB au jour 150, et une augmentation de la concentration d’E2 sérique durant le deuxième et troisième tiers de la gestation clonée coïncident avec les anomalies de gestation déjà reportées. Ces changements du profil hormonal associés aux anomalies phénotypiques du placenta compromettent le déroulement normal de la gestation clonée et gênent le développement et le bien-être fœtal. Sur la base des observations faites sur le placenta de gestation clonée, le mécanisme moléculaire pouvant expliquer la disparition de l’épithélium du placenta (l’interface entre le tissue maternel et le placenta) a été étudié. L’étude a identifié des changements dans l’expression de deux protéines d’adhérence (E-cadhérin et β-catenin) de cellules épithéliales pouvant être associées aux anomalies du placenta chez les gestations clonées. Le tissu de cotylédons provenant de gestations clonées et contrôles a été analysé par Western blot, RT-PCR quantitatif, et par immunohistochimie. Les résultats présentaient une diminution significative (p<0.05) de l’expression des dites protéines dans les cellules trophoblastiques chez les gestations clonées. Le RT-PCR quantitatif démontrait que les gènes CCND1, CLDN1 et MSX1 ciblés par la voie de signalisation de la Wnt/β-catenin étaient significativement sous exprimés. La diminution de l’expression des protéines E-cadherin et β-catenin avec une réduction de l’activation de la protéine β-catenin durant le période d’attachement de l’embryon peut potentiellement expliquer l’absence totale ou partielle de l’attachement des membranes fœtales au tissu maternel et éventuellement, l’insuffisance placentaire caractéristique des gestations clonées chez la vache. La caractérisation morphologique et fonctionnelle du placenta durant les gestations clonées à haut risque est essentielle pour évaluer le statut de la gestation. Les résultats de la présente étude permettront de prédire le développement et le bien-être fœtal de façon critique à travers un protocole standardisé et permettre des interventions médicales pour améliorer le taux de succès des gestations clonées chez les bovins.

Aberrant E-cadherin, beta-catenin, and glial fibrillary acidic protein (GFAP) expression in canine choroid plexus tumors

Expression of E-cadherin and beta-catenin has been widely studied in various human and canine epithelial tumors and has been correlated with dedifferentiation, invasiveness, and metastasis. Choroid plexus tumors (CPTs) are of epithelial origin, and the most important prognostic factor in human medicine is the tumor grade. Limited information is available regarding E-cadherin and beta-catenin expression in human CPTs, and no information is found in the veterinary literature. In the current study, 42 canine CPTs (19 choroid plexus papillomas and 23 choroid plexus carcinomas) were retrospectively reviewed, and the intensity and cellular staining pattern of E-cadherin and beta-catenin were correlated with histological features, paying special attention to grade, invasion, and metastasis. In addition, cytokeratin and glial fibrillary acidic protein (GFAP) antibodies were evaluated as markers for canine CPTs. It was found that loss of E-cadherin and beta-catenin expression was uncommon in canine CPTs. Rather, membranous expression of both molecules was increased in CPTs compared to normal choroid plexus (NCP), regardless of tumor grade. Additionally, aberrant cytoplasmic or nuclear expression of both E-cadherin and beta-catenin was often observed in CPTs. GFAP was frequently expressed in CPTs in contrast to NCP. None of these parameters were correlated with malignancy, and therefore, do not appear to be useful for prognostic information. Nevertheless, a panel of antibodies including E-cadherin and GFAP might be useful to support the diagnosis of CPTs and help to differentiate them from other tumors, such as ependymomas and metastatic epithelial tumors.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin.

Plakoglobin interacts with both classical and desmosomal cadherins. It is closely related to Drosophila aramadillo (arm) gene product; arm acts in the wingless (wg)-signaling pathway to establish segment polarity. In Xenopus, homologs of wg--i.e., wnts, can produce anterior axis duplications by inducing dorsal mesoderm. Studies in Drosophila suggest that wnt acts by increasing the level of cytoplasmic armadillo protein (arm). To test whether simply increasing the level of plakoglobin mimics the effects of exogenous wnts in Xenopus, we injected fertilized eggs with RNA encoding an epitope-tagged form of plakoglobin; this induced both early radial gastrulation and anterior axis duplication. Exogenous plakoglobin accumulates in the nuclei of embryonic cells. Plakoglobin binds to the tail domain of the desmosomal cadherin desmoglein 1. When RNA encoding the tail domain of desmoglein was coinjected with plakoglobin RNA, both the dorsalizing effect and nuclear accumulation of plakoglobin were suppressed. Mutational analysis indicates that the central arm repeat region of plakoglobin is sufficient to induce axis duplication and that this polypeptide accumulates in the nuclei of embryonic cells. These data show that increased plakoglobin levels can, by themselves, generate the intracellular signals involved in the specification of dorsal mesoderm.

Immunohistochemical expression of E-cadherin and beta-catenin in ameloblastomas and tooth germs

OBJECTIVE: The aim was to analyze the expression of E-cadherin and beta-catenin in ameloblastomas and tooth germs to determine their roles in cell differentiation processes and invasiveness compared with odontogenesis. STUDY DESIGN: Twenty-one ameloblastoma cases (16 solid and 5 unicystic tumors) and 5 tooth germs were submitted to the immunohistochemical detection of E-cadherin and beta-catenin. Immunoreactivity was evaluated using descriptive and semiquantitative analysis, investigating the location and intensity of staining. The Fisher exact test was performed, and P values of <.05 were considered to indicate statistical significance. RESULTS: There was no statistically significant difference in the expression of E-cadherin and beta-catenin between solid and unicystic ameloblastomas (P = .59; P = .63; respectively). The same was found when comparing solid and unicystic ameloblastomas with the tooth germs for both E-cadherin (P = .53; P = .44; respectively) and beta-catenin (P = .12; P = .16; respectively). Nuclear staining of beta-catenin was observed in only 4 cases (3 solid and 1 unicystic tumor). CONCLUSION: The results showed no differences in the expression of E-cadherin or beta-catenin between tooth germs and solid and unicystic ameloblastomas. The expression of these molecules seems mainly to be related to the process of cell differentiation.