The Notch ligand delta-like 3 promotes tumor growth and inhibits Notch signaling in lung cancer cells in mice
San‐Ming DengXianchun YanLiang LiangLi WangYuan LiuJuanli DuanZiyan YangTianfang ChangBai RuanQijun ZhengHua Han
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Lewis lung carcinoma
Notch 1
NFAT
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Cyclin-dependent kinase 8
RUNX2
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This paper's purpose is to analyze temporally and spatially differential roles of Notch-1, -2 and -3 in the neurogenesis of mouse embryos. In Northern blot hybridization, Notch-1 and -3 were expressed in the nervous tissue and increased in the level from 12 to 14 day post coitum (dpc). Notch-2 was more weakly expressed than Notch-1 and -3 on 12 and 14 dpc. In in situ hybridization, Notch-1 and -3 expressions appeared early on 6 dpc. On 12, 14, and 15 dpc, high level of Notch-1 expression was observed in the ventricular zone, and Notch-3 was diffusely expressed in the ventricular, intermediate and marginal zones of the brain, and Notch-2 expression was in the lower level. These findings suggest that Notch-1, -2 and -3 play individu-ally complementary and combinatorial roles in the neurogenesis of mouse embryos.
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Notch signaling provides an important cue in mammalian developmental. It is a key player in T cell development and function. Notch ligands, such as Delta-like ligands (DLL) 1, 3, 4 and Jagged 1, 2, can impact Notch signaling positively or negatively, by trans-activation or cis-inhibition. Trans and cis interactions are receptor-ligand interaction on two adjacent cells and interaction on the same cell respectively. The former sends an activation signal and the latter, a signal for inhibition of Notch. However, earlier reports suggested that Notch is activated in the absence of Notch ligand-expressing APCs in a purified population of CD4 T cells. Thus the role of ligands in Notch activation, in a purified population of CD4 T cells, remains obscure. In this study we demonstrate that mature CD4 T cells are capable of expressing Notch ligands on their surface very early upon activation with soluble antibodies against CD3 and CD28. Moreover, signaling solely through CD28 induces Notch ligand expression and CD3 signaling inhibits ligand expression, in contrast to Notch which is induced by CD3 signaling. Additionally, by using decoys mimicking the Notch extracellular domain, we demonstrated that DLL1, DLL4, and JAG1, expressed on the T cells, can cis-interact with the Notch receptor and inhibit activation of Notch. Thus, our data indicate a novel mechanism of the regulation of Notch ligand expression on CD4 T cells and its impact on activated Notch.
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Abstract Notch-1 is a transmembrane receptor whose activation upon ligand binding or calcium depletion leads to translocation of the Notch-1 intracellular domain (NICD) to the nucleus where it activates transcription of Notch target genes. Targeting Notch signaling, such as with inhibitors of γ-secretase that is required for Notch-1 activation, is currently in early clinical development. It is critical to characterize the expression of Notch-1, an important member of Notch family of receptors in human cancers with a validated assay suitable to monitor pharmacodynamic responses to Notch inhibitors and to carry out clinical validation investigation of Notch-1 as predictive/prognostic biomarker. This could improve our understanding of pathobiology of Notch signaling and in turn increase the odds of success of targeting Notch signaling. The aims of this study were to develop an immunohistochemistry assay for Notch-1/NICD (activated Notch-1), and to evaluate expression levels and patterns of Notch-1/NICD in paraffin-embedded tumor samples. Human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, and colorectal cancer HCT-116 cells were treated with and without a calcium chelator EDTA for 10 min and embedded in paraffin after formalin fixation. Expression levels and patterns of Notch-1 were examined by Western blot, and immunocytochemistry as well as immunohistochemistry and were quantitated using an Automated Cellular Imaging System. There was an increase in Notch-1/NICD expression in cells treated with EDTA versus without EDTA in MCF-7 (staining index: 28 vs. 10) and MDA-MB-231 (81 vs. 62) cells by immunocytochemistry and confirmed by Western blot. Interestingly, membranous/cytoplasmic Notch-1 was in part translocated into the nucleus in MCF-7 and MDA-MB-468 cells following EDTA treatment, suggesting Notch-1 activation and specific detection of Notch-1 in distinct cellular compartments. In addition, a predominant cytoplasmic/membranous expression of Notch-1 was observed in MDA-MB-231 and HCT-116 cells with and without EDTA treatment. Low to intermediate levels of Notch-1 with a predominantly cytoplasmic localization, relative to moderate to high levels in cell lines, were detected in 13% (6/45), 4% (2/48), 16% (7/45) of tumor cells in carcinomas of the breast, lung, and ovary. Nuclear/cytoplasmic staining of Notch-1 was observed in one case of lung cancer. In addition, Notch-1 expression was found in a significant fraction of endothelial cells of human solid tumors. We have established a fit-for-use immunohistochemistry assay with an antibody that specifically detects Notch-1 in formalin-fixed and paraffin-embedded human tumors. Its application could facilitate the evaluation of pharmacodynamic responses to various Notch targeting agents undergoing clinical development and subsequent clinical validation studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 746. doi:1538-7445.AM2012-746
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Το αντικείμενο της παρούσας διατριβής ήταν η μελέτη της αναστολής των σηματοδοτικών μονοπατιών του υποδοχέα του επιδερμικού αυξητικού παράγοντα (EGFR) και του Notch καθώς και της αλληλεπίδρασής τους στον μη-μικροκυτταρικό καρκίνο του πνεύμονα (ΜΜΚΠ).Στα πλαίσια της μελέτης της διπλής στόχευσης των δύο αυτών μονοπατιών, πραγματοποιήθηκαν in vitro πειράματα σε δύο κυτταρικές σειρές ΜΜΚΠ, Η23 και Η661, που διαφέρουν μεταξύ τους ως προς την μεταστατική ικανότητα καθώς και ως προς τα N1ICD (ενδοκυττάριο τμήμα του Notch-1 υποδοχέα), EGFR και HER2 πρωτεϊνικά επίπεδα. Χρησιμοποιήθηκαν ο μη αντιστρεπτός αναστολέας των υποδοχέων της ErbB οικογένειας, αφατινίμπη και ο αναστολέας γ-σεκρετάσης, DAPT. Τα αποτελέσματα της διατριβής παρέχουν ενδείξεις ότι η συλλογική κυτταρική μετανάστευση και η έκφραση πρωτεολυτικών μορίων αναστέλλονται καλύτερα ενώ οι δια-κυτταρικές συνδέσεις ενισχύονται σε μεγαλύτερο βαθμό, στα πιο μεταστατικά Η661 κύτταρα, από την ταυτόχρονη αναστολή των EGFR και Notch μονοπατιών σε σύγκριση με τις αντίστοιχες μονοθεραπείες, φανερώνοντας το πιθανό όφελος μιας συνδυαστικής θεραπείας στην αντιμετώπιση του ΜΜΚΠ. Στα πλαίσια της μελέτης του ιδιαίτερου ρόλου του Notch-1 στον ΜΜΚΠ και της συσχέτισής του με την πρωτεΐνη του EGFR, εφαρμόστηκε γονιδιακή αποσιώπηση του Notch-1 με τη χρήση της τεχνολογίας της RNA παρεμβολής (small interfering RNA-siRNA) στα Η23 και Η661 κύτταρα. Τα αποτελέσματα της διατριβής φανερώνουν ότι το Notch-1 ελέγχει την μεταναστευτική και διηθητική ικανότητα των κυττάρων ΜΜΚΠ. Επιπλέον, προτείνεται ότι το Notch-1 πιθανά λειτουργεί ως ένας κρίσιμος τελεστής στη σταθεροποίηση της πρωτεΐνης του EGFR.
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Notch signaling is indicated as novel therapeutic targets to prevent recurrence of breast cancer. LncRNAs were identified as downstream target of Notch pathway. However, the exact mechanisms involved in Notch signaling, lncRNAs and breast cancer remain to be explained.This original research aimed to determine the prognostic implications of Notch-1 for breast cancer, and explain mechanisms involved in regulation of lnRNA GAS5 by Notch-1, and identify the function of this mechanism on breast cancer.Thirty breast cancer patients were included from The First Affiliated Hospital of Anhui Medical University (China) since January 2006 in this study. The mRNA level by RT-PCR and protein level of Notch-1 by western blot in tumor tissues and adjacent normal tissues were evaluated and 5-year survival analysis was applied to examine the significance of Notch-1. The levels of ten reported lncRNAs were determined by RT-PCR, and subsequently linear analysis was applied to analyze the relationship between these four unique lncRNAs and protein level of Notch-1, which identified the most relevant lncRNA GAS5 with Notch-1 in breast cancer. Subsequently, Notch1-siRNA was applied to influence the expression of Notch-1 in T47D, then the level of RSA5 was measured by RT-PCR, and CCK-8 assay was applied to measure the proliferation of T47D cells.High level of Notch-1 provided a poor prognosis in breast cancer. Interference of Notch-1 significantly suppressed proliferation of T47D cell (P < 0.05), and significantly increased the level of GAS5.Notch-1 promotes breast cancer cells proliferation by regulating LncRNA GAS5.
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ABSTRACT: Members of the Notch gene family have been shown to play an important role in the control of cell fate in many developmental systems. We hypothesized that the fate of the male germ line stem cells may also be mediated through the Notch signaling pathway. We therefore sought to determine whether the components of the Notch pathway are expressed in the mouse testis. Western blot analysis revealed the expression of three Notch receptors (Notch 1, Notch 2, and Notch 3), Notch ligands (Jagged 1, Jagged 2, and Delta 1), and presenilin 1 (PS1) in neonatal mouse testis. We then examined their cellular localization by immunohistochemical analysis of cocultures of spermatogonia and Sertoli cells. The 3 Notch receptors were found to be expressed in spermatogonia. Sertoli cells expressed only Notch 2 receptor. Among the Notch ligands, Delta 1 and Jagged 1 were localized exclusively in spermatogonia and Sertoli cells, respectively. PS1 was apparent in both spermatogonia and Sertoli cells. The presence of Notch receptors and Notch ligands in spermatogonia and Sertoli cells indicates that these cells are capable of responding to and eliciting Notch signaling during the process of spermatogenesis.
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The aim of this study was to investigate the influence of micro-ribonucleic acid-34a (miR-34a) on preeclampsia through the Notch signaling pathway.The expressions of miR-34a, Notch-1, Notch-2, and Notch-3 in the placenta of 39 preeclampsia patients and 42 normal patients were detected by immunohistochemistry and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The correlations between miR-34a expression with the expressions of Notch-1, Notch-2 and Notch-3 were analyzed, respectively. Besides, placental trophoblasts were isolated from preeclampsia patients and cultured in vitro. The expressions of miR-34a, Notch-1, Notch-2 and Notch-3 in placental trophoblasts were analyzed. Furthermore, the influences of miR-34a on the protein expressions of Notch-1, Notch-2, Notch-3, and hairy and enhancer of split-1 (Hes-1) in the Notch signaling pathway were analyzed by Luciferase reporter gene assay and Western blotting. The role of Notch in trophoblast invasion was investigated through the Notch inhibitors. In addition, its influence on the expression of urokinase-type plasminogen activator (uPA) was studied by miR-34a overexpression.The expressions of miR-34a and Notch-1 were correlated with preeclampsia in the placentas of preeclampsia patients and normal patients to a certain degree. The expression of miR-34a in preeclamptic placenta was significantly higher than that of the normal placenta (p<0.05). However, Notch-1 expression was markedly lower in preeclamptic placenta (p<0.05). No significant differences were found in the expressions of Notch-2 and Notch-3 between the two types of placentas (p>0.05). MiR-34a had a remarkable negative correlation with Notch-1 expression in the Notch family (p<0.001, r=-0.5775). RT-PCR results revealed that the mRNA expression of miR-34a in placental trophoblasts of patients with preeclampsia was notably higher than that of normal people (p<0.01). However, Western blotting demonstrated that the protein expressions of Notch-1, Notch-2 and Notch-3 exhibited the opposite results. Additionally, the protein expression of Notch-1, Notch-2, Notch-3 and Hes-1 in trophoblasts transfected with pre-miR-34a was significantly decreased. The treatment with Notch inhibitors markedly reduced the trophoblast invasion. Furthermore, miR-34a overexpression or intracellular domain of Notch (ICN) overexpression regulated uPA expression.MiR-34a regulates uPA system through the Notch signal transduction, thereby regulating the invasion of placental trophoblasts in patients with preeclampsia.
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