hes1-protein--human and Barrett-Esophagus

Cdx2 expression in esophageal stem cells induced by reflux bile acids may be an important factor for development of Barrett's esophagus, whereas Notch signaling is a molecular signaling pathway that plays an important role in the determination of cell differentiation. ATOH1 (a factor associated with Notch signaling) plays an important role in differentiation of stem cells into goblet cells. However, the relationship between the Notch signaling pathway and Cdx2 expression in the development of Barrett's esophagus has not been explored. The aim of this study was to investigate the interrelationship between Notch signaling and Cdx2 in esophageal epithelial cells. The expressions of Cdx2, MUC2, and intracellular signaling molecules related to Notch signaling (Notch1, Hes1, and ATOH1) were examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining with biopsy specimens obtained from esophageal intestinal metaplasia (IM) with goblet cells (IM⁺) and columnar epithelium not accompanied by goblet cells (IM⁻). For in vitro experiments, we employed human esophageal epithelial cell lines (OE33, OE19, and Het-1A). After forced Cdx2 expression by applying a Cdx2 expression vector to the cells, changes in the expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 were analyzed by real-time PCR and western blot analysis. Changes in expressions of Notch1, Hes1, ATOH1, Cdx2, and MUC2 in cells were analyzed following stimulation with bile acids in the presence or absence of Cdx2 blocking with Cdx2-siRNA. Suppressed Hes1 and enhanced ATOH1 and MUC2 expressions were identified in IM⁺ specimens. Forced expression of Cdx2 in cells suppressed Hes1, and enhanced ATOH1 and MUC2 expressions, whereas bile acids suppressed Hes1, and enhanced ATOH1, Cdx2, and MUC2 expressions. On the other hand, these effects were blocked by siRNA-based Cdx2 downregulation. Enhanced expression of Cdx2 by stimulation with bile acids may induce intestinal differentiation of esophageal columnar cells by interaction with the Notch signaling pathway.

Topics: Adenocarcinoma; Aged; Barrett Esophagus; Basic Helix-Loop-Helix Transcription Factors; CDX2 Transcription Factor; Cell Line, Tumor; Cholic Acid; Deoxycholic Acid; Epithelial Cells; Esophageal Neoplasms; Esophagus; Female; Gene Expression; Gene Silencing; Goblet Cells; Homeodomain Proteins; Humans; Male; Metaplasia; Mucin-2; Real-Time Polymerase Chain Reaction; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Transcription Factor HES-1; Transfection

Esophageal adenocarcinoma is often considered to arise from a clonal stem-like population of cells, which is potentially responsible for its poor prognosis. Transforming growth factor β (TGF-β) and Notch signaling pathways play important roles in regulating self-renewal of stem cells and cell-fate determination. Both pathways are frequently implicated in gastrointestinal carcinogenesis. However, their contributions to esophageal adenocarcinoma remain unclear.. We evaluated TGF-β and Notch signaling components in normal esophagus, Barrett's esophagus, and adenocarcinoma tissues and cell lines via immunohistochemical analysis and immunoblotting; Hes-1 transcription was assayed using a Hes-1 luciferase reporter.. We observed loss of Smad4 (P<.05) and β2 spectrin (β2SP) (P<.01) in 5/10 Barrett's esophagus and 17/22 adenocarcinoma tissue sections. Concomitantly, dramatically raised levels of Notch signaling components Hes1 and Jagged1 occurred in adenocarcinoma tissues and cell lines compared with normal tissues. In normal esophagus, Oct3/4-positive cells are located in the basal layer (2-3 per cluster), representing a pool of progenitor cells. We observed an expansion of this pool of Oct3/4 positive cells in esophageal adenocarcinoma (15 per cluster). Furthermore, a panel of SOXs proteins documented for stem cell markers exhibit increased expression in tumor cells, indicating expansion of putative cancer stem cells. Finally, we observed growth inhibition in BE3 cells with a γ-secretase inhibitor, but not in SKGT-4 cells. Unlike SKGT-4 cells, BE3 cells have activated Notch signaling with disruption of TGF-β signaling.. Our findings demonstrated a potential therapeutic value for targeted therapy in esophageal adenocarcinoma in the setting of loss of β2SP/TGF-β with concomitant constitutively active Notch signaling.

Topics: Adenocarcinoma; Barrett Esophagus; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 3 Subunit; Cyclin-Dependent Kinase 4; Esophageal Neoplasms; Esophagus; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Octamer Transcription Factor-3; Receptors, Notch; Serrate-Jagged Proteins; Signal Transduction; Transcription Factor HES-1; Transforming Growth Factor beta

Barrett's esophagus (BE) is the predominant risk factor for the development of esophageal adenocarcinoma. BE is characterized by intestinal metaplasia with goblet cells. Reflux of bile acids is known to induce intestinal metaplasia, but the mechanisms are unclear. Inhibition of Notch signaling accompanied by increased Hath1 and induction of caudal homeobox 2 (CDX2) may be involved in development of intestinal goblet cells.. Esophageal adenocarcinoma cell lines OE19 and OE33 were exposed for up to 8 hours to DCA (100-300 microM), and for up to 24 hours with and without the gamma-secretase inhibitor, DAPT (20 microM). Notch signaling components and CDX2 levels were measured by real-time PCR (for mRNA) and by Western blot analysis (for proteins).. DCA induced a time and concentration dependent decrease in Notch pathway components mRNAs in OE33 and in the proteins in both cell lines. CDX2 mRNA and Hath1 protein were increased in OE19 by 3-fold. Inhibition of Notch pathway by DAPT decreased downstream Notch signaling mRNAs and proteins in both cell lines and increased Hath1 and CDX2 proteins only in OE19.. Bile acid inhibition of Notch signaling in esophageal cells is correlated with an increase in Hath1 and CDX2 and may be one of the key processes contributing to the formation of BE.

Topics: Barrett Esophagus; Basic Helix-Loop-Helix Transcription Factors; Bile Acids and Salts; CDX2 Transcription Factor; Cell Line, Tumor; Deoxycholic Acid; Dipeptides; Esophagus; Gene Expression Regulation; Homeodomain Proteins; Humans; Receptors, Notch; RNA, Messenger; Signal Transduction; Transcription Factor HES-1