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CONTROL ID: 1287104 CURRENT CATEGORY: Gastrointestinal Oncology PRESENTATION TYPE: AGA Institute Oral or Poster PRESENTER: Mitsuteru Natsuizaka PRESENTER (E-MAIL ONLY): [email protected] Abstract TITLE: A functional interplay between TGF-β and Notch signaling determines esophageal cancer cell fates AUTHORS (LAST NAME, FIRST NAME): Natsuizaka, Mitsuteru1, 2; Naganuma, Seiji1, 2; Kagawa, Shingo1, 2; Ohashi, Shinya1, 3; Whelan, Kelly A.1, 2; Chang, Sanders1, 2; Rhim, Andrew D.1, 2; Klein-Szanto, Andres J.4; Diehl, J. Alan5, 2; Herlyn, Meenhard6; Nakagawa, Hiroshi1, 2 INSTITUTIONS (ALL): 1. Gastroenterology Division, University of Pennsylvania, Philadelphia, PA, United States. 2. Abramson Cancer Center, Philadelphia, PA, United States. 3. Division of Gastroenterology and Hepatology, Digestive Disease Center, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka, Japan. 4. Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA, United States. 5. Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, United States. 6. Wistar Institute, Philadelphia, PA, United States. ABSTRACT BODY: Introduction: Notch signaling regulates cell fates. In esophageal squamous cell carcinoma (ESCC), Notch-mediated squamous-cell differentiation may be suppressed in the invasive front of tumors, thereby allowing epithelial-mesenchymal transition (EMT) induced by transforming growth factor (TGF)-β (Cancer Res. 2011;71:6836-47) to occur. We investigated how TGF-β cooperates with Notch signaling to facilitate EMT and generate a unique subset of cells with high CD44 expression, contributing to disease progression. Methods: Transformed human esophageal cells expressing EGFR, p53R175H and cyclin D1 (EPC2T) and ESCC cell line TE11 were stably transduced with ICN1, an active form of Notch1, or short hairpin RNA to knockdown either Notch1 or Notch3 in a regulatable manner (Tet-On system). Compound E, a γ-secretase inhibitor and SB-431542, a TGF-β type I receptor kinase inhibitor was used for pharmacological inhibition of Notch and TGF-β signaling, respectively. 8xCSL-luciferase reporter was used to assess the Notch-mediated transcriptional activity. Quantitative RT-PCR, Western blotting, immunohistochemistry or immunofluorescence and fluorescence-activated cell sorting (FACS) were done to determine gene expression. Primary tumor tissues annotated with known clinical outcomes were analyzed. Results: Within EPC2T and TE11, FACS revealed two discrete subsets of cells: CD44High-CD24-/Low (CD44H) and CD44Low-CD24High (CD44L), which were isolated and grown separately. CD44L cells have clear epithelial features expressing E-cadherin. By contrast, CD44H cells display a spindle-shaped morphology and express genes such as N-cadherin and ZEB1. In purified CD44L cells, TGF-β activated Notch1 to induce EMT, giving rise to CD44H cells with concurrent suppression of Notch3. TGF-β-mediated conversion of CD44L to CD44H cells was greatly augmented by ICN1 while being antagonized sharply by SB-431542, Compound E or Notch1 knockdown. By contrast, Notch3 knockdown alone induced CD44H cells. Interestingly, TGF-β selectively prevented ICN1 from inducing Notch3 and squamous-cell differentiation markers while enhancing ICN1 to induce EMT related genes. In primary tumors, the active form of Notch1 (ICN1Val1744) was detected as intense nuclear staining of tumor cells in the invasive fronts in 56% of ESCC (n=171), where CD44 was often upregulated along with ZEB1, a transcription factor essential in EMT. Nuclear ICN1Val1744 was significantly associated with a poor 5-year survival in postsurgical ESCC patients (n=115). Conclusions: These data indicate that generation of CD44H cells involves TGF-β-mediated Notch1 activation and concurrent Notch3 inhibition, leading to a robust shift in the spectrum of the Notch target genes, providing novel mechanistic insights into a crosstalk between Notch and TGF-β signaling in the regulation of ESCC disease progression.