Volume 134, Issue 5, Pages 1555-1566 (May 2008) Polysialylated NCAM Represses E-Cadherin-Mediated Cell-Cell Adhesion in Pancreatic Tumor Cells  Susanne C. Schreiber, Klaudia Giehl, Caroline Kastilan, Cornelia Hasel, Martina Mühlenhoff, Guido Adler, Doris Wedlich, Andre Menke  Gastroenterology  Volume 134, Issue 5, Pages 1555-1566 (May 2008) DOI: 10.1053/j.gastro.2008.02.023 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 NCAM and ST8Sia-II/IV expression in pancreatic carcinomas. (A) mRNA concentration of NCAM was increased in 8 out of 12 samples of pancreatic carcinomas, expression of ST8Sia-II in 10 out of 12 samples, and of ST8Sia-IV in 8 out of 12 samples. The postsurgical histopathologic pTNM classification and stage grouping are shown in Supplemental Table T1 (see Supplementary Table T1 online at www.gastrojournal.org). (B) Three out of 5 pancreatic carcinoma cell lines exhibited expression of NCAM mRNA, and most carcinoma cell lines showed expression of ST8Sia-II/IV mRNA. NCAM protein was detected in the NCAM-mRNA positive cell lines after immunoprecipitation of NCAM from 200 μg cell lysate. PSA was detected after restaining of the blot with a PSA-specific antibody (lower panel). (C) Immunohistochemical analyses of NCAM and PSA localization in IMIM-PC1, BxPC-3, and AsPC-1 pancreatic carcinoma cells using NCAM- and PSA-specific antibodies. Bar, 15 μm. (D) In protein lysates from PANC-1 cells stably expressing EGFP-K-Ras(V12), EGFP-K-Ras(N17), EGFP-H-Ras, or EGFP-N-Ras(V12), only K-Ras(V12)-expressing cells exhibit increased NCAM-expression. Gastroenterology 2008 134, 1555-1566DOI: (10.1053/j.gastro.2008.02.023) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Polysialylated NCAM is up-regulated by K-Ras(V12). (A and B) PANC-1 and HEK293 cells stably expressing EGFP-K-Ras(V12) show elevated concentration of PSA-NCAM. Two different K-Ras(V12)-expressing PANC-1 and HEK293 cell clones were included, and 50 μg total protein lysate were analyzed using NCAM- and PSA-specific antibodies. (C and D) Expression of K-Ras(V12) resulted in elevated NCAM, ST8Sia-II, and ST8Sia-IV mRNA concentrations in PANC-1 and HEK293 cell clones. Amplification of GAPDH mRNA served as control. (E and F) Endo-N treatment of AsPC-1 and PANC-1-K-Ras(V12) cells removed PSA from NCAM as shown by PSA staining. SH-SY5Y neuroblastoma cells served as positive control. Gastroenterology 2008 134, 1555-1566DOI: (10.1053/j.gastro.2008.02.023) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Membrane localization of PSA-NCAM correlates with enhanced cell migration. (A) NCAM and PSA are localized at the cell membrane of K-Ras(V12)-expressing PANC-1 cells by confocal microscopy. The second and fourth panels show overlays of NCAM or PSA staining (red) with EGFP staining (green). Bar, 15 μm. (B) Transwell migration assays revealed an enhanced migration of K-Ras(V12)-expressing PANC-1 cells. (C) Cell aggregation of K-Ras(V12)-expressing, NCAM-positive PANC-1 cells was reduced compared with EGFP-transfected cells. Cadherin dependence was measured by addition of EDTA and EGTA (E+E). Data: Means ± SEM. Gastroenterology 2008 134, 1555-1566DOI: (10.1053/j.gastro.2008.02.023) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 PSA-NCAM interacts with the E-cadherin adhesion complex. (A) NCAM was coimmunoprecipitated with E-cadherin from 3 mg PANC-1-K-Ras(V12) lysates. The blot was restained for E-cadherin. (B) Coimmunoprecipitation assays with lysates from endo-N-treated cells show that E-cadherin was not coprecipitated with NCAM in the absence of PSA. The blots were restained for NCAM. (C) Knock down of NCAM, ST8Sia-II/IV, or of both by specific siRNAs resulted in reduced amounts of polysialylated NCAM in PANC-1-K-Ras(V12) lysates. The blot was restained for β-actin to confirm equal loading. (D) Knock down of NCAM, ST8Sia-II/IV, or both reduced the amount of E-cadherin coimmunoprecipitated with NCAM from 3 mg lysate. Blots were restained for NCAM and PSA to prove the knock down. (E) PSA-NCAM was coimmunoprecipitation with E-cadherin from AsPC-1 lysates. Removal of PSA by endo-N-treatment or siRNA transfection strongly reduced the binding of the NCAM to E-cadherin. Gastroenterology 2008 134, 1555-1566DOI: (10.1053/j.gastro.2008.02.023) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 PSA-NCAM is responsible for low cell-cell aggregation. (A) Cell-cell aggregation assays were performed with EGFP- and EGFP-K-Ras(V12)-expressing cells after treatment with endo-N (+) or buffer only (−). Removal of PSA from NCAM restores E-cadherin-mediated cell-cell adhesion in K-Ras(V12)-expressing cells. The involvement of E-cadherin was determined by addition of inhibitory E-cadherin antibody (shaded bars) and by addition of EDTA and EGTA (open bars). (B) Down-regulated expression of NCAM (shaded bars) or ST8Sia-II/IV (open bars) by siRNA revealed up-regulation of cell-cell aggregation in PANC-1-K-Ras(V12) cells (left graph). Cell migration was reduced in NCAM- or ST8Sia-II/IV-siRNA transfected cells as estimated by transwell migration assays (right graph). (C) Cell-cell aggregation capacity of AsPC-1 cells was increased after transfection with NCAM or ST8Sia-II/IV siRNAs. The involvement of E-cadherin was determined by addition of inhibitory E-cadherin antibody (light gray). All data are means ± SEM of 3 independent assays. Gastroenterology 2008 134, 1555-1566DOI: (10.1053/j.gastro.2008.02.023) Copyright © 2008 AGA Institute Terms and Conditions