Volume 130, Issue 6, Pages 1696-1706 (May 2006) Trefoil Factor Family-1 Mutations Enhance Gastric Cancer Cell Invasion Through Distinct Signaling Pathways  Xianyang Yio, Matthew Diamond, Jie–Yu Zhang, Harel Weinstein, Lu–Hai Wang, Lawrence Werther, Steven Itzkowitz  Gastroenterology  Volume 130, Issue 6, Pages 1696-1706 (May 2006) DOI: 10.1053/j.gastro.2006.01.040 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Computational and molecular modeling of TFF1. (A) Rotated 90° relative to the other frames, which are oriented such that the Loop 1 region is facing out of the page. The 3 loops of the trefoil domain are labeled with roman numerals. (A–C) Wild-type protein, (D) the A10D mutant, (E) the E13K mutant. (A, B) Surfaces of the 3 Loop 1 residues found mutated by Park et al14 are labeled and colored. (C–E) Colored by electrostatic potential, calculated with GRASP,39 where positivity is indicated by blue and negativity by red. All models were based on the 1PS2 structure by Polshakov et al36 before being energetically minimized in explicit solvent with NAMD.38 GRASP39 and InsightII (Accelrys) were used for molecular visualization. Gastroenterology 2006 130, 1696-1706DOI: (10.1053/j.gastro.2006.01.040) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Gastric cancer cell proliferation in response to wtTFF1 and mutant TFF1 proteins. (A) AGS cells were treated with either wild-type TFF1 or TFF1 mutants A10D, E13K, or C58S at a dose of 260 mg/mL (18 μmol/L; similar to previous reports) for 48 hours. After this, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagents were applied and optical density was measured at 570 nm. Each condition was run in triplicate. Bars represent mean ± SD. Differences between wild-type TFF1 and BSA, A10D, E13K, and C58S were analyzed by the Student t test. **P < .01. (B) Summary of 7 separate experiments showing that for both AGS and KATO-III cells, wtTFF1 produces an approximate 30% decrease in proliferation compared with BSA controls (**P < .01), whereas mutant TT1 proteins lose this effect. Gastroenterology 2006 130, 1696-1706DOI: (10.1053/j.gastro.2006.01.040) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 AGS cell apoptosis in response to wtTFF1 and mutant TFF1 proteins (40 mcg/mL). Caspase-3 activity of AGS cells treated with wild-type TFF1, A10D, or E13K (each at 40 mcg/mL) for 24 hours, followed by etoposide (1 mmol/L) treatment for 24 hours. Each assay was run in triplicate. Bars represent mean ± SD. Differences were analyzed by the Student t test. **P < .01. Gastroenterology 2006 130, 1696-1706DOI: (10.1053/j.gastro.2006.01.040) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Invasion of AGS cells in response to wtTFF1 and mutant TFF1 proteins. (A) Invasion of AGS cells through Matrigel (Becton Dickinson) in the modified transwell assay. (B) AGS cell invasion in response to BSA, wild-type TFF1, A10D, and E13K (40 mcg/mL). Data from 7 separate experiments are combined and expressed as an invasion index, whereby the degree of invasion in response to BSA alone was set at 1.0 for each experiment. The bars represent the mean ± SD. Differences were analyzed by the Student t test. ***P < .01. Gastroenterology 2006 130, 1696-1706DOI: (10.1053/j.gastro.2006.01.040) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Invasion of AGS cells in response to wtTFF1, A10D, and E13K in the presence and absence of signaling inhibitors. (A) PLC inhibitor U73122 (2.5 μmol/L), (B) PI3-kinase inhibitor LY294002 (20 μmol/L), and (C) ROCK inhibitor Y27632 (20 μmol/L). For each condition, invasion without inhibitor (open bars) is expressed as 100% invasion, to permit comparison with that observed with inhibitor (closed bars). Each experiment was run in duplicate; bars represent mean ± SD. Differences were analyzed by the Student t test. *P < .05; **P < .01; ***P < .001. Gastroenterology 2006 130, 1696-1706DOI: (10.1053/j.gastro.2006.01.040) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Western blot of Akt activation in AGS cells treated with medium alone, wtTFF1, A10D, or E13K (40 mcg/mL) for 5 minutes and 30 minutes and then blotted with antibody to serine 473-phosphorylated Akt (top) and total Akt (bottom). This experiment was repeated twice with similar results. Gastroenterology 2006 130, 1696-1706DOI: (10.1053/j.gastro.2006.01.040) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions