Volume 146, Issue 5, Pages 1397-1407 (May 2014) Activating Mutations in PTPN3 Promote Cholangiocarcinoma Cell Proliferation and Migration and Are Associated With Tumor Recurrence in Patients  Qiang Gao, Ying–Jun Zhao, Xiao–Ying Wang, Wei–Jie Guo, Song Gao, Lin Wei, Jie–Yi Shi, Guo–Ming Shi, Zhi–Chao Wang, Yuan–Nv Zhang, Ying–Hong Shi, Jie Ding, Zhen–Bin Ding, Ai–Wu Ke, Zhi Dai, Fei–Zhen Wu, Hui Wang, Zhao–Ping Qiu, Zhi–Ao Chen, Zhen– Feng Zhang, Shuang–Jian Qiu, Jian Zhou, Xiang–Huo He, Jia Fan  Gastroenterology  Volume 146, Issue 5, Pages 1397-1407 (May 2014) DOI: 10.1053/j.gastro.2014.01.062 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Summary of commonly mutated genes in 124 ICC samples. The heat map shows genes (rows) and tumors (columns) with mutations (dark yellow). The number of events per tumor and per gene is indicated at the top and to the left, respectively. The etiologic and sequencing features for each tumor are shown by blue boxes. Gastroenterology 2014 146, 1397-1407DOI: (10.1053/j.gastro.2014.01.062) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Schematic representation of somatic PTPN3 mutations. (A) Distribution of somatic mutations in PTPN3. (Upper labels) Mutations reported in the literature (see Supplementary Table 10). (Lower labels) Mutations identified in this study, which are all missense and localize to or near the FERM domain, leaving the PDZ and PTPc domains unaffected. FERM, band 4.1, ezrin, radixin, moesin homology domain; PDZ, a common modular protein-interaction domains identified initially in PSD-95, DLG, and ZO-1; PTPc, protein tyrosine phosphatase, catalytic domain. (B) Three-dimensional view of mutation sites based on structural models of the PTPN3 FERM domain. (D, E) and (F) show a detailed view of the residues A90, A211, and L232, respectively. (C) The full-length model of PTPN3 protein shows that residue L384 is located in a disorder region between the FERM and PDZ domain. Gastroenterology 2014 146, 1397-1407DOI: (10.1053/j.gastro.2014.01.062) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Effects of PTPN3 knockdown in cholangiocarcinoma cells. (A) Small interfering RNA (siRNA)-mediated PTPN3 silencing enhances cell proliferation in all 3 lines containing WT PTPN3 (RBE, HCCC-9810, and Huh28). Also shown is Western blot analysis of PTPN3 in cells transfected with PTPN3 siRNA compared with control siRNA (Si-Ctrl). β-actin indicates protein loading in each line. Western blotting was performed 48 hours after siRNA treatment. (B) The bar graphs summarize cell cycle analysis of PTPN3-knockdown cholangiocarcinoma cells compared with Si-Ctrl. (C) Colony formation activity of cells treated with Si-Ctrl or PTPN3 siRNA. The bar graphs show quantification of the colony formation assay. (D) Knockdown of PTPN3 markedly reduces cell migration in the 3 cholangiocarcinoma cells compared with Si-Ctrl. Representative migration images and statistics in bar graphs are shown. All results are the mean ± SE of 3 independent experiments in triplicate. *P < .05, **P < .01, and ***P < .005 compared with the respective controls (Mann–Whitney U test). Gastroenterology 2014 146, 1397-1407DOI: (10.1053/j.gastro.2014.01.062) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Functional consequence of PTPN3 mutations in cholangiocarcinoma cells. (A) The proliferation assays of RBE cells stably transduced with WT or mutant PTPN3 compared with vector control. WT and the 4 mutant PTPN3 strongly enhance RBE cell proliferation compared with vector (P < .01), while PTPN3L232R and PTPN3L384H are more potent than WT in promoting proliferation (P < .05). (B) Cell cycle analysis of lentivirus-mediated stable expression of WT or mutant PTPN3 in RBE cells compared with vector control. (C) Colony formation activity of RBE cells expressing WT or mutant PTPN3 compared with lentivirus-vector control. The bar graphs show quantification of the colony formation assay. (D) Migration assays of RBE cells expressing WT or mutant PTPN3 compared with lentivirus-vector control. The graphs show the rates of migration at 24 hours. (E) Western blot analysis of RBE cells infected with WT or mutant PTPN3 lentivirus compared with lentivirus-vector control cells. GAPDH indicates protein loading control. All results are the mean ± SE of 3 independent experiments in triplicate. *P < .05, **P < .01, and ***P < .005 compared with the respective controls (Mann–Whitney U test). Gastroenterology 2014 146, 1397-1407DOI: (10.1053/j.gastro.2014.01.062) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 Clinical relevance of PTPN3 somatic mutations and protein expression in patients with ICC. (A) Kaplan–Meier curve shows that patients with ICC who had PTPN3 mutations had a significantly high risk of recurrence than those with WT PTPN3 (log-rank test). (B) Kaplan–Meier curve showing TTR in patients with ICC with WT or mutant PTPN3 (log-rank test). (C) Quantitative reverse-transcription polymerase chain reaction shows no differences in PTPN3 mRNA expression between tumors with PTPN3 mutations (n = 42) or tumors with the L384H mutation (n = 16) and WT tumors (n = 29) (Mann–Whitney U test). (D) Representative immunohistochemical pictures showing PTPN3 protein expression in ICC tumors and normal intrahepatic bile duct. Scale bars = 100 μm. (E) Kaplan–Meier curve showing decreased TTR in patients with high PTPN3 expression versus low expression (log-rank test). Gastroenterology 2014 146, 1397-1407DOI: (10.1053/j.gastro.2014.01.062) Copyright © 2014 AGA Institute Terms and Conditions