Jhih-Yun Ho1,2, Hsin-Ying Lu3,4,5, Hsing-Hsien Cheng1,2, Yu-Chieh Kuo1, Yu-Lin Amy Lee6, Chia-Hsiung Cheng7,8,9. 1. Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, 250 Wuxing Street, Taipei, 11031, Taiwan. 2. Graduate Institute of Medical Science, College of Medicine, Taipei Medical University, 11031, Taipei, Taiwan. 3. Division of Cardiovascular Surgery, Department of Surgery, Wan Fang Hospital, Taipei Medical University, 11031, Taipei, Taiwan. 4. Department of Physical Medicine and Rehabilitation, Wan Fang Hospital, Taipei Medical University, 11031, Taipei, Taiwan. 5. Taipei Heart Institute, Taipei Medical University, 11031, Taipei, Taiwan. 6. Departments of Medicine and Pediatrics, Duke University Hospital, Durham, NC, 27704, USA. 7. Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, 250 Wuxing Street, Taipei, 11031, Taiwan. chcheng@tmu.edu.tw. 8. Graduate Institute of Medical Science, College of Medicine, Taipei Medical University, 11031, Taipei, Taiwan. chcheng@tmu.edu.tw. 9. Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, 11031, Taipei, Taiwan. chcheng@tmu.edu.tw.
Abstract
PURPOSE: Nuclear factor (NF)-κB signaling in cancer cells has been reported to be involved in tumorigenesis. Phosphorylation and degradation of inhibitor of NF-κBα (IκBα) is a canonical pathway of NF-κB signaling. Here, we aimed to identify and characterize noncanonical activation of NF-κB signaling by ubiquitin-conjugating enzyme E2S (UBE2S) in lung adenocarcinoma cells. METHODS: TCGA and the Human Atlas Protein Database were used to analyze the survival rate of lung adenocarcinoma patients in conjunction with UBE2S expression. In addition, PC9, H460, H441 and A549 lung adenocarcinoma cells were used in this study. PC9 and H460 cells were selected for further analysis because they expressed different UBE2S protein levels. Specific IKK inhibitors, PS1145 and SC514, were used to assess IκBα phosphorylation. Western blot analysis was used to assess protein levels in PC9 and H460 cells. A scratch wound-healing assay was used to analyze the migrative abilities of PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells were used to analyze their effects on downstream protein levels. Immunoprecipitation, immunofluorescent staining, glutathione S transferase (GST) pull-down and in vitro binding assays were used to analyze the interaction between UBE2S and IκBα. A luciferase assay was used to analyze activation of NF-κB signaling regulated by UBE2S. An in vivo zebrafish xenograft model was used to assess metastasis of PC9 cells regulated by UBE2S. RESULTS: We found that UBE2S expression in lung adenocarcinoma patients was negatively related to survival rate. The protein level of UBE2S was higher in PC9 cells than in H460 cells, which was opposite to that observed for IκBα. PC9 cells showed a higher UBE2S expression and migrative ability than H460 cells. Phosphorylation of IκBα was not changed by treatment with the IKK-specific inhibitors PS1145 and SC514 in PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells revealed that the protein levels of IκBα were inversely regulated. Immunoprecipitation, immunofluorescent staining, GST pull-down and in vitro binding assays revealed direct binding of UBE2S with IκBα. Nuclear P65 protein levels and luciferase assays showed that NF-κB signaling was regulated by UBE2S. The expression of epithelial-to-mesenchymal (EMT) markers and the migrative ability of lung adenocarcinoma cells were also regulated by UBE2S. A zebrafish xenograft tumor model showed a reduction in the metastasis of PC9 cells that was induced by UBE2S knockdown. CONCLUSIONS: Higher UBE2S expression in lung adenocarcinomas may lead to increased binding with IκBα to activate NF-κB signaling and promote adenocarcinoma cell metastasis. UBE2S may serve as a potential therapeutic target for lung adenocarcinomas.
PURPOSE: Nuclear factor (NF)-κB signaling in cancer cells has been reported to be involved in tumorigenesis. Phosphorylation and degradation of inhibitor of NF-κBα (IκBα) is a canonical pathway of NF-κB signaling. Here, we aimed to identify and characterize noncanonical activation of NF-κB signaling by ubiquitin-conjugating enzyme E2S (UBE2S) in lung adenocarcinoma cells. METHODS: TCGA and the Human Atlas Protein Database were used to analyze the survival rate of lung adenocarcinoma patients in conjunction with UBE2S expression. In addition, PC9, H460, H441 and A549 lung adenocarcinoma cells were used in this study. PC9 and H460 cells were selected for further analysis because they expressed different UBE2S protein levels. Specific IKK inhibitors, PS1145 and SC514, were used to assess IκBα phosphorylation. Western blot analysis was used to assess protein levels in PC9 and H460 cells. A scratch wound-healing assay was used to analyze the migrative abilities of PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells were used to analyze their effects on downstream protein levels. Immunoprecipitation, immunofluorescent staining, glutathione S transferase (GST) pull-down and in vitro binding assays were used to analyze the interaction between UBE2S and IκBα. A luciferase assay was used to analyze activation of NF-κB signaling regulated by UBE2S. An in vivo zebrafish xenograft model was used to assess metastasis of PC9 cells regulated by UBE2S. RESULTS: We found that UBE2S expression in lung adenocarcinoma patients was negatively related to survival rate. The protein level of UBE2S was higher in PC9 cells than in H460 cells, which was opposite to that observed for IκBα. PC9 cells showed a higher UBE2S expression and migrative ability than H460 cells. Phosphorylation of IκBα was not changed by treatment with the IKK-specific inhibitors PS1145 and SC514 in PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells revealed that the protein levels of IκBα were inversely regulated. Immunoprecipitation, immunofluorescent staining, GST pull-down and in vitro binding assays revealed direct binding of UBE2S with IκBα. Nuclear P65 protein levels and luciferase assays showed that NF-κB signaling was regulated by UBE2S. The expression of epithelial-to-mesenchymal (EMT) markers and the migrative ability of lung adenocarcinoma cells were also regulated by UBE2S. A zebrafish xenograft tumor model showed a reduction in the metastasis of PC9 cells that was induced by UBE2S knockdown. CONCLUSIONS: Higher UBE2S expression in lung adenocarcinomas may lead to increased binding with IκBα to activate NF-κB signaling and promote adenocarcinoma cell metastasis. UBE2S may serve as a potential therapeutic target for lung adenocarcinomas.
Authors: Frederik C Roos; Andrew J Evans; Walburgis Brenner; Bill Wondergem; Jeffery Klomp; Pardeep Heir; Olga Roche; Christian Thomas; Heiko Schimmel; Kyle A Furge; Bin T Teh; Joachim W Thüroff; Christian Hampel; Michael Ohh Journal: Am J Pathol Date: 2011-02 Impact factor: 4.307
Authors: L Hu; X Li; Q Liu; J Xu; H Ge; Z Wang; H Wang; Z Wang; C Shi; X Xu; J Huang; Z Lin; R O Pieper; C Weng Journal: Oncogene Date: 2016-09-05 Impact factor: 9.867
Authors: Donato Tedesco; Jianhuan Zhang; Lan Trinh; Guita Lalehzadeh; Rene Meisner; Ken D Yamaguchi; Daniel L Ruderman; Harald Dinter; Deborah A Zajchowski Journal: Neoplasia Date: 2007-07 Impact factor: 5.715