Shanshan Wang1, Taishu Wang1, Qianyi Yang1, Shaoxuan Cheng1, Fang Liu1, Guoheng Yang1, Fuqiang Wang1, Ruilin Wang1, Dian Yang1, Mingyu Zhou1, Chengen Duan1, Yingqiu Zhang1, Han Liu1, Zhaoxia Dai2,3, Kang Tian4,5,6, Shuyan Liu7,8. 1. Second Affiliated Hospital, Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China. 2. Second Affiliated Hospital, Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China. daizhaoxia@dmu.edu.cn. 3. The Second Department of Thoracic Medical Oncology, The Second Hospital of Dalian Medical University, 467 Zhongshan Road, Shahekou District, 116027, Dalian, Liaoning Province, P. R. China. daizhaoxia@dmu.edu.cn. 4. Department of Bone and Joint, First Affiliated Hospital, Dalian Medical University, Dalian, China. dmu-tiankang@outlook.com. 5. Biomaterials Innovation Research Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA. dmu-tiankang@outlook.com. 6. Department of Orthopedic Sports Medicine, First Affiliated Hospital, Dalian Medical University, 222 Zhongshan Road, 116044, Dalian, Liaoning Province, P. R. China. dmu-tiankang@outlook.com. 7. Second Affiliated Hospital, Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China. liusy@dmu.edu.cn. 8. Institute of Cancer Stem Cell, Dalian Medical University, 9 West Sec. Lvshun South Road, 116044, Dalian, Liaoning Province, P. R. China. liusy@dmu.edu.cn.
Abstract
PURPOSE: The epidermal growth factor receptor (EGFR) represents a top therapeutic target in the treatment of non-small cell lung cancer. EGFR expression is intricately modulated by receptor endocytosis, during which EGFR ubiquitylation and deubiquitylation play fundamental roles to govern receptor fate. This study aims to uncover novel aspects of the endocytic regulation of EGFR trafficking by deubiquitylases. METHODS: The expression and ubiquitylation of EGFR in non-small cell lung cancer cells treated with deubiquitylase inhibitors were assessed by immunoblotting, immunoprecipitation and mass spectrometry analyses. The intracellular EGFR distribution was investigated using immunofluorescence and confocal microscopy assays, and colocalizations with endocytic compartments were examined using GFP-tagged Rab proteins as markers. The influence of the proteasomal deubiquitylase inhibitor b-AP15 on EGF- and HSP90 inhibitor-induced EGFR downregulation was evaluated by immunoblotting. The anticancer effects of b-AP15 were assessed by cell proliferation, colony formation and flow cytometry assays, as well as xenograft animal models. RESULTS: We found that b-AP15 caused a dramatically enhanced ubiquitylation of EGFR in lung cancer cells. Treatment with b-AP15 decreased cell surface EGFR levels and accumulated EGFR on recycling endosomes marked with Rab4A and Rab11A. b-AP15 effectively repressed EGF- and HSP90 inhibitor-induced EGFR degradation. Lung cancer cells exposed to b-AP15 showed markedly reduced cell propagation and significantly increased cell apoptosis. Furthermore, b-AP15 effectively inhibited tumor xenograft growth in nude mice. CONCLUSION: Proteasomal USP14 and UCHL5 act collectively to promote cell surface recovery of EGFR. Inhibition of proteasomal deubiquitylase activity induces increased EGFR ubiquitylation and retention on recycling endosomes. The USP14 and UCHL5 dual inhibitor b-AP15 elicits potent tumor-suppressive effects to deter cell proliferation and induce apoptotic cell death in lung cancer.
PURPOSE: The epidermal growth factor receptor (EGFR) represents a top therapeutic target in the treatment of non-small cell lung cancer. EGFR expression is intricately modulated by receptor endocytosis, during which EGFR ubiquitylation and deubiquitylation play fundamental roles to govern receptor fate. This study aims to uncover novel aspects of the endocytic regulation of EGFR trafficking by deubiquitylases. METHODS: The expression and ubiquitylation of EGFR in non-small cell lung cancer cells treated with deubiquitylase inhibitors were assessed by immunoblotting, immunoprecipitation and mass spectrometry analyses. The intracellular EGFR distribution was investigated using immunofluorescence and confocal microscopy assays, and colocalizations with endocytic compartments were examined using GFP-tagged Rab proteins as markers. The influence of the proteasomal deubiquitylase inhibitor b-AP15 on EGF- and HSP90 inhibitor-induced EGFR downregulation was evaluated by immunoblotting. The anticancer effects of b-AP15 were assessed by cell proliferation, colony formation and flow cytometry assays, as well as xenograft animal models. RESULTS: We found that b-AP15 caused a dramatically enhanced ubiquitylation of EGFR in lung cancer cells. Treatment with b-AP15 decreased cell surface EGFR levels and accumulated EGFR on recycling endosomes marked with Rab4A and Rab11A. b-AP15 effectively repressed EGF- and HSP90 inhibitor-induced EGFR degradation. Lung cancer cells exposed to b-AP15 showed markedly reduced cell propagation and significantly increased cell apoptosis. Furthermore, b-AP15 effectively inhibited tumor xenograft growth in nude mice. CONCLUSION: Proteasomal USP14 and UCHL5 act collectively to promote cell surface recovery of EGFR. Inhibition of proteasomal deubiquitylase activity induces increased EGFR ubiquitylation and retention on recycling endosomes. The USP14 and UCHL5 dual inhibitor b-AP15 elicits potent tumor-suppressive effects to deter cell proliferation and induce apoptotic cell death in lung cancer.
Authors: Sara Sigismund; Elisabetta Argenzio; Daniela Tosoni; Elena Cavallaro; Simona Polo; Pier Paolo Di Fiore Journal: Dev Cell Date: 2008-08 Impact factor: 12.270
Authors: Sara Sigismund; Stefano Confalonieri; Andrea Ciliberto; Simona Polo; Giorgio Scita; Pier Paolo Di Fiore Journal: Physiol Rev Date: 2012-01 Impact factor: 37.312
Authors: Cyriac Kandoth; Michael D McLellan; Fabio Vandin; Kai Ye; Beifang Niu; Charles Lu; Mingchao Xie; Qunyuan Zhang; Joshua F McMichael; Matthew A Wyczalkowski; Mark D M Leiserson; Christopher A Miller; John S Welch; Matthew J Walter; Michael C Wendl; Timothy J Ley; Richard K Wilson; Benjamin J Raphael; Li Ding Journal: Nature Date: 2013-10-17 Impact factor: 49.962