RESUMEN
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.
