Cushings disease tumours bear activating mutations in the ubiquitin-specific-protease 8 gene (USP8) gene in 40-60% of cases. We have previously observed that patients with USP8 mutant tumours have smaller tumour size and show a better response to high dose (8mg) dexamethasone stimulation test compared to those with wild type USP8 (1). The aim of this study was to define the role of USP8 on glucocorticoid receptor (GR) in corticotroph tumour cells. We did experiments in immortalized AtT-20 corticotroph tumour overexpressing USP8 wild type or mutants (Pro720Arg, Ser718Pro, Ser718del). Cells overexpressing the USP8 mutants showed better GR transcriptional response to dexamethasone treatment (determined by MMTV luciferase assay) compared to those transfected with wild type USP8. Furthermore, GR nuclear translocation determined as GR content in the nuclear fraction after dexamethasone treatment (10nM, 2 hours) was more prominent in cells overexpressing USP8 mutants than wild type. In addition, although GR protein levels were decreased after long-term exposure to dexamethasone (12 hours) in wild type cells, cells overexpressing USP8 mutants retained GR showing protection from treatment-induced degradation. USP8 knockdown increased GR ubiquitination and coimmunoprecipitation experiments showed that all USP8 forms physically interact with GR, indicating a direct effect of USP8 on GR. Introducing a mutation in the putative ubiquitination site lysine 419 halved the fraction of ubiquitinated GR and potentiated GR activity on POMC promoter and MMTV. These data show that USP8 deubiquitinates and rescues GR activity. USP8 is mainly a cytoplasmatic protein, but its mutants are also localized in the nucleus. Therefore, an intriguing hypothesis is that their closer topological association with GR may render USP8 mutant corticotroph cells more sensitive to the physiological negative glucocorticoid feedback. Resistance to the glucocorticoid negative feedback is a hallmark in Cushings disease. Our data of a regulatory role of USP8 mutants on GR may explain the smaller size and increased glucocorticoid sensitivity of the USP8 mutant tumours and reveals a novel action of USP8 in corticotroph tumour pathophysiology.
Reference: 1. Perez-Rivas et al., J Clin Endocrinol Metab, 2015, 100(7):E997E1004.
18 - 21 May 2019
European Society of Endocrinology