Endocrine Abstracts

The tumorigenesis of Cushing’s disease is characterized by somatic mutations in the USP8 gene in almost half of the cases. USP8 encodes for ubiquitin specific protease 8, a deubiquitinase that rescues proteins involved in the regulation of ACTH synthesis in corticotroph cells. In the present study we tested the antisecretory and antiproliferative efficacy of a commercially available specific USP8 inhibitor (IC50 3.1 μM USP8; >90 μM USP7) in immortalized murine corticotroph tumour AtT-20 cells and human corticotroph tumours in primary cell culture (n=11). The USP8 inhibitor decreased POMC transcription and promoter activity and ACTH secretion in a dose response manner starting from 10 nM (% suppression at 1 μM 58±2, 53±12 and 59±7 respectively). Knocking down USP8 abolished the inhibitory effect on POMC promoter activity, confirming the specificity of the targeted treatment. Treatment of the human corticotroph tumours in vitro decreased ACTH secretion beyond the arbitrarily set cut-off of 20%in all cases at 1.5–3 μM concentration (% suppression 33±15 and 44±17 respectively), 9 out of 11 cases at the 1 μM (% 34±15) and 7 out of 11 at the 0.1 μM (% 24±16). No toxicity was observed in any of these concentrations. In AtT-20 cells the USP8 inhibitor (1 μM) decreased cell number (% suppression 35%±1) without affecting cell volume and without cytotoxicity. The treatment decreased cell viability at 1 μM (but not at lower concentrations; % suppression 30±3). No changes in the apoptosis markers PARP and cleaved caspase 3 were observed under these conditions. All human corticotroph tumours responded to 3 μM by decreasing cell viability (% suppression 46±18) and 6 out of 11 to 1 μM treatment (% 30±24). Co-treatment with EGF, an ACTH secretagogue whose receptor is the best characterized target of USP8, in EGFR-overexpressing AtT-20 cells shifted the antiproliferative and antisecretory response to the USP8 inhibitor (% suppression 36±3 vs 27±2 and 64±6 vs 57±7 at 1 μM respectively), indicating that in part the effect of USP8 inhibition is mediated via its inhibitory crosstalk with the EGFR signalling. Altogether these data show that pharmacological USP8 inhibition can effectively suppress ACTH synthesis in vitro without accompanying cytotoxicity and indicate its potential for the management of ACTH hypersecretion in Cushing’s disease.