Endocrine Abstracts

The somatostatin analogue pasireotide is approved for the treatment of Cushing’s disease, where it achieves biochemical normalization in ~40% of patients. Predicting pasireotide resistance would help avoid unnecessary treatment. Pasireotide mediates its antisecretory action by binding to somatostatin receptor 5 (SSTR5). Almost half of corticotroph tumours carry mutations in the ubiquitin specific protease 8 (USP8) gene that encodes for a deubiquitinase. Previous analysis has suggested a correlation between USP8 mutational status and SSTR5 expression. In the present study, we determine the impact of USP8 mutational status on pasireotide’s antisecretory response in vitro and propose a potential mechanism through which USP8 may control SSTR5 expression. We treated 17 consecutive human corticotroph tumours with 10 nM pasireotide and determined ACTH secretion with a radioimmunoassay, arbitrarily setting physiologically relevant suppression 20% (compared to vehicle control). We assessed 50 archival human corticotroph tumours for USP8 mutational status by Sanger sequencing and SSTR5 expression by immunohistochemistry (UMB4 rabbit monoclonal antibody). We detected USP8 mutations in the mutational hotspot of exon 14 in 9/17 (59%) fresh and 21/50 paraffin-embedded corticotroph tumours (42%). Pasireotide treatment suppressed ACTH secretion beyond the 20% cut-off in 11 out of 17 cases (3 USP8wt and 8 mutant). We observed more than two-fold inhibitory action in mutant corticotroph tumours (overall % ACTH suppression USP8mut 42.6 ± 24.7 vs wt 18.9 ± 10.9; independent t-test P = 0.032). The SSTR5 immunoreactivity score was significantly higher in USP8 mutant corticotroph tumours compared with wt (mean 0.543 ± 0.338 vs 0,228 ± 0.263, t-test P = 0.001). AtT-20 cells overexpressing the most frequently found in Cushing’s disease USP8 mutants p.Pro720Arg, p.Ser718del and Ser718Pro, showed improved response to pasireotide’s inhibitory action on hPOMC promoter activity and increased endogenous Sstr5 but not Sstr2- transcript levels two-fold. The murine Sstr5 promoter has two binding sites for the activating protein 1 (AP-1). USP8 mutants stimulate AP-1 transcriptional activity, indicating an unexpected mechanism through which activated USP8 regulates SSTR5 levels at transcriptional level. In conclusion, the present study shows that USP8 mutant corticotroph tumours have higher SSTR5 expression and are more prone to respond favourably to the antisecretory action of pasireotide.