Cushings Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in excess of cortisol release by the adrenal glands. Pasireotide is the pituitary-targeted drug approved to treat adult patients. Its mechanism of action seems to rely on the preferential binding to the highly expressed somatostatin receptor in corticotroph tumors, SST5. Recently, somatic mutations in the deubiquitinase USP8 gene have been reported in 40% of patients affected by CD, resulting in epidermal growth factor receptor (EGFR) signaling hyperactivation and ACTH secretion. Although mutated tumors were found to express significantly higher levels of SSTR5, the interplay between USP8 and SST5 has never been explored so far. The aim of this study is to investigate whether modulation of USP8 activity could affect SST5 physiology. First, by immunoprecipitation experiments we found an enhanced SST5 ubiquitination in murine corticotroph tumor AtT-20/D16v-F2 cells incubated with the chemical inhibitor of USP8, RA-9, compared to basal (2.1±0.4-fold increase vs basal, P<0.05). On the contrary, pasireotide did not significantly modify the ubiquitination status of the receptor. Interestingly, a significant SST5 protein expression levels increase was only observed in cells pre-treated with RA-9 and exposed to 10 nM pasireotide for 72 h (+36%±12.1, % vs basal, P<0.01), this effect being reverted by cycloheximide treatment (−49.3%±23, % vs basal, P<0.05). Accordingly, cells transfection of the catalytically active USP8-C40, followed by pasireotide incubation, resulted in a reduction of SST5 expression level (−38.7%±18.4% vs basal, P<0.05). In AtT-20/D16v-F2 cells, similar inhibitory actions on p-ERK1/2 were triggered by pasireotide, RA-9 and by their co-incubation (−72.1%±16.1, −60.2%±16.7, and −66.4%±14.6,% P<0.05 vs basal), whereas RA-9 was able to reduce p-ERK1/2 levels (−48.5±9.2% vs basal, P<0.05) in human tumoral corticotroph primary cultures regardless in vitro responsiveness to pasireotide. Altogether, our data demonstrate that the expression of agonist-activated SST5 is negatively modulated by USP8 in AtT-20/D16v-F2 and that RA-9 is effective in reducing ERK phosphorylation in mouse and human ACTH-secreting pituitary tumor cells.
18 - 21 May 2019
European Society of Endocrinology