Ubiquitin-Specific Protease 8 Mutant Corticotrope Adenomas Present Unique Secretory and Molecular Features and Shed Light on the Role of Ubiquitylation on ACTH Processing

Background: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. Objectives: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. Methods: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. Results: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. Conclusions: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more "typical" corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas