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

Abstract

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.

Sexual Dimorphism in Cellular and Molecular Features in Human ACTH-Secreting Pituitary Adenomas

“…Interestingly, exome sequencing recently identified two somatic mutations, i.e., USP8 and USP48, which occurred with far greater frequency in corticotrope adenomas from women with Cushing's disease [10,11], thus indicating that the corticotrope adenoma itself may harbour features which contribute to gender-dependent differences in Cushing's disease. In fact, we and others observed different molecular signatures in adenomas carrying USP8 variants compared to USP8-wildtype adenomas [12,48,49], and this carried over into increased POMC synthesis and ACTH secretion [12] and changes in intracellular signalling [50].…”

“…USP8 sequencing was performed in 111 adenoma specimens and 29 proved carriers of somatic mutations; in detail, 25 adenomas out of 84 specimens tested in culture carried USP8 variants as did 5 out of 38 specimens used in microarray analysis. All variants have been previously described [10,12]. As expected, the proportion of USP8 variant carriers was greater in adenomas from female compared to male patients (32.2% vs. 4.2%, p < 0.005); in fact, only a single adenoma from a male patient presented an USP8 mutation (i.e., p.P720R); thus, no subanalysis according to USP8 variant status could be performed.…”

“…Overexpression of SSTR5 has recently been reported among USP8-mutated corticotrope adenomas compared to wild-type adenomas [49]; however, USP8 mutations were found in adenomas from female patients only, thus this finding could be gender-rather than USP8-variant specific. In fact, findings reported so far on USP8 variant adenomas [12,[48][49][50] were collected almost exclusively in female patients; only two specimens were obtained from male patients, the remainder (80 USP8 variant adenomas in the four series) in female patients. A multicentre effort is clearly required to discern sex-independent, USP8-determined features.…”

“…RNA was extracted from formalin-fixed paraffin-embedded adenomatous samples using Recover All Total Nucleic Acid Isolation Kit (Life Technologies, Carlsbad, CA, USA), as previously described [12,13]. RNA (300 ng) was analysed on Human HT_12 v4 Bead Chip (Whole Genome DASL High Throughput assay, Illumina, San Diego, CA, USA) and fluorescence data captured into HiScan, a high-resolution laser imager (Illumina).…”

“…RNA was obtained from formalin-fixed or fresh specimens and carried out as previously described [12,103].…”