The adenomas in Cushing’s disease frequently exhibit mutations in exon 14, within a binding motif for the regulatory protein 14-3-3 located between the catalytic domain (DUB), responsible for ubiquitin hydrolysis, and the WW-like domain that mediates autoinhibition, resulting in constantly active USP8. The exact molecular mechanism of deubiquitinase activity disruption in Cushing’s disease remains unclear. To address this, Sanger sequencing of USP8 was performed to identify mutations in corticotropinomas. These mutations were subjected to computational screening, followed by molecular dynamics simulations to assess the structural alterations that might change the biological activity of USP8. Eight different variants of the USP8 gene were identified both within and outside the “hotspot” region. Six of these had previously been reported in Cushing’s disease, while two were detected for the first time in our patients with CD. One of the two new variants, initially classified as benign during screening, was found in the neighboring SH3 binding motif at a distance of 20 amino acids. This variant demonstrated pathogenicity patterns similar to those of known pathogenic variants. All USP8 variants identified in our patients caused conformational changes in the USP8 protein in a similar manner. The identified mutations, despite differences in annotation results—including evolutionary conservation assessments, automated predictor data, and variations in localization within exon 14—exhibit similar patterns of protein conformational change. This suggests a pathogenic effect that contributes to the development of CD.

In the current work, we aimed to evaluate the association of clinical data of Cushing’s disease (CD) patients with USP8 mutation status and to study USP8-related molecular mechanisms connected to the regulation of corticotropinoma growth and activity. 35 CD patients were enrolled; the sequencing of exon 14 in USP8 revealed variants in eighteen adenomas, two of which were described for the first time in CD. USP8 variants were more common in women (94% vs. 76%; p = 0.001), and microadenomas and tumor recurrence were prevalent in the USP8-mutant group (44% vs. 29%; p = 0.04 and 44% vs. 22%; p = 0.0015). Preoperative ACTH and serum cortisol did not differ in the USP8-WT and USP8-mutant patients. All USP8-mutant adenomas were SST5-positive, and 73% of them were double-positive (SST5+/SST2+). A total of 50% of USP8-WT adenomas were double-negative (SST5−/SST2−), and 40% of them were SST5-positive. Analysis of transcriptome was performed for nine USP8-mutant and six USP8-WT adenomas and revealed the that the bidirectional dysregulation of Wnt signaling, including both the agonist RSPO2 and antagonist SFRP1, in the USP8-mutant corticotropinomas was downregulated. These alterations may indicate the existence of regulatory connections between USP8 enzyme activity, Wnt signaling, EGFR signaling and somatostatin receptors’ trafficking, which can explain, at least in part, the clinical manifestations of CD in patients with corticotropinomas harboring USP8 variants.

ACTH-secreting pituitary neuroendocrine tumors (ACTH-NETs) drive endogenous hypercortisolism through autonomous ACTH production, resulting in increased risks of cardiovascular, metabolic and infectious complications, and higher mortality. The role of somatic mutations in the development of ACTH-NETs has been shown. The most frequently detected are variants in the ubiquitin-specific protease 8 (USP8) gene. The USP8 enzyme mediates the removal of ubiquitin tags from proteins, preventing their degradation; it also regulates the intracellular localization of proteins and signal transduction pathways. The prevalence of somatic USP8 mutations in ACTH-NETs is highly variable across studies, ranging from 12 to 60%. It has been shown that mutations in the USP8 gene lead to hyperfunction of the enzyme, which may be associated with the overactivation of oncogenic signaling pathways, including MAPK, JAK/STAT3, and AKT/mTOR, as well as dysregulation of the Wnt pathway. Clinical data indicate that ACTH-NETs with somatic mutations in the USP8 gene are smaller in size, more prone to recurrence, and more frequently express somatostatin receptor subtypes 5 and 2 compared to tumors in which USP8 mutations are not found. The molecular mechanisms underlying these effects require further investigation.