Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease

Abstract

Context Cushing's disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation.Objective To investigate the impact of USP8 mutations on proteins deregulated in CD.Design 108 pituitary adenomas (75 corticotroph (58 USP8 wildtype (WT) and 17 USP8 mutated), 14 somatotroph and 19 non-functioning) were investigated by immunohistochemistry. All evaluated proteins (USP8, AVP receptor 1b & 2, CRH receptor, CREB, p27/kip1, cyclin E, HSP90, TR4, EGFR, HDAC2, GR, Cables1) were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot.Results Whereas most of the investigated proteins were not differentially expressed, the cell cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0±1.0 vs 1.1±1.1 in wt adenomas; p=0.004). In contrast, the chaperone HSP90 was higher expressed (0.5±0.4 vs. 0.2±0.4; p=0.29) and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5±0.40.9 vs. 0.70.5±0.40.7; P=0.014).Accordingly, AtT20 cells transfected with USP8 P720R mutant had higher pCREB levels than WT transfected cells (1.3 ±0.14 vs 1 ±0.23; p=0.13). ConclusionsWe could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and phosphorylated CREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.

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].…”

“…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.…”

The New Genetic Landscape of Cushing’s Disease: Deubiquitinases in the Spotlight

“…Increased EGFR activity results in ERK1/2 activation [33] which, in a parallel manner to the physiological pathway involving the CRHR stimulation by CRH followed by cAMP dependent PKA activation-lead to activation and binding of the nuclear receptor Nur77 to the Nur responsive element in the promoter of POMC leading to an increased ACTH secretion [15,34]. However, EGFR is not the only substrate of USP8 in CD, recent studies showed several proteins deregulated in the context of USP8 mutations making them putative substrates of USP8 in CD and all with a possible role in either ACTH production or cell cycle [34,35]. Most importantly, it has also been shown in other diseases that USP8 deubiquitinates SMO in the SHH signaling pathway and this mechanism might be relevant in CD as well [36].…”

Novel Insights into Pituitary Tumorigenesis: Genetic and Epigenetic Mechanisms

“…Recently, SSTR5 expression has been shown to be higher in USP8 -mutated tumors ( 17 , 159 ), potentially allowing the mutation status to be used as a predictor of response to pasireotide (a second generation SSA with greater affinity for SSTR5 ( 167 )). In vitro studies found increased expression of pCREB and protein kinase A Cα on immunoblotting in AtT20 cells transfected with mutant USP8 ( 160 ).…”