| Summary: | Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases <i>USP8</i> and <i>USP48</i>, and in <i>BRAF</i> genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in <i>USP8</i>, <i>USP48</i>, and <i>BRAF</i> hotspot regions. Seven patients were found to carry <i>USP8</i> somatic mutations in the well-characterized 14-3-3 protein binding motif (<i>n</i> = 5 P720R, <i>n</i> = 1 P720Q, <i>n</i> = 1 S718del); 2 patients were mutated in <i>USP48</i> (M415I); no mutation was identified in <i>BRAF</i>. In addition, a novel <i>USP8</i> variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of <i>USP8</i> G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the <i>USP8</i> G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, <i>p</i> < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, <i>p</i> < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, <i>p</i> < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in <i>USP8</i> (13.3% vs. 36.5% incidence of all published mutations) and <i>USP48</i> (3.3% vs. 13.3% incidence) hotspot regions. A novel <i>USP8</i> variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.
|
|---|