Recurrent gain-of-function USP8 mutations in Cushing's disease

Abstract

Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.

Figure 1

Recurrent USP8 mutations in Cushing's disease. (A) The number of somatic mutations (top) and the mutational status of the indicated gene (bottom) in each patient, as revealed by whole-exome sequencing and Sanger sequencing. The total number of each type of somatic mutation in 12 cases is shown on the top right. Patients carrying the indicated mutated gene are marked in pink (bottom). (B) Schematic diagram of USP8 domains and landscape of USP8 alterations in the 14-3-3 binding motif and its nearby region. These alterations were identified in 108 patients with Cushing's disease by Sanger sequencing. The number of cases with the indicated USP8 mutation among 108 patients is denoted in parentheses. The purple bar represents the 14-3-3 binding motif of USP8, and its amino acid sequence is shown on the bottom left. MIT, microtubule interaction and transport domain; Rhod, rhodanese-like domain; DUB, deubiquitinating domain.

Figure 2

Molecular characterization of USP8 mutants. (A) USP8 mutants fail to bind 14-3-3 protein. Cell lysates from 293T cells expressing Flag-tagged WT-USP8 or the indicated USP8 mutants were incubated with GST-14-3-3ε immobilized on Glutathione-Sepharose beads. GST pull-down or input samples were immunoblotted with anti-Flag antibody. (B) Reduced EGFR ubiquitination in USP8 mutants. Cell lysates from HeLa cells stably expressing Flag-tagged WT-USP8, the indicated mutant or the vector only (Ctrl) were immunoprecipitated (IP) using anti-EGFR antibody, and the immunoprecipitation products were analyzed by immunoblotting with anti-EGFR and ubiquitin (Ub) antibodies. (C) Slower EGFR degradation in HeLa cells expressing USP8 mutant relative to WT. Serum-starved HeLa cells stably expressing Flag-tagged USP8 protein were treated with EGF (20 ng/ml) in the presence of cycloheximide (25 μg/ml) for 3 h. Immunoblot analysis was performed to determine p-EGFR, EGFR and Flag-USP8 protein levels. (D) Enhanced EGF-induced Erk phosphorylation in USP8 P720R mutant. HeLa cells stably expressing WT, USP8 P720R mutants or the control were treated with EGF (20 ng/ml) for the indicated times. Cell lysates were subjected to immunoblot analysis for total Erk and phospho-Erk (p-Erk). The densitometric ratio of p-Erk1/2 to total-Erk1/2 is shown between panels. Three independent experiments were repeated with similar results.

Figure 3

USP8 mutation enhances EGFR protein expression and POMC mRNA abundance in ACTH-secreting PAs. (A) Immunohistochemical staining for USP8 and EGFR in a representative normal pituitary, USP8 WT and mutated PAs. Scale bar, 50 μm. Inset, high magnification. In both WT and mutated groups, the percentage of USP8- and EGFR-positive cases was calculated (right). On top of the column, actual number of mutated samples over total analyzed. P values were calculated by Pearson's χ² test. (B) Immunoblot analysis of USP8, EGFR and actin levels in USP8 WT and mutated PAs (left). Each lane represents one case. The relative protein levels of USP8 and EGFR are normalized to actin and shown on the right. (C) Relative mRNA levels of USP8, EGFR and POMC in USP8 WT and mutated PAs, assessed by RT-qPCR analysis and normalized to the housekeeper gene HPRT. In B and C, a total of 6 WT and 8 USP8-mutated cases are analyzed and the number is indicated by n. Each symbol represents an individual case and lines indicate the median. P values were calculated by the Mann-Whitney U-test. (D) Suppression of ACTH secretion in primary human ACTH-secreting tumor cells after USP8 knockdown using lentivirus-mediated shRNA. (E) Decreased ACTH secretion in primary tumor cells treated with Gefitinib (1 μM) for 48 h. In D and E, ACTH levels in culture media were measured with radioimmunoassy (RIA) and presented as % of control media. Error bar indicates SEM of 3 replicates. *P < 0.05 compared to control (Mann-Whitney U-test). One WT and two USP8-mutated PAs are shown.

Figure 4

Clinical characteristics of patients with Cushing's disease in relation to USP8 mutational status. (A) Frequency of USP8 mutations in male and female patients with Cushing's disease. (B) Representative contrast-enhanced T1 weighted MRI of PAs with or without USP8 mutations. Tumor bulk is indicated by arrows. The WT tumor is very large and invasively extends into the sphenoidal sinus and the cavernous sinus, whereas the mutant tumor is markedly smaller in size and diffusely distributed within the sella. (C) Percentage of cases with invasion in both USP8 WT and mutated groups. Invasive adenomas were defined as fulfilling 1 of 2 conditions: (1) Hardy's modified classification grade III, IV and/or stage C, D and E; (2) Knosp classification grade III and IV. In A and C, the actual number of mutated cases over the total cases analyzed is shown on top of each column. P values were calculated by Pearson's χ² test. (D) Different maximal diameter between USP8 WT and mutated groups. Each symbol (dot) represents an individual case. Lines indicate the median with interquartile range (25% and 75%). The number of patients with each genotype is indicated by n. P values were calculated by the Mann-Whitney U-test.

Figure 5

Mechanisms of USP8 mutation-mediated ACTH hyperproduction. The deubiquitinating enzyme USP8 can be phosphorylated allowing for association with 14-3-3 protein, which subsequently inhibits its activity. USP8 mutant fails to bind 14-3-3 protein, leading to an elevated USP8 activity. USP8 deubiquitinates numerous targets and protect them from degradation. EGFR is an crucial USP8 target, and deregulation of EGFR leads to increased MAPK signaling and subsequently promotes POMC transcription partially through inducing the degradation of p27(Kip1), perhaps other regulators such as Brg1 and HDAC2. USP8 also regulates other RTKs such as c-MET and ErbB3, both of which potentially play a role in ACTH production. Finally, through regulating Smoothened expression and cellular location, USP8 mutation activates Hedgehog signaling, resulting in ACTH secretion. Therefore, inhibiting USP8 and/or EGFR activity represents a potential therapeutic approach for Cushing's disease.