USP8 Mutations Research Articles

Defining the Genomic Landscape of Diffuse Sclerosing Papillary Thyroid Carcinoma: Prognostic Implications of RET Fusions.

Diffuse sclerosing papillary thyroid carcinoma (DSPTC) is an aggressive histopathologic subtype of papillary thyroid carcinoma. Correlation between genotype and phenotype has not been comprehensively described. This study aimed to describe the genomic landscape of DSPTC comprehensively using next-generation sequencing (NGS), analyze the prognostic implications of different mutations, and identify potential molecular treatment targets. Tumor tissue was available for 41 DSPTC patients treated at Memorial Sloan Kettering Cancer Center between 2004 and 2021. After DNA extraction, NGS was performed using the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets platform, which sequences 505 critical cancer genes. Clinicopathologic characteristics were compared using the chi-square test. The Kaplan-Meier method and log-rank statistics were used to compare outcomes. The most common mutation was RET fusion, occurring in 32% (13/41) of the patients. Other oncologic drivers occurred in 68% (28/41) of the patients, including 8 BRAFV600E mutations (20%) and 4 USP8 mutations (10%), which have not been described in thyroid malignancy previously. Patients experienced RET fusion-positive tumors at a younger age than other drivers, with more aggressive histopathologic features and more advanced T stage (p=0.019). Patients who were RET fusion-positive had a significantly poorer 5-year recurrence-free survival probability than those with other drivers (46% vs 84%; p=0.003; median follow-up period, 45 months). In multivariable analysis, RET fusion was the only independent risk factor for recurrence (hazard ratio [HR], 7.69; p=0.017). Gene-sequencing should be strongly considered for recurrent DSPTC due to significant prognostic and treatment implications of RET fusion identification. The novel finding of USP8 mutation in DSPTC requires further investigation into its potential as a driver mutation.

Relevance of USP8 mutations in the pathogenesis of pituitary tumours of the corticotroph lineage

Relevance of USP8 mutations in the pathogenesis of pituitary tumours of the corticotroph lineage

Pituitary Neuroendocrine Tumor Organoids Generated from Induced Pluripotent Stem Cells Reveal the Dysregulation of Cell Lineage Differentiation in Cushing’s Disease

Background: Cushing’s Disease (CD) is an endocrine disease typically caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET). Corticotroph subtype PitNETs subsequently stimulate the adrenal glands to overproduce causing a wide range of detrimental effects on health, including increased stroke rates, diabetes, obesity, depression, anxiety, and a threefold increase in the risk of death from cardiovascular disease. In some instances, CD is a manifestation of genetic mutation syndromes that include MEN1, FIPA, CDH23 and Carney complex. In other cases, somatic mutations underlie the development of CD including mutations in the deubiquitinase gene USP8, identified in 35-62 % of sporadic corticotroph PitNETs and USP48 as a driver mutation in USP8 wild-type tumors. There are currently no in vitro models to study the impact of genetic mutations on the pathways leading to PitNET development in humans. Objective: To define the cellular complexity of corticotroph subtype PitNETs at a spatial single-cell resolution. Methodology: Organoids were generated from a CD patient expressing a CDH23 mutation (hPitNETO iPSCDH23) as reported by our group (Mallick et al., 2023, Translational Research). CRISPR-Cas9 gene editing of iPSCs was also used to model the development of corticotroph PitNETs expressing USP48 and USP8 mutations. Human PitNET organoids (PitNETOs) were generated from tumor tissue harvested during transsphenoidal surgery. Cell lineage trajectory analysis was performed on scRNA-seq data collected during the iPSC differentiation program. ScRNA-seq was used to compare the transcriptomic profile and cell composition of CD patient PitNETOs to the patient’s own tissue using the NanoString CosMxTM spatial transcriptomic analysis. Results: Organoids generated from a CD patient expressing a CDH23 mutation (hPitNETO iPSCDH23) exhibited tumor behavior consistent with the expected pathology and function of sparsely granulated corticotroph PitNETs. Cell lineage trajectories of hPitNETO iPCCtrl control cultures revealed multiple branching paths from the stem-like (SOX2/PAX7+) base cluster leading to clusters enriched for markers of pituitary cell lineages (lactotroph, somatotroph, thyrotroph, gonadotroph and corticotroph). Trajectory analysis for hPitNETO iPSCDH23 showed dysregulation in the normal pituitary cell lineage differentiation leading to the development of poorly differentiated corticotrophs (PAX7+/TPit+) and PAX7+/stem cells. CosMxTM spatial gene mapping revealed a cell population co-expressing PAX7 and stem cell markers SOX2 and CD44 that was abundant in Crooke cell and sparsely granulated TPit+ PitNET subtypes. An increase in the PAX7/SOX2/CD44 PitNET cell population correlated with a higher Knosp grade, tumor size and invasiveness, when compared to densely granulated PitNETs. Conclusions: Defining the complexity of the PitNET microenvironment will provide insights into the pathogenesis of CD and potentially lead to the stratification of high-risk patients for disease recurrence. 1R01DK133325. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

OR20-04 Genetic Profiling Using A Gene Panel Identifies Pathogenic Variants And Copy Number Variations In Pituitary Adenomas

Abstract Disclosure: T. Silva: None. D. Mohan: None. J. Mebarak: None. D. Meredith: None. W. Bi: Speaker; Self; Stryker. R. Carroll: None. U.B. Kaiser: None. A. Marcondes Lerario: None. A. Abreu: None. Introduction: Pituitary adenomas (PA) are the second most common central nervous system neoplasm and arise from different cell lineages. PA are benign tumors, although a subset of them may present with aggressive behavior, resulting in significant morbidity. Several genetic alterations have been implicated in PA tumorigenesis, such as USP8 mutations in Cushing disease (CD) and GNAS mutations in somatotroph adenomas (SA). However, the genetic profiles of PA are not completely understood. The aim of this study was to identify genetic alterations associated with PA using a gene panel. Methods: Targeted next generation exome sequencing (OncoPanel) was performed to identify single nucleotide variants (SNV) and copy number variations (CNV) associated with PA. OncoPanel version 1 included 275 genes, version 2 had 300 genes, and the third, current version 447 genes. USP8 was included only in the most recent version. Patients undergoing surgery at our institution between 2013-2020, who agreed to participate in the OncoPanel initiative and who had samples with good quality DNA were enrolled. Classification of PA into subtypes was performed by combining clinical and histopathological data. A strict algorithm was used to assess SNV pathogenicity. Chi-square, Mann Whitney and Kruskal-Wallis tests were used for statistical analyses. Results: Our cohort included 171 patients with following PA subtypes: TPIT lineage - 18 CD, 14 silent ACTH; PIT-1 lineage - 29 SA, 21 lactotroph and 4 silent poorly differentiated PIT-1 adenomas; SF-1 lineage - 77 gonadotroph and 8 null cell adenomas (negative immunostaining for hormones and TF). GNAS mutations were found in 28% of SA and USP8 mutations in 50% of CD screened for USP8. Pathogenic variants in MECOM were associated with silent ACTH, in FGFR4 and NF2 with CD, and in NOTCH2 with lactotroph adenomas, whereas pathogenic variants in TP53, EGFR, MEN1, BRCA1, FH, ATR, BLM, and XPC were identified in multiple PA subtypes. The number of CNV varied significantly between different subtypes of adenomas and were higher in lactotroph (median, 46), poorly differentiated PIT-1 (41), SA (26) and silent ACTH (17) compared to CD (3), gonadotroph (1) and null cell (0.5) (p< 0.001). MIB-1 proliferation index >3% was associated with higher CNV burden (p=0.022). Interestingly, copy number loss of chromosome 16 and 1q combined were exclusively identified in SA without GNAS mutations. Conclusion: OncoPanel analyses identified pathogenic variants in PA, some well recognized variants such as USP8 and GNAS, and some rare events such as NF2 in CD. In addition, our findings highlight subtype-specific patterns of genomic instability. The association between high CNV burden and the MIB-1 proliferation index may help to predict adenoma behavior. Genomic profile using a gene panel has the potential to better classify PA and contribute to clinical management. Presentation: Saturday, June 17, 2023

The clinicopathological features and prognosis of silent corticotroph tumors: an updated systematic review and meta-analysis.

Data on silent corticotroph tumor (SCT) are still heterogeneous and controversial. In this study, we aimed to compare the demographic, clinicopathological manifestations, postoperative complications, and patient outcomes of SCTs with other non-functioning pituitary neuroendocrine tumor (NFT) and functioning corticotroph tumor (FCT) or so-called Cushing disease adenoma. We searched PubMed and Web of Science for data of interest. Odds ratio (OR), mean difference (MD), hazard ratio (HR), and their 95% confidence intervals (CI) were pooled using the random-effect model. Twenty-nine studies with 985 SCTs were included in meta-analyses. In comparison to other NFTs, SCTs were more commonly associated with female gender, younger age, cavernous sinus invasion, apoplexy, and radiotherapy administration. Postoperatively, SCT patients were more likely to experience hypocortisolism, new-onset visual disturbances, and a higher risk for tumor progression than other NFTs. We did not find any significant differences between SCT type I and type II. Compared to FCTs, SCTs were more likely male, older age, and had larger tumor sizes. The prevalence of a USP8 mutation was significantly higher in FCT than in SCT. SCT was demographically, clinicopathologically, and prognostically distinct from other NFTs and FCTs. These tumors should be considered high-risk; appropriate treatment decisions and more stringent follow-up should be tailored to improve patient outcomes.

Feasibility analysis of ACTH adenoma model in USP8-/- mice.

Patients with adrenocorticotropic hormone (ACTH)-secreting pituitary tumours (35% to 60%) present with somatic mutations in the USP8 gene. USP8 mutations lead to enhanced deubiquitination of the epidermal growth factor receptor (EGFR) and result in an imbalance in EGFR signalling, accompanied by excessive activation of ACTH production and cell growth. USP8 emerged as a novel and exciting candidate gene for Cushing's disease. In this study, USP8 mutant mouse models (USP8+/- and USP8-/-) were established, their phenotypes were analysed and identified, biochemical indexes were detected, pituitary and adrenal tissue specimens were taken for HE staining and immunohistochemical identification of hormones, and the differences between the 2 groups of mutant mice and wild type mice were analysed and compared. Compared with the control group (wild type), immunofluorescence assay results for USP8+/- mice and USP8-/- mice showed increased pituitary ACTH expression, which was statistically different (p < 0.05), and there were no significant differences in body weight, plasma ACTH, 24-hour urinary free cortisol, and immunohistochemical results. Higher blood glucose in USP8-/- mice than in USP8+/+ mice was observed. The heart rates of USP8-/- mice were higher than those of USP8+/- mice and USP8+/+ mice. HE staining and tissue fibre staining were done, and no significant pathological changes were seen in the 3 groups of pituitary and adrenal tissues. USP8 knockout mice have the potential to form an animal model of ACTH adenoma.

The expression of glucocorticoid and mineralocorticoid receptors in pituitary tumors causing Cushing's disease and silent corticotroph tumors.

Pituitary neuroendocrine corticotroph tumors commonly cause Cushing's disease (CD) that results from increased adrenocorticotropic hormone (ACTH) secretion by the pituitary tumor and consequent increase of cortisol levels in blood. However, in some patients, corticotroph tumors remain clinically non-functioning. Cortisol secretion is regulated by the hypothalamic-pituitary-adrenal axis and includes a negative feedback between cortisol and ACTH secretion. Glucocorticoids reduce ACTH level both by hypothalamic regulation and acting on corticotrophs via glucocorticoid (GR) and mineralocorticoid (MR) receptors. The aim of the study was to determine the role of GR and MR expression at mRNA and protein levels in both functioning and silent corticotroph tumors. Ninety-five patients were enrolled, including 70 with CD and 25 with silent corticotroph tumors. Gene expression levels of NR3C1 and NR3C2 coding for GR and MR, respectively, were determined with qRT-PCR in the two tumor types. GR and MR protein abundance was assessed with immunohistochemistry. Both GR and MR were expressed in corticotroph tumors. Correlation between NR3C1 and NR3C2 expression levels was observed. NR3C1 expression was higher in silent than in functioning tumors. In CD patients NR3C1 and NR3C2 levels were negatively correlated with morning plasma ACTH levels and tumor size. Higher NR3C2 was confirmed in patients with remission after surgery and in densely granulated tumors. Expression of both genes and GR protein was higher in USP8-mutated tumors. Similar relationship between USP8 mutations and expression levels were observed in analysis of silent tumors that also revealed a negative correlation between GR and tumor size and higher NR3C1 expression in densely granulated tumors. Although the associations between gene/protein expression and patients clinical features are not strong, they consistently show an evident trend in which higher receptor expression corresponds to more favorable clinical characteristics.

The hallmarks of cancer… in pituitary tumors?

Over 20years ago, Hanahan and Weinberg published a seminal review that addressed the biological processes that underly malignant transformation. This classical review, along with two revisions published in 2011 and 2022, has remain a classic of the oncology literature. Since many of the addressed biological processes may apply to non-malignant tumorigenesis, we evaluated to what extent these hallmarks pertain to the development of pituitary adenomas.Some of the biological processes analyzed in this review include genome instability generated by somatic USP8 and GNAS mutations in Cushing's diseases and acromegaly respectively; non-mutational epigenetic reprograming through changes in methylation; induction of angiogenesis through alterations of VEGF gene expression; promotion of proliferative signals mediated by EGFR; evasion of growth suppression by disrupting cyclin dependent kinase inhibitors; avoidance of immune destruction; and the promotion of inflammation mediated by alteration of gene expression of immune check points. We also elaborate further on the existence of oncogene induced senescence in pituitary tumors. We conclude that a better understanding of these processes can help us dilucidated why pituitary tumors are so resistant to malignant transformation and can potentially contribute to the development of novel anticancer treatments.

The Expression of Cell Cycle-Related Genes in USP8-Mutated Corticotroph Neuroendocrine Pituitary Tumors and Their Possible Role in Cell Cycle-Targeting Treatment.

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes' expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

RF01 | PMON168 Hedgehog-Induced Somatostatin Receptor Expression is Inhibited by the Activation of the Glucocorticoid Receptor Signaling Pathway in Human Pituitary Adenoma Organoids

Abstract Background Cushing's disease (CD) is an endocrine disorder caused by an ACTH-secreting pituitary adenoma that is associated with increased morbidity and mortality. Because somatostatin inhibits pituitary ACTH secretion, the somatostatin receptor subtypes (SSTR) 2 and 5 have been targets of medical therapies for CD. The SSTR5 is most consistently and strongly expressed in corticotroph pituitary tumors but the SSTR dynamics in CD during treatment and responses to cortisol level changes are far from being understood. Studies in adult stomach demonstrated that the Hedgehog (Hh) signaling pathway is critical for the regulation of somatostatin and SSTR signaling. While Hh signaling is known to be essential during the embryonic development of the pituitary and in the adult gland, the mechanism by which Hh signaling regulates SSTR expression in CD is unknown. Hypothesis Activation of glucocorticoid receptor (NR3C1, GR) results in the inhibition of Hh transcription factor GLI1 leading to reduced SSTR expression. Methods We developed a human pituitary adenoma organoid model (hPITOiPSC) from induced pluripotent stem cells treated with the glucocorticoid receptor (GR) antagonist mifepristone, and co-treated them with or without SSTR agonists pasireotide or octreotide. In a separate series of experiments, the role of Hh transcription factor GLI1 was identified using hPITOiPSC treated with mifepristone with or without GANT61 (GLI inhibitor) or ketoconazole (Smoothened, SMO inhibitor). CRISPR-Cas9 gene editing of hPITOiPSC pituitary organoids were used to model the development of pituitary corticotroph adenomas in the presence of BRAF, USP48 and USP8 mutations. Human pituitary adenoma tissue harvested fresh during pituitary surgery was used to generate pituitary corticotroph subtype adenoma organoids (hPITOs). Dose responses using standard of care drugs were performed using the hPITOs. Results 1) At baseline SSTR2 was abundantly expressed within hPITOiPSC. Mifepristone induced significant increases in ACTH secretion and POMC expression that correlated with induced SSTR2 and 5. Mifepristone-induced SSTR2 and 5 expression was significantly inhibited in the presence of GANT61, whereas SMO inhibitor ketoconazole had a minimal effect on mifepristone-induced SSTR2/5, POMC expression and ACTH secretion. Dexamethasone alone significantly inhibited both GLI1 and SSTR2 and 5. 2) While pituitary organoids that were differentiated from control iPSCs (iPSCCtrl) expressed all major hormone-producing cell lineages, there was a significant increase in ACTH expression with loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing mutated BRAF, USP48 and USP8. Organoids expressing a mutation in BRAF had significantly higher SSTR2 expression levels compared to controls. 3) HPITOs generated from pituitary adenomas of CD patients showed differential organoid responses to pasireotide, mifepristone, and ketoconazole. Conclusion Within pituitary adenomas, the SSTR is a transcriptional target of Gli1, and this response is blocked by the activation of the GR. These data form the basis of combination therapy for CD with mifepristone and pasireotide. Presentation: Saturday, June 11, 2022 1:37 p.m. - 1:42 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Concurrent mutations of germline GPR101 and somatic USP8 in a pediatric giant pituitary ACTH adenoma: a case report

BackgroundCushing’s disease (CD) is rare in pediatric patients. It is characterized by elevated plasma adrenocorticotropic hormone (ACTH) from pituitary adenomas, with damage to multiple systems and development. In recent years, genetic studies have shed light on the etiology and several mutations have been identified in patients with CD.Case presentationA girl presented at the age of 10 years and 9 months with facial plethora, hirsutism and acne. Her vision and eye movements were impaired. A quick weight gain and slow growth were also observed. Physical examination revealed central obesity, moon face, buffalo hump, supra-clavicular fat pads and bruising. Her plasma ACTH level ranged between 118 and 151 pg/ml, and sella enhanced MRI showed a giant pituitary tumor of 51.8 × 29.3 × 14.0 mm. Transsphenoidal pituitary debulk adenomectomy was performed and immunohistochemical staining confirmed an ACTH-secreting adenoma. Genetic analysis identified a novel germline GPR101 (p.G169R) and a somatic USP8 (p. S719del) mutation. They were hypothesized to impact tumor growth and function, respectively.ConclusionsWe reported a rare case of pediatric giant pituitary ACTH adenoma and pointed out that unusual concurrent mutations might contribute to its early onset and large volume.

Impact of USP8 mutations on corticotroph tumor cells responsiveness to pasireotide

Impact of USP8 mutations on corticotroph tumor cells responsiveness to pasireotide

Transcription Factor ASCL1 Acts as a Novel Potential Therapeutic Target for the Treatment of the Cushing's Disease.

The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. In this multiomics study, we combined RNA sequencing (RNA-seq) with Sanger sequencing to depict transcriptional dysregulation under different gene mutation backgrounds. Furthermore, we evaluated the potential of achaete-scute complex homolog 1 (ASCL1), a pioneer transcription factor, as a novel therapeutic target for treatment of CD and its possible downstream pathway. RNA-seq was adopted to investigate the gene expression profile of CD, and Sanger sequencing was adopted to detect gene mutations. Bioinformatics analysis was used to depict transcriptional dysregulation under different gene mutation backgrounds. The function of ASCL1 in hormone secretion, cell proliferation, and apoptosis were studied in vitro. The effectiveness of an ASCL1 inhibitor was evaluated in primary CD cells, and the clinical relevance of ASCL1 was examined in 68 patients with CD. RNA-seq in AtT-20 cells on Ascl1 knockdown combined with published chromatin immunoprecipitation sequencing data and dual luciferase assays were used to explore downstream pathways. ASCL1 was exclusively overexpressed in USP8-mutant and wild-type tumors. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in AtT-20 cells. An ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. Our findings help to clarify the pathogenesis of CD and suggest that ASCL1 is a potential therapeutic target the treatment of CD. The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. Moreover, few effective medical therapies are currently available for the treatment of CD. Here, using a multiomics approach, we first report the aberrant overexpression of the transcription factor gene ASCL1 in USP8-mutant and wild-type tumors of CD. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in mouse AtT-20 cells. Notably, an ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. Importantly, ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. Thus, our findings improve understanding of CD pathogenesis and suggest that ASCL1 is a potential therapeutic target the treatment of CD.

Responsiveness to DDAVP in Cushing's disease is associated with USP8 mutations through enhancing AVPR1B promoter activity.

To clarify the characteristics of Cushing's disease (CD) patients who respond to the desmopressin (DDAVP) test and its underlying mechanisms. Forty-seven patients with CD who underwent DDAVP testing were included. Patients were divided into two groups: DDAVP test (+) (adrenocorticotropic hormone [ACTH] levels increased by ≥ 1.5-fold during the DDAVP test) and DDAVP test (-) (ACTH levels increased by < 1.5-fold). AVP receptor expression levels in these tumors were quantified using quantitative RT-PCR and immunohistochemistry. AVP receptor promoter activity was analyzed using a dual-luciferase reporter assay system. Females (96.9%) and USP8 mutants (85.7%) were more prevalent in the DDAVP test (+) than in the DDAVP test (-). Indeed, the ACTH and cortisol responsiveness to DDAVP was greater in USP8 mutation positive tumors than that in USP8 wild type tumors (3.0-fold vs. 1.3-fold, 1.6-fold vs. 1.1-fold, respectively). Responsiveness to DDAVP was correlated with the expression levels of AVPR1B, but not with those of AVPR2. Comparably, Avpr1b promoter activity was enhanced by the overexpression of mutant USP8 compared to the wild type. We found that the responsiveness of ACTH to DDAVP in CD was greater in tumors with USP8 mutations. The present data suggest that USP8 mutations upregulate the AVPR1B promoter activity. Additionally, we showed that the DDAVP test can predict the presence of USP8 mutations.

Molecular mechanisms involved in somatostatin receptor regulation in corticotroph tumors: the role of cytoskeleton and USP8 mutations.

Adrenocorticotropic hormone (ACTH)-secreting pituitary tumors mainly express somatostatin receptor 5 (SSTR5) since SSTR2 is downregulated by the elevated levels of glucocorticoids that characterize patients with Cushing's disease (CD). SSTR5 is the molecular target of pasireotide, the only approved pituitary tumor-targeted drug for the treatment of CD. However, the molecular mechanisms that regulate SSTR5 are still poorly investigated. This review summarizes the experimental evidence supporting the role of the cytoskeleton actin-binding protein filamin A (FLNA) in the regulation of SSTR5 expression and signal transduction in corticotroph tumors. Moreover, the correlations between the presence of somatic USP8 mutations and the expression of SSTR5 will be reviewed. An involvement of glucocorticoid-mediated β-arrestins modulation in regulating SSTRs expression and function in ACTH-secreting tumors will also be discussed.

USP8, USP48, and BRAF mutations differ in their genotype-phenotype correlation in Asian Indian patients with Cushing's disease.

To estimate the prevalence of USP8, USP48, and BRAF mutations in patients with Cushing's disease (CD) from the Indian subcontinent, and determine their genotype-phenotype correlation. We prospectively recruited 46 patients with CD who underwent surgery between September 2015 and July 2019 at our institute. Fresh frozen tumour tissue was obtained in all patients. Using Sanger sequencing, the presence of somatic USP8 mutations was documented and the frequency of USP48 and BRAF mutations in USP8 wild-type corticotroph adenomas was determined. Clinical, hormonal, and surgical data were then compared between USP8-, USP48- and BRAF-variant carriers and patients with wild-type tumours. Signature USP8 mutations were detected in 17 (37%) patients. Of the 29 USP8 wild-type adenomas, 4 (13.8%) harboured USP48 mutations, one of them being a splice-site mutation that has previously not been described. BRAF mutations were not found in any of the 29 patients. Corticotroph adenomas with USP8 mutations had a higher incidence of Crooke's hyaline change than wild-type tumours (70.6 vs. 37.9%, p = 0.032). Adenomas with USP48 mutations had a higher rate of cavernous sinus invasion than their wild-type counterparts (50 vs. 4%, p = 0.042). No other significant phenotypic difference could be established between mutant and wild-type tumours. The prevalence of USP8 mutations in our series of patients with CD was 37%. The prevalence of USP48 mutations in USP8 wild-type adenomas was 13.8%, including a novel splice-site mutation. BRAF mutations were not found in any USP8 wild-type tumour. USP8-mutants showed significantly more Crooke's hyaline change and USP48-mutants were more likely to demonstrate cavernous sinus invasion.

Consecutive adrenal cushing´s syndrome and cushing´s disease in a patient with somatic CTNNB1, USP8, and NR3c1 mutations

Consecutive adrenal cushing´s syndrome and cushing´s disease in a patient with somatic <i>CTNNB1, USP8, and NR3c1</i> mutations

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant.

Simple SummaryCushing’s Disease (CD) is a rare but severe endocrine disorder due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, and pathogenetics remained a puzzling issue for a long time. The recent identification of somatic mutations in the 14-3-3 protein binding motif of ubiquitin specific peptidase 8 gene (USP8), present in a consistent subgroup of ACTH-secreting pituitary tumors, have represented a major advance in the understanding of CD pathogenesis. In our cohort of 60 patients we found an incidence of 11.7% of USP8 recurrent somatic mutations whereas a novel USP8 variant (G664R) located upstream the canonical USP8 mutational hotspot was identified in one case. This alteration has never been reported by previous records. The present study provides USP8 G664R variant in vitro functional characterization in AtT-20 cells and demonstrates its possible implication in ACTH-secreting tumor pathogenesis, contributing to enlarge the genetic landscape of CD.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 USP8 and USP48, and in BRAF 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 USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 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, p < 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, p < 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, p < 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 USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 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.

Consecutive development of adrenal Cushing´s syndrome and Cushing´s disease in a female patient with somatic CTNNB1, USP8, and NR3C1 mutations

Consecutive development of adrenal Cushing´s syndrome and Cushing´s disease in a female patient with somatic CTNNB1, USP8, and NR3C1 mutations