Sexual Dimorphism in Cellular and Molecular Features in Human ACTH-Secreting Pituitary Adenomas.

Background. Cushing\u2019s disease presents gender disparities in prevalence and clinical course. Little is known, however, about sexual dimorphism at the level of the corticotrope adenoma itself. The aim of the present study was to evaluate molecular features of ACTH-secreting pituitary adenomas collected from female and male patients with Cushing\u2019s disease. (2) Methods. We analyzed 153 ACTH-secreting adenomas collected from 31 men and 122 women. Adenomas were established in culture and ACTH synthesis and secretion assessed in basal conditions as well as during incubation with CRH or dexamethasone. Concurrently, microarray analysis was performed on formalin-fixed specimens and differences in the expression profiles between specimens from male and female patients identified. (3) Results. ACTH medium concentrations in adenomas obtained from male patients were significantly lower than those observed in adenomas from female patients. This could be observed for baseline as well as modulated secretion. Analysis of corticotrope transcriptomes revealed considerable similarities with few, selected differences in functional annotations. Differentially expressed genes comprised genes with known sexual dimorphism, genes involved in tumour development and genes relevant to pituitary pathophysiology. (4) Conclusions. Our study shows for the first time that human corticotrope adenomas present sexual dimorphism and underlines the need for a gender-dependent analysis of these tumours. Differentially expressed genes may represent the basis for gender-tailored target therapy

Role of the ubiquitin/proteasome system on ACTH turnover in rat corticotropes

Purpose: A large number of studies has investigated proopiomelanocortin processing in anterior pituitary corticotropes but little is known on proopiomelanocortin/ACTH degradation within these cells. The ubiquitin-proteasome system is an intracellular protein degradation pathway which has garnered considerable interest in recent times, given its role in maintenance of protein homeostasis. Aim of the present study was to evaluate the role of the ubiquitin-proteasome system in proopiomelanocortin/ACTH turnover in pituitary corticotropes. Methods: Rat anterior pituitary primary cultures were treated with 0.01\u2013100 nM MG132, a proteasome inhibitor, or 0.1\u2013100 nM K48R, an inhibitor of polyubiquitylation, for 4 and 24 h and ACTH concentrations in medium and cell lysates estimated by immunometric assay. Co-immunoprecipitation for ubiquitin and ACTH was carried out to establish ubiquitin-tagged protein products. Results: Inhibition of proteasome-mediated degradation with MG132 lead to an increase in ACTH concentrations, both as regards secretion and cell content. Likewise, inhibition of polyubiquitylation was associated with increased ACTH secretion and cell content. Ubiquitin/ACTH co-immunoprecipitation revealed that proopiomelanocortin was a target of ubiquitylation. Conclusions: We provide the first evidence that the ubiquitin-proteasome system is involved in proopiomelanocortin/ACTH degradation in corticotropes. Indeed, proopiomelanocortin is a target of ubiquitylation and modulation of ubiquitin-proteasome system affects ACTH turnover. This study shows that regulation of ACTH proteolytic degradation may represent a means to control ACTH secretion

Gene expression profiling in human corticotrope tumors reveals distinct, neuroendocrine profiles

ACTH-secreting pituitary adenomas give rise to a severe endocrinological disorder, i.e., Cushing's disease, with multifaceted clinical presentation and treatment outcomes. Experimental studies suggested that disease variability is inherent to the pituitary tumor, thus pointing to the need for further studies into tumor biology. Aim of the present study was to evaluate transcriptome expression pattern in a large series of ACTH-secreting pituitary adenoma specimens, in order to identify molecular signatures of these tumors. Gene expression profiling of formalin-fixed paraffin-embedded specimens from 40 human ACTH-secreting pituitary adenomas revealed significant expression of genes involved in protein biosynthesis and ribosomal function, in keeping with neuroendocrine cell profile. Unsupervised cluster analysis identified three distinct gene profile clusters and several genes were uniquely overexpressed in a given cluster, accounting for different molecular signatures. Of note, gene expression profiles were associated with clinical features such as age and size of the tumor. Altogether, our study shows that corticotrope tumors are characterized by neuroendocrine gene expression profile and present subgroup-specific molecular features

Ubiquitin-Specific Protease 8 Mutant Corticotrope Adenomas Present Unique Secretory and Molecular Features and Shed Light on the Role of Ubiquitylation on ACTH Processing

Background: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. Objectives: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. Methods: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. Results: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. Conclusions: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more "typical" corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas

Effect of retinoic acid on human adrenal corticosteroid synthesis

Aims Retinoic acid has recently yielded promising results in the treatment of Cushing's disease, i.e., excess cortisol secretion due to a pituitary corticotropin (ACTH)-secreting adenoma. In addition to its effect on the tumoral corticotrope cell, clinical results suggest an additional adrenal site of action. Aim of this study was to evaluate whether retinoic acid modulates cortisol synthesis and secretion by human adrenals in vitro. Main methods Primary cultures from 10 human adrenals specimens were incubated with 10 nM, 100 nM and 1 \u3bcM retinoic acid with and without 10 nM ACTH for 24 h. Cortisol levels were measured by radioimmunoassay and CYP11A1, STAR and MC2R gene expression analyzed by real-time PCR. Key findings Retinoic acid increased cortisol secretion (149.5 \ub1 33.01%, 151.3 \ub1 49.45% and 129.3 \ub1 8.32% control secretion for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05) and potentiated STAR expression (1.51 \ub1 0.22, 1.56 \ub1 0.15 and 1.59 \ub1 0.14 fold change over baseline, for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05). Concurrently, retinoic acid markedly blunted constitutional and ACTH-induced MC2R expression (0.66 \ub1 0.11, 0.62 \ub1 0.08 and 0.53 \ub1 0.07 fold change over baseline, for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05; 0.71 \ub1 0.10, 0.51 \ub1 0.07 and 0.51 \ub1 0.08 fold change over ACTH alone, for 10 nM, 100 nM and 1 \u3bcM respectively, p < 0.05). No effect on CYP11A1 was observed. Significance Retinoic acid stimulates cortisol synthesis and secretion in human adrenals and at the same time markedly blunts ACTH receptor transcription. These results reveal a novel, adrenal effect of retinoic acid which may contribute to its efficacy in patients with Cushing's disease

Benzene and 2-ethyl-phthalate induce proliferation in normal rat pituitary cells

Purpose: Endocrine disruptors are known to modulate a variety of endocrine functions and increase the risk for neoplasia. Epidemiological data reported increased prevalence of pituitary tumors in high industrial areas while genotyping studies showed that mutations in the aryl hydrocarbon receptor (AhR) interacting protein (AIP)\u2014chaperone to the dioxin ligand AhR\u2014gene are linked to predisposition to pituitary tumor development. Aim of the present study was to establish whether endocrine pollutants can induce cell proliferation in normal rat pituitary cells. Methods: Pituitary primary cultures were incubated with 250, 650 and 1250 pM benzene or 2-ethyl-phthalate for up to 96 h and viability, energy content and cell proliferation assessed. Expression of pituitary tumor transforming gene (PTTG), cyclin D1 (Ccnd1), AhR and AIP was quantified by RT-qPCR. Results: Incubation with benzene or 2-ethyl-phthalate increased viability and energy content in pituitary cells. The endocrine disruptors also increased cell proliferation as well as Ccnd1 and PTTG expression. Increased AhR and AIP expression was observed after incubation with the two pollutants. Conclusions: Our findings indicate that benzene and 2-ethyl-phthalate activate AhR/AIP expression and stimulate proliferation in normal rat pituitary cells. This study is the first demonstration that pollutants can induce normal pituitary cells to proliferate and provides a link between epidemiological and genomic findings in pituitary tumors

Responses to corticotrophin-releasing hormone and dexamethasone in a large series of human adrenocorticotrophic hormone-secreting pituitary adenomas in vitro reveal manifold corticotroph tumoural phenotypes

Patients with Cushing's disease are known to present a variable secretory response to stimulatory and inhibitory challenges. Evaluation of the secretory behaviour of pituitary adrenocorticotrophic hormone (ACTH)-secreting adenomas in vitro aids in the comprehension of its behaviour in vivo; however, given the small size of these tumours and the consequent paucity of material available to in vitro studies, a comprehensive study on the secretory behaviour of human corticotroph tumours has not yet been performed. The present study aimed to assess the spectrum of responses to the two main corticotroph modulators, corticotrophin-releasing hormone (CRH) and dexamethasone, in a large series of human ACTH-secreting pituitary tumours. Seventy-two ACTH-secreting pituitary tumours were collected during surgery and established in culture. Specimens were incubated with 10 nm CRH and/or 10 nm dexamethasone for 4 h and 24 h. Secretion in unstimulated, control wells was set at 100% and changes in ACTH concentrations by at least 20% were considered as responses. Parallel experiments in 12 rat anterior pituitary primary cultures were evaluated. A marked ACTH increase was observed during incubation with CRH in 70% of tumoural specimens at 4 h (range 124-3500% of control wells) and in 57% at 24 h (range 122-3323%). Dexamethasone reduced ACTH secretion in almost 50% of tumours (range 78-2% of control at 4 h; 76-3% at 24 h), whereas it did not affect ACTH medium levels in 30% of specimens and induced a paradoxical ACTH increase in 20% of tumours (range 130-327% of control at 4 h; 156-348% at 24 h). By comparison, CRH uniformly increased ACTH levels in rat anterior pituitary primary cultures (mean 745 \ub1 84% at 4 h; 347 \ub1 25% at 24 h), whereas dexamethasone decreased ACTH levels by 40-50% in all experiments. In conclusion, the present study of a large series of human ACTH-secreting pituitary tumours in vitro revealed a considerable variability in the responses to CRH and dexamethasone. This finding indicates the existence of multiple corticotroph tumoural phenotypes and may account for the different responses to physiological and pharmacological modulators in vivo

Clinical characteristics and surgical outcome in USP8-mutated human adrenocorticotropic hormone-secreting pituitary adenomas

Purpose: somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been described in patients with Cushing\u2019s disease (CD). The aim of the study is to verify whether USP8 mutation may predict early and late outcome of pituitary surgery in patients with CD operated at a single institution. Methods: We performed a retrospective genetic analysis of 92 adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Specimens were screened for USP8 hotspot mutations in the exon 14 with Sanger sequencing. Hormonal and surgical data were compared between USP8 variant carriers and wild-type tumors. Results: USP8 variants were detected in 22 adenomas (23.9%) with higher prevalence in women (28.9% vs. 5.3% in men; p < 0.05). No significant difference in hormonal levels and tumoral features in relation to USP8 status was observed. Interestingly, USP8-variant carriers were more likely to achieve surgical remission than wild-type adenomas (100% vs. 75.7%; p = 0.01). Conversely, recurrence of CD occurred in 23% of USP8-mutated patients and in 13% of patients with wild-type adenoma. The recurrence-free survival did not differ significantly between the two groups (p = 0.42). Conclusions: ACTH-secreting pituitary adenomas carrying somatic USP8 mutations are associated with a greater likelihood of surgical remission in patients operated by a single neurosurgeon. Recurrence rates are not related with USP8-variant status

Effect of SOM230 on human and rat adrenal secretion in vitro

SOM230, a recently developed somatostatin analogue, has been tested in patients with Cushing's disease with promising results. Experimental evidence indicates a pituitary site of action but no data is yet available on any direct adrenal effect on cortisol secretion. This aspect of the analogue's action appears worth investigating as some patients with Cushing's syndrome have been known to experience adrenal insufficiency soon after initiation of SOM230 treatment. Aim of the current study was to evaluate whether SOM230 modulates corticosteroid secretion by normal adrenals in vitro. Methods: Primary cultures from 6 normal human adrenals and 6 rat adrenals were incubated with 10-100 nM SOM230 with and without 10 nM ACTH. Cortisol/corticosterone levels in medium were measured after 4 and 24 hours. Results: 10 nM SOM230 significantly increased corticosteroid levels after 24h incubation in both human (131.4 \ub1 1.55% of baseline, p<0.05) and rat (138.1 \ub1 15.99%, p<0.05) adrenals; lesser effects were observed with 100 nM SOM (120.4 \ub1 9.33% p<0.05; 117.3 \ub1 15.49% N.S., for human and rat adrenals, respectively). The corticosteroid secretory response to ACTH was unaffected by SOM230 co-incubation (at 24h human adrenals: cortisol 105.6 \ub1 7.16% and 108.4 \ub1 6.99% of ACTH-stimulated wells for 10 nM and 100 nM SOM230, NS, respectively; rat adrenals: corticosterone 98.3 \ub1 12.11 and 91.8 \ub1 12.55% of ACTH-stimulated wells for 10 nM and 100 nM SOM230, respectively, NS.) Conclusions: SOM230 exerted an unexpected, moderate stimulatory effect on adrenal corticosteroid secretion in vitro. This argues against an additional, direct adrenal inhibitory effect of SOM230 in patients with Cushing's syndrome

AZA-Deoxycytidine stimulates proopiomelanocortin gene expression and ACTH secretion in human pituitary ACTH-secreting tumors

It is well known that methylation plays an important role in regulating tissue expression of proopiomelanocortin (POMC) and recent studies have shown that demethylation can occur also in vitro in neuroendocrine tumors. Aim of the present study was to evaluate whether inhibition of methylation modulates POMC expression and ACTH secretion by human corticotrope tumors. Twenty two ACTH-secreting pituitary tumors were incubated with 5-AZA-2'-deoxycytidine (AZA), an inhibitor of DNA-methyltransferases, with or without 10 nM corticotropin-releasing hormone (CRH). Both dose response (100 nM-10 mu M AZA) and time course (4-96 h) experiments were carried out for measurement of ACTH secretion and POMC gene expression. Incubation with AZA increased constitutive POMC expression and ACTH secretion by human corticotrope adenomas. The effect appeared most notable at 24 and 48 h with 1 mu M AZA. Incubation with AZA did not exert an additional stimulatory effect on CRH-stimulated POMC and ACTH. The present study shows that AZA increases POMC gene expression and ACTH secretion in human pituitary ACTH-secreting tumors. This can be taken to indicate that mechanisms set into motion by AZA play a role in the regulation of ACTH secretion/POMC expression in tumoral corticotropes and paves the way to further studies in Cushing's disease