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Pituitary adenomas constitute about 15% of intracranial tumors, and about one-third of secretory pituitary adenomas produce ACTH. Corticotroph adenomas, a subset of pituitary adenomas staining positive for ACTH, are further categorized into functional (FCA) and silent (SCA) adenomas. FCAs result in central Cushing’s disease (CD) due to the resulting excess of cortisol secretion stimulated by ACTH secretion through hormone disruption while SCAs exhibit mass effects and show increased aggression as compared to its functional counterpart. Obesity and cardiovascular disease, resulting from hypercortisolism in functional adenomas, increase patient morbidity while the invasive nature of silent adenomas increases mortality. Trans sphenoidal surgery (TSS) is the best available treatment option for cotricotroph adenomas, but tumor recurrence is common. We sought to identify differential genetic drivers of sporadic FCAs and SCAs in order to better characterize these tumors and develop novel treatment options. We examined 17 adenomas including 12 FCA and 5 SCA as well as 2 corticotroph hyperplasia (CH) tissue samples. We performed next generation sequencing using OncoPanel versions 2 and 3 on patients operated on at Brigham and Woman’s Hospital between 2008 and 2018 and determined to have a corticotroph adenoma. 3 of 4 FCA patients screened for USP8 mutations contained variants previously described in CD including USP8S718P and USP8S719del. 3 of the 12 FCA patients screened for mutations in ARID1B contained novel variants and 1 patient contained a variant previously described in large intestine adenocarcinomas. Additionally, SNPs were commonly identified in genes responsible for epigenetic regulation implicating histone modification as a therapeutic target. We identified recurrent copy number variants (CNV) in both FCAs and SCAs. Gains of 6p, 20q and 21q were frequently observed in FCAs alongside less common losses in 11p and 19q. Significant amplifications of chromosome 12 were detected in SCAs with single nucleotide deletions in chromosome 10. Furthermore, we report diverging genomic disruption between subtypes associated with functional hormone status. Our data identifies novel genetic drivers in subclasses of corticotroph adenomas and indicate distinct genomic profiles associated with hormone secretion and clinical presentation. Further research is required to better elucidate the role of these genetic variants and how they influence tumorigenesis and hormone production.

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which controls the neuroendocrine response to stress. Following stressful stimuli, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus act synergistically to stimulate adrenocortiotrophin hormone (ACTH) secretion from corticotroph cells. ACTH is released into the circulation where it stimulates the secretion of glucocorticoids from the adrenal cortex. The HPA axis is kept in fine balance through an elegant negative feedback system where elevation of plasma glucocorticoids results in inhibition at the level of both the pituitary and the hypothalamus. During acute stress, glucocorticoids can be beneficial however chronic elevation of glucocorticoids can have many adverse effects on health. Corticotroph cells are electrically excitable and have been shown to fire single-spike action potentials as well as complex bursting patterns. Stimulation of corticotrophs with physiological concentrations of CRH/AVP results in a robust increase in firing frequency and a transition from spiking to bursting. Intracellular Ca2+ increases to a greater extent during bursting which has been proposed to drive hormone secretion. There is evidence to suggest that large conductance calcium- and voltage-gated potassium (BK) channels promote bursting behaviour in anterior pituitary cells. Glucocorticoids have been shown to regulate ACTH secretion and also modulate BK channel activity. However, the effects of glucocorticoids on native corticotroph excitability are currently unknown. The aim of this study was to first characterise the electrical properties of corticotrophs under basal conditions and following exposure to CRH/AVP. Secondly, to investigate the regulation of corticotroph excitability by glucocorticoids. Finally, establish the role of the BK channel in regulating bursting behaviour and CORT regulation in corticotroph cells. Corticotroph cells were acutely isolated by trypsin digestion from mice aged 2-5 months constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Mice used for pituitary cell culture were male unless otherwise stated. Cells were maintained in a serum free media and electrophysiological recordings obtained 24-96 hours post-isolation. Current clamp recordings were obtained from corticotrophs using the perforated patch technique. Although spontaneous activity of corticotroph cells was variable, they displayed predominantly single-spike action potentials under basal conditions. Stimulation with physiological concentrations of CRH and AVP (0.2 nM and 2 nM respectively) resulted in a membrane depolarisation accompanied by an increase in firing frequency and a transition to bursting. Individually, CRH and AVP were able to increase corticotroph excitability. However, only CRH was able to drive an increase in bursting suggesting that bursting is primarily regulated through the cAMP/PKA pathway. Experiments were performed to investigate the modulation of corticotroph activity by glucocorticoid negative feedback. Acute exposure (< 10 min) to corticosterone resulted in a decrease in spontaneous activity as well as shortening the response to CRH/AVP. Pretreatment of corticotrophs with 100 nM corticosterone (90 min) resulted in a membrane hyperpolarisation and a decrease in spontaneous firing frequency. Following corticosterone pretreatment, CRH/AVP failed to induce a significant transition from spiking to bursting. Increasing the pretreatment time to 150 minutes resulted in a further suppression of both spontaneous and CRH/AVPevoked activity. Fast activation of BK channels during the upstroke of an action potential has been proposed to promote bursting behaviour in other pituitary cells. Corticotrophs treated with a BK channel blocker (1 μM paxilline) or isolated from BK-/- mice showed no significant difference in basal activity but displayed a reduction in CRH/AVPevoked bursting activity. In both cases, bursting was significantly reduced but not completely abolished. Corticosterone treatment of BK-/- cells resulted in a further decrease in both firing frequency and bursting behaviour. Taken together, these results suggest that although BK channels play an important role in bursting, they are not the only component. Comparisons of male and female corticotrophs revealed subtle differences in their properties. Following CRH/AVP stimulation, male cells displayed a high degree of bursting activity whereas female cells exhibited predominantly an increase in singlespike action potential frequency. Treatment of female corticotrophs with corticosterone (150 min) resulted in a significant reduction in firing frequency but no measurable change in bursting behaviour. BK-/- cells from female mice showed no difference in bursting activity following CRH/AVP compared to wild types. This data suggests that modulation of firing frequency is the more important component in female corticotroph cells. In conclusion, CRH/AVP is proposed to drive ACTH secretion in male corticotroph cells through an increase in bursting activity. Corticosterone pretreatment suppresses both spontaneous and CRH/AVP-evoked activity. It is possible that corticosterone regulates corticotroph excitability through two mechanisms. Corticosterone suppresses bursting activity following CRH/AVP stimulation through multiple targets which might include the BK channel. Additionally, corticosterone reduces firing frequency through a mechanism independent of BK channels. It is important to further characterise the physiology of corticotroph cells and how ACTH secretion is regulated through their electrical excitability. This would lead to a greater understanding of the role of corticotrophs in the HPA axis. Further study of corticotrophs could potentially lead to pharmacological manipulation of the stress response and novel treatments for stress-related disorders.

La maladie de Cushing est caractérisée par une sécrétion excessive de l’hormone adrénocorticotrope (ACTH) à partir des tumeur corticotropes de l'hypophyse. Un excès d'ACTH entraîne un hypercortisolisme et provoque des symptômes tels que diabète, hypertension, obésité et les maladies cardiovasculaires entraînant un risque accru de mortalité si la maladie n’est pas traitée. Les tumeurs corticotropes sont caractérisées par la perte du rétro-contrôle négatif exercé par les glucocorticoïdes (GCs) sur la proopiomélanocortine (POMC) qui est le précurseur de l’ACTH : c'est la caractéristique majeure de la maladie de Cushing. Les causes de la résistance aux GC dans les adénomes corticotropes sont encore mal connues. Des études récentes ont montré une surexpression du récepteur du facteur de croissance épidermique (EGFR) dans les adénomes corticotropes provoquant une augmentation de l'activité du gène POMC et de la sécrétion d'ACTH. Les principaux objectifs de ce travail étaient de comprendre la relation entre la signalisation dérégulée de EGF et la résistance aux GCs. Dans le présent travail, nous avons identifié la voie JAK/STAT3 comme la principale voie de signalisation EGFR qui active la transcription du gène POMC. De plus, nous montrons que l'activation de la signalisation EGFR entraîne une résistance du promoteur POMC aux GCs et que l’activation de STAT3 est responsable de cette résistance. STAT3 affecte le mécanisme de transrepression de GR sans affecter le recrutement de GR au promoteur POMC. L’utilisation d’un inhibiteur de STAT3 restaure la répression de la transcription du promoteur POMC par les GCs. Nous avons aussi trouvé que 50% des adénomes corticotropes humains montrent une surexpression de la forme active de STAT3. Nous avons aussi étudié les mécanismes sous le contrôle des GCs qui régulent la prolifération cellulaire et qui pourraient être dérégulés dans la maladie de Cushing. CABLES1 est un régulateur négatif du cycle cellulaire et son expression est sous le contrôle des GCs. L’expression de CABLES1 est perdue dans 55 % des adénomes hypophysaires corticotropes, mais la cause de cette perte est encore mal comprise. Dans ce travail, nous avons identifié quatre variants faux-sens dans le gène CABLES1, deux chez de jeunes adultes (c.532G > A, c.718C > T) et deux chez des enfants (c.935G > A, et c.1388A > G) atteints de la maladie de Cushing. Les quatre variants touchent une région de la protéine CABLES1 qui est proche du motif de liaison de la kinase-3 dépendante des cyclines (CDK3). Ces variants ont perdu la capacité d’inhiber la croissance de cellules corticotropes tumorales (AtT20). Les quatre variantes sont donc des mutations de perte de fonction. En résumé, nos travaux révèlent le rôle important de STAT3 dans la résistance aux GC et ainsi, le blocage de l'action de STAT3 peut être une nouvelle stratégie pour le traitement de la maladie de Cushing. Nous avons aussi supporté un rôle de CABLES1 en tant que nouveau gène prédisposant aux tumeurs hypophysaires. / Cushing’s disease (CD) is characterized by excess secretion of adrenocorticotropic hormone (ACTH) from corticotroph tumors of the pituitary gland. Excessive ACTH leads to hypercortisolism that causes disabling symptoms such as diabetes, hypertension, obesity and cardiovascular disease resulting in an increased risk of mortality if it is not treated. Corticotroph tumors are characterized by the loss of glucocorticoid (GC) feedback repression of the proopiomelanocortin (POMC) that encodes the precursor of ACTH: this is the hallmark of CD. The causes of GC resistance in corticotroph adenomas of CD patients remain unknown. Recent findings showed overexpression of epidermal growth factor receptor (EGFR) in corticotroph adenomas causing increased POMC activity and ACTH secretion. The main objectives of this work were to understand the relationship between deregulated EGF signaling and GC resistance in the tumorigenesis of CD. In the present work, we identified the JAK/STAT3 pathway as the main EGFR pathway activating transcription of the POMC gene. We found that sustained activation of EGFR signaling or overactivation of STAT3 causes unresponsiveness of the POMC promoter to GCs and that activated STAT3 is responsible for GC resistance. STAT3 affects the transrepression mechanism of GR without affecting GR recruitment to the POMC promoter. The use of STAT3 inhibitor restores the repressive effect of GC on POMC transcription. Importantly, 50% of human corticotroph adenomas showed overexpression of activated STAT3. We also studied the mechanisms under the control of GCs that regulate cell proliferation and that could be deregulated in CD. CABLES1 is a negative cell cycle regulator, its expression is under the control of GC. CABLES1 expression is lost in 55 % of corticotroph adenomas and the underlying reasons remain unclear. In this work, we identified the presence of four missense variants in CABLES1 gene, two in young adults (c.532G > A, c.718C > T) and two in children (c.935G > A, and c.1388A > G) with CD. The four variants are close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein. The variants have lost the ability to inhibit growth of corticotropinoma cells (AtT20). The four variants are thus loss of function mutations. In summary, our work revealed the important role of STAT3 in GC resistance and further indicates that inhibition of STAT3 action may be a novel strategy for CD treatment. We also provided evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene.