Annabel Berthon

WNT/β-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production

Primary aldosteronism (PA) is the main cause of secondary hypertension, resulting from adrenal aldosterone-producing adenomas (APA) or bilateral hyperplasia. Here, we show that constitutive activation of WNT/β-catenin signalling is the most frequent molecular alteration found in 70% of APA. We provide evidence that decreased expression of the WNT inhibitor SFRP2 may be contributing to deregulated WNT signalling and APA development in patients. This is supported by the demonstration that mice with genetic ablation of Sfrp2 have increased aldosterone production and ectopic differentiation of zona glomerulosa cells. We further show that β-catenin plays an essential role in the control of basal and Angiotensin II-induced aldosterone secretion, by activating AT1R, CYP21 and CYP11B2 transcription. This relies on both LEF/TCF-dependent activation of AT1R and CYP21 regulatory regions and indirect activation of CYP21 and CYP11B2 promoters, through increased expression of the nuclear receptors NURR1 and NUR77. Altogether, these data show that aberrant WNT/β-catenin activation is associated with APA development and suggest that WNT pathway may be a good therapeutic target in PA.

Macronodular Adrenal Hyperplasia due to Mutations in an Armadillo Repeat Containing 5 (ARMC5) Gene: A Clinical and Genetic Investigation

CONTEXT Inactivating germline mutations of the probable tumor suppressor gene, armadillo repeat containing 5 (ARMC5), have recently been identified as a genetic cause of macronodular adrenal hyperplasia (MAH). OBJECTIVE We searched for ARMC5 mutations in a large cohort of patients with MAH. The clinical phenotype of patients with and without ARMC5 mutations was compared. METHODS Blood DNA from 34 MAH patients was genotyped using Sanger sequencing. Diurnal serum cortisol measurements, plasma ACTH levels, urinary steroids, 6-day Liddles test, adrenal computed tomography, and weight of adrenal glands at adrenalectomy were assessed. RESULTS Germline ARMC5 mutations were found in 15 of 34 patients (44.1%). In silico analysis of the mutations indicated that seven (20.6%) predicted major implications for gene function. Late-night cortisol levels were higher in patients with ARMC5-damaging mutations compared with those without and/or with nonpathogenic mutations (14.5 ± 5.6 vs 6.7 ± 4.3, P < .001). All patients carrying a pathogenic ARMC5 mutation had clinical Cushings syndrome (seven of seven, 100%) compared with 14 of 27 (52%) of those without or with mutations that were predicted to be benign (P = .029). Repeated-measures analysis showed overall higher urinary 17-hydroxycorticosteroids and free cortisol values in the patients with ARMC5-damaging mutations during the 6-day Liddles test (P = .0002). CONCLUSIONS ARMC5 mutations are implicated in clinically severe Cushings syndrome associated with MAH. Knowledge of a patients ARMC5 status has important clinical implications for the diagnosis of Cushings syndrome and genetic counseling of patients and their families.

Analysis of the role of Igf2 in adrenal tumour development in transgenic mouse models.

Adrenal cortical carcinomas (ACC) are rare but aggressive tumours associated with poor prognosis. The two most frequent alterations in ACC in patients are overexpression of the growth factor IGF2 and constitutive activation of Wnt/β-catenin signalling. Using a transgenic mouse model, we have previously shown that constitutive active β-catenin is a bona fide adrenal oncogene. However, although all these mice developed benign adrenal hyperplasia, malignant progression was infrequent, suggesting that secondary genetic events were required for aggressive tumour development. In the present paper, we have tested IGF2 oncogenic properties by developing two distinct transgenic mouse models of Igf2 overexpression in the adrenal cortex. Our analysis shows that despite overexpression levels ranging from 7 (basal) to 87 (ACTH-induced) fold, Igf2 has no tumour initiating potential in the adrenal cortex. However, it induces aberrant accumulation of Gli1 and Pod1-positive progenitor cells, in a hedgehog-independent manner. We have also tested the hypothesis that Igf2 may cooperate with Wnt signalling by mating Igf2 overexpressing lines with mice that express constitutive active β-catenin in the adrenal cortex. We show that the combination of both alterations has no effect on tumour phenotype at stages when β-catenin-induced tumours are benign. However, there is a mild promoting effect at later stages, characterised by increased Weiss score and proliferation. Formation of malignant tumours is nonetheless a rare event, even when Igf2 expression is further increased by ACTH treatment. Altogether these experiments suggest that the growth factor IGF2 is a mild contributor to malignant adrenocortical tumourigenesis.

Primary Aldosteronism and ARMC5 Variants.

CONTEXT Primary aldosteronism is one of the leading causes of secondary hypertension, causing significant morbidity and mortality. A number of genetic defects have recently been identified in primary aldosteronism, whereas we identified mutations in ARMC5, a tumor-suppressor gene, in cortisol-producing primary macronodular adrenal hyperplasia. OBJECTIVE We investigated a cohort of 56 patients who were referred to the National Institutes of Health for evaluation of primary aldosteronism for ARMC5 defects. METHODS Patients underwent step-wise diagnosis, with measurement of serum aldosterone and plasma renin activity followed by imaging, saline suppression and/or oral salt loading tests, plus adrenal venous sampling. Cortisol secretion was also evaluated; unilateral or bilateral adrenalectomy was performed, if indicated. DNA, protein, and transfection studies in H295R cells were conducted by standard methods. RESULTS We identified 12 germline ARMC5 genetic alterations in 20 unrelated and two related individuals in our cohort (39.3%). ARMC5 sequence changes in 6 patients (10.7%) were predicted to be damaging by in silico analysis. All affected patients carrying a variant predicted to be damaging were African Americans (P = .0023). CONCLUSIONS Germline ARMC5 variants may be associated with primary aldosteronism. Additional cohorts of patients with primary aldosteronism and metabolic syndrome, particularly African Americans, should be screened for ARMC5 sequence variants because these may underlie part of the known increased predisposition of African Americans to low renin hypertension.

PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors.

Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKAs role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway.

Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease

Context: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have been recently identified as the most common genetic alteration in patients with Cushing disease (CD). However, the frequency of these mutations in the pediatric population has not been extensively assessed. Objective: We investigated the status of the USP8 gene at the somatic level in a cohort of pediatric patients with corticotroph adenomas. Design and Methods: The USP8 gene was fully sequenced in both germline and tumor DNA samples from 42 pediatric patients with CD. Clinical, biochemical, and imaging data were compared between patients with and without somatic USP8 mutations. Results: Five different USP8 mutations (three missense, one frameshift, and one in-frame deletion) were identified in 13 patients (31%), all of them located in exon 14 at the previously described mutational hotspot, affecting the 14-3-3 binding motif of the protein. Patients with somatic mutations were older at disease presentation [mean 5.1 ± 2.1 standard deviation (SD) vs 13.1 ± 3.6 years, P = 0.03]. Levels of urinary free cortisol, midnight serum cortisol, and adrenocorticotropic hormone, as well as tumor size and frequency of invasion of the cavernous sinus, were not significantly different between the two groups. However, patients harboring somatic USP8 mutations had a higher likelihood of recurrence compared with patients without mutations (46.2% vs 10.3%, P = 0.009). Conclusion: Somatic USP8 gene mutations are a common cause of pediatric CD. Patients harboring a somatic mutation had a higher likelihood of tumor recurrence, highlighting the potential importance of this molecular defect for the disease prognosis and the development of targeted therapeutic options.

Adrenal cortex tissue homeostasis and zonation: A WNT perspective.

The adrenal cortex plays essential roles in the control of sodium and water homeostasis, stress response, inflammation and metabolism, through secretion of glucocorticoids and mineralocorticoids. Coordinated production of these hormones relies on functional zonation of the cortex, characterised by expression of Cyp11b2 under the control of angiotensin II and plasma potassium level in zona glomerulosa (ZG) and Cyp11b1 under the control of ACTH in zona fasciculata (ZF). The mechanisms involved in the establishment of functional zonation and its maintenance during centripetal cortex cell renewal are still poorly understood. Here, we hypothesise that the hormonal and signalling pathways that control adrenal cortex function are also involved in cortical zonation. In particular, we summarise evidence on the role of WNT/β-catenin signalling in ZG differentiation and how tight control of its activity is required to shape the adult cortex. In this context, we discuss the potential role of known WNT regulators and the possibility of a reciprocal cross-talk between PKA and WNT signalling.

Adrenocortical Cancer and IGF2: Is the Game Over or Our Experimental Models Limited?

Adrenocortical cancer (ACC) is a rare (0.5‐2 cases per million per year) but aggressive disease with low survival rates of 16 to 47%, depending on the clinical series. Although mitotane alone or in combination with chemotherapy can improve patients’ survival, there is no efficient treatment of metastatic ACC, which is found in almost half of the patients at diagnosis. Identification of themolecularunderpinningsofACCmayopenthewayto targetedtherapyofthisaggressivecancer.Overthelast20 years, considerable concerted effort has led to the identification of molecular alterations in patients’ samples that may drive the tumorigenic process. One of the breakthroughsinthislongprocesswasthediscoverybyGicquel etal(1)in1994thatthegrowthfactorIGF2wasmarkedly overexpressed in about 80‐90% of adrenocortical carcinomas compared with adenomas or healthy adrenals (1). This seminal report was followed by a number of largerscale studies that confirmed thatIGF2was the most upregulated gene in ACC. These studies also showed overexpressionoftheIGF2andIGF-Ireceptor(IGF-IR)andof one IGF binding protein (IGFBP2) in adrenal carcinomas (reviewed in Ref. 2).

PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development

Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates β-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling.

From β-catenin to ARM-repeat proteins in adrenocortical disorders.

Armadillo-containing proteins (ACPs) are a large family of evolutionary conserved proteins, characterized by the tandem repeat copy of a 42 amino acids motif, which forms a 3 dimensional protein-protein interaction domain. This permits ACPs to interact with plenty of partners and consequently, most of these proteins have several independent cellular roles. Perhaps the most well-known protein of this family is β-catenin, which is crucial in the regulation of development and adult tissue homeostasis through its 2 independent functions, acting in cellular adhesion in addition to being a transcriptional co-activator. APCs have important functions in many tissues, but here we summarize the adrenocortical role of 2 well-described ACPs, β-catenin (CTNNB1), Adenomatous Polyposis Coli (APC), and discuss the possible role in the adrenal cortex of the most recently discovered, Armadillo-repeat containing 5 (ARMC5).