Review 869

Protein Kinase A Alterations in Adrenocortical Tumors

Authors

S. Espiard1, 2, 3, B. Ragazzon1, 2, 3, J. Bertherat1, 2, 3, 4

Affiliations

1

Key words ▶ cAMP ● ▶ PKA ● ▶ adrenal tumors ● ▶ Cushing’s syndrome ●

Abstract

 INSERM U1016, Institut Cochin, Paris, France  CNRS UMR8104, Paris, France 3  Université Paris Descartes, Sorbonne Paris Cité, Paris, France 4  Center for Rare Adrenal Diseases, Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Paris, France

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Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing’s syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a

Introduction received 08.06.2014 accepted 15.07.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1385908 Published online: August 8, 2014 Horm Metab Res 2014; 46: 869–875 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence J. Bertherat INSERM U1016 Institut Cochin Paris France Tel.:  + 33/1/58411 895 Fax:  + 33/1/46338 060 [email protected]

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The cAMP/PKA signaling pathway is essential for the development, maintenance, and function of the adrenal cortex. In the mature gland, activation of the cAMP signal by the pituitary hormone adrenocorticotrophin (ACTH) is required for the survival of the adrenocortical cells, as well as steroid synthesis and secretion [1, 2]. In the absence of ACTH, as observed for instance in some pituitary diseases, the adrenal cortex is atrophic and the patient present with cortisol and adrenal androgens deficiency. On the contrary, chronic oversecretion of ACTH, as for instance in Cushing’s disease due to pituitary corticotroph tumor, lead to adrenal cortex hyperplasia and cortisol excess [3]. ACTH stimulates the cAMP pathway after binding to its specific 7-transmembrane receptor coupled to Gs protein. After stimulation of the Gs protein, the synthesis of the second messenger cAMP by the enzyme Adenyl Cyclase (AC) is stim-

tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40 % of cortisolsecreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders.

ulated. The intracellular targets of cAMP are mainly Protein Kinase A (PKA) and EPAC (Exchange Protein Activated by cAMP). PKA is a central component of the cAMP pathway. It is a heterotetramer formed by 2 regulatory subunits and 2 catalytic subunits. Four genes encode the regulatory subunits (RIα, RIβ, RIIα, RIIβ) [4]. At least 4 catalytic subunits have been identified (Cα, Cβ, Cγ, PRKX). Two types of PKA, PKAI and PKAII, have been distinguished on the basis of their biochemical properties. They have different structures and functions, although overlaps exist. After activation of its synthesis, cAMP binds to the regulatory subunit of PKA (2 cAMP molecules per R subunit) leading to the dissociation and activation of the catalytic subunits. The activated catalytic subunits then phosphorylate many downstream cytoplasmic and nuclear targets. One main nuclear target of PKA is the transcription factor CREB (cAMP Response ▶  Fig. 1a, b) [5]. Binding Protein) ( ●

Espiard S et al. PKA in Adrenocortical Tumors  …  Horm Metab Res 2014; 46: 869–875

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

2

870 Review genes of the catalytic subunits Cα (PRKACA) and Cβ (PRKACB) can also be altered in tumors of the adrenal cortex [11, 12]. This review will summarize the various alterations of PKA that have been observed in adrenocortical tumors. The studies on the mechanisms of the cAMP pathway dysregulation as well as adrenal tumorigenesis caused by these PKA alterations will be presented and discussed.

Alterations of the Regulatory Subunits of PKA in Adrenocortical Tumors

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PRKAR1A

Alterations of both the regulatory subunits R1A (encoded by PRKAR1A) and R2B (encoded by PRKAR2B) of PKA have been ▶  Fig. 1c, d). However, the observed in adrenocortical tumors ( ● most investigated subunit to date is PRKAR1A. This is easily explained by the discovery almost 15 years ago that PRKAR1A is the Carney complex gene 1 [10]. Carney Complex (CNC: OMIM #160980), described in 1985, is an autosomal dominantly inherited multiple endocrine neoplasia responsible for spotty skin pigmentation (lentigine), skin or cardiac myxomas, schwannomas, and a variety of endocrine tumors [13]. The most frequent endocrine tumor in CNC is Primary Pigmented Nodular Adrenocortical Dysplasia (PPNAD) causing adrenal Cushing’s syndrome (CS). Pituitary GH-secreting adenomas, thyroid tumors (benign and malignant), and testicular tumors are also observed in CNC (see reviews, [14] and [15]). PPNAD is observed in more than 60 % of patients with CNC and can be isolated in some patients without other manifestations of CNC nor familial history [16]. It is a bilateral benign subtype of adrenocortical tumors with typical bilateral pigmented nodules responsible for an ACTH-independent CS [17].

Fig. 1  cAMP/PKA signaling pathway and alterations of the regulatory subunits in adrenocortical tumors. a In nonactivated cells, protein kinase A exists as an inactive tetramer, with the catalytic subunits bound to a dimer of regulatory subunits. b The extra cellular ligand ACTH binds to its specific 7-transmembrane G-protein coupled receptor (GPCR) resulting in a conformational change of the GPCR that activates Gs protein and then adenylyl cyclase. Adenylyl cyclase transforms ATP to cAMP. Elevation in the cellular cAMP levels lead to the activation of the tetrameric enzyme PKA, composed of 2 regulatory and 2 catalytic subunits. When PKA is activated, the catalytic subunits of the PKA are free and can phosphorylate a series of targets that regulate downstream effectors enzymes, ion channels, in particular the transcription factor CREB which than activate the transcription of cAMP-regulated genes. c PRKAR1A mutations (yellow lightning), described in CNC and PPNAD, lead to the inactivation of R1α and suppression of its inhibitory action on the catalytic subunit. d In cortisol producing adrenocortical adenomas (ACAs), a decrease of RIIβ protein levels associated with increased PKA activity has been shown. AC: Adenylyl cyclase; C: Catalytic subunits of protein kinase (Cα or Cβ subunits); cAMP: Cyclic AMP; CREB: Cyclic AMP response element binding protein; PDE: Phosphodiesterase; PKA: Protein kinase A (PKA 1: type 1; PKA 2: type 2); R: Regulatory subunits of protein kinase (RIα or RIIβ subunits). (Color figure available online only).

Espiard S et al. PKA in Adrenocortical Tumors  …  Horm Metab Res 2014; 46: 869–875

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Many molecular defects in the cAMP/PKA pathway have been identified in various types of endocrine tumors, including those of the adrenal cortex. The first genetic defect of the cAMP signaling reported was the occurrence of somatic activating mutations of the Gs protein in GH-secreting adenomas [6]. The mutations lead to constitutive activation of the AC activity. It was later reported in thyroid toxic adenomas and in the McCune-Albright syndrome. In the latter, the mutation is acquired during embryonic development, and when the mutation is present in the adrenal cortex Cushing’s syndrome due to bilateral adrenal tumors is observed. Activating mutations of the genes of 7 transmembrane receptors have been observed in endocrine tumors: that is, activating mutations of the TSH receptor in thyroid tumors. Activating mutations of the ACTH receptor gene (MC2R) have been rarely reported in adrenal Cushing. On the opposite, inactivation mutation of MC2R or its accessory protein MRAP2 causes cortisol deficiency. More recently, inactivation mutations of the phosphodiesterases PDE11A4 and PDE8B have been found in patients with micronodular adrenal hyperplasia causing Cushing’s syndrome [7]. Phosphodiesterases are enzymes that hydrolyze cAMP and cGMP [8]. Although these various molecular defects will lead to stimulation of the cAMP signal, the precise role of downstream targets of the cAMP pathway in endocrine tumorigenesis is still a matter of debate. It was therefore of particular interest to study PKA, which is one of the main effector of cAMP. Despite some limited studies on the PKA in adrenal tumors developed more than 30 years ago [9], the clear demonstration of the involvement of PKA in endocrine tumors came from the identification of the first Carney Complex Gene (CNC1), as the regulatory subunit RIα of PKA (PRKAR1A) gene located at chromosome 17q22–24 [10]. Since this first report of a PKA genetic defect in adrenal tumors almost 15 years ago, it has been reported very recently that the

The 17q22-2 locus was identified by linkage analysis leading to the identification of PRKAR1A as the CNC1 gene [10]. PRKAR1A was previously identified as the Tissue Extinguishing Factor by a functional cloning approach for its property to inhibit PKA signaling. Indeed PRKAR1A encode the PKA regulatory subunit 1A (RIα) whose invalidation leads to excess of PKA activity. More than two-third of families with CNC present a germline heterozygous inactivating mutation of PRKAR1A [18]. The RIα protein consists of 384 amino acids organized in a dimerization/docking domain at the amino terminal, followed by a PKA inhibitor site, 2 tandem binding domains for cAMP at the carboxyl terminus (cAMP:A and cAMP:B), and a linker region that contains the main docking site for the C subunit. The PRKAR1A gene is composed of 11 exons, 10 of which (2–11) are coding, with a total coding region of 1 143 bp. Most of the mutations are uniquely identified in single families [19]. They can be located in all the coding exons and adjacent introns of the gene. To date, more than 100 PRKAR1A mutations have been identified in 387 unrelated families of various ethnic origins. About 80 % of these mutations result in the generation of a premature stop codon caused by nonsense, frame shift, and splice variants located before the last exon of PRKAR1A. The mutant mRNA is then degraded by a mechanism of nonsense mediated mRNA decay (NMD). Consequently, the mutant mRNA and protein cannot be detected in the normal tissue nor in the tumors of the patients. The inactivation by this mechanism of a single allele might result in haploinsufficiency. In tumors from CNC patients, the wild type allele can also be eliminated by allelic loss (LOH), suggesting that PRKAR1A may act as a tumor suppressor. The second group, consisting of the less frequent mutations, leads to a mutant mRNA that escape NMD [20, 21]. These mutations do not create a premature stop codon or are located in the 3′-end of the gene and alter the last exon. Most of them might therefore give rise to an altered protein. The first mutation reported that causes a truncated protein stimulates PKA activity [20]. Interestingly, this mutation alters the cellular traffic of PKA [22]. Tumors from patients with such mutation usually do not present LOH, suggesting that this mutant could have a dominant negative effect over the wild type allele. Some relatively large deletions in the PRKAR1A gene have been described. The inframe deletion (c.178_348del171/p.Glu60_Lys116del) that eliminate exon 3 has been found in patients and also confirmed in vitro models producing a shorter protein that lack the binding site for C subunits and part of the linker region [23, 24]. Most of the expressed proteins are truncated compared with normal RIα, but they can also be elongated due to frameshift mutations that abolish the wild-type stop codon and generate a new one further downstream in the 3′-untranslated open reading frame. Recently, it has been nicely demonstrated by structural studies that some expressed PRKAR1A mutants alters the interface acting as cAMP sensor in the R1A subunit leading to structural changes altering this function [25]. Few genotype/phenotype correlations have been described for PRKAR1A mutations. Interestingly, patients presenting isolated PPNAD (iPPNAD) without other manifestations of CNC nor familial history can also present a germline inactivating mutations of PRKAR1A [16]. Most of these patients with iPPNAD bear a small intronic deletion generating a splice variant that skips exon7 (c.709-7_709-2del) [26, 27]. They can also have the c.1a > G/p. Met1Val substitution affecting the initiation codon of the protein [28]. A possible explanation may be that these 2 mutations lead to a less severe RIα insufficiency in vivo, affecting mainly

the adrenal gland that could be more sensitive to cAMP/PKA signaling alteration. When a given tumor suppressor gene is responsible for a familial disease transmitted through the inheritance of a germline defect, it is expected that the same defect might be found restricted to the tumor DNA as a somatic alteration in sporadic tumors. Somatic inactivating mutations of PRKAR1A have been found in adrenocortical adenomas. Interestingly, these mutations have been observed in tumors presenting a phenotype close to PPNAD: small secreting benign adenomas presenting a paradoxical response of cortisol to dexamethasone suppression [29, 30]. Furthermore, in adrenal tumors with no PRKAR1A mutation, the PRKAR1A expression can nevertheless be reduced. In adrenocortical cancer, allelic losses of the 17q22–24 locus are observed. Despite the fact that genes other than PRKAR1A might be implicated in these LOH, this could participate in the reduced expression of R1A observed in some of these tumors. Many in vitro studies have been developed to elucidate the implication of the consequences of the inactivation of PRKARIA on PKA activity and adrenocortical tumor development. Animal models were also used to investigate the consequence on the whole organism. Transcriptome analysis by the serial analysis of gene expression (SAGE) method has shown that PKA-regulated genes as well as genes controlled by the transcription factor CREB, including the genes involved in steroidogenesis, are stimulated in PPNAD tissue from patients presenting a PRKAR1A germline mutation [31]. The effects of PRKAR1A inactivation have been also investigated in in vitro experiments on different endocrine cells and nonendocrine cells. Inactivation of PRKAR1A by siRNA stimulates PKA activity [32, 33]. Using FRET (Förster resonance energy transfer) reporter genes to monitor PKA activity and cAMP levels in intact cells, it has been observed that PRKAR1A inactivation stimulates PKA activity but also cAMP production. Activation of PKA activity is more pronounced in the cytoplasmic and the outer mitochondria regions than at the cell membrane level [34]. Lymphocytes B from CNC patients that carry germline PRKAR1A mutations display an increased MAPK pathway activity [35]. Extracellular signal-regulated kinase 2 (ERK2) is activated in these lymphocytes with PRKAR1A haploinsufficiency. In adrenocortical cells (H295R), the PRKAR1A inactivation confers resistance to TGFβ induced apoptosis by decreasing the expression of SMAD3 that mediates the TGFβ receptor signaling [32]. In the H295R adrenocortical cells, 8-chloroadenosine-cAMP (8ClcAMP) stimulation of PKA activity decreases RIα levels and increases RIIβ levels, resulting in cell cycle arrest in the G2 phase parallel with the accumulation of cyclin B and the inactivation of CDC2 kinase [33]. PRKARIA inactivation could also alter cell migration and autophagy. In mouse embryonic fibroblasts (MEF) inactivated for PRKAR1A, cell morphology and cell migration differs from wild type MEF [36, 37]. Hyperpigmentation in PPNAD nodules is due to an accumulation of lipofuscin and is a consequence of autophagic deficiency. Lipofuscin accumulates with aging and influences cell function. RIα deficiency causes deregulation of autophagy as demonstrated in PRKAR1A–/– MEF. Mavrakis et al. showed that the number of autophagosomes is reduced in PRKAR1A–/– MEF [38]. Depletion of R1A in mammalian cells and tissues activates mTOR and causes autophagic deficiency [38]. Several observations suggest the involvement of the Wnt/βcatenin signaling pathway in PPNAD and CNC. By immunohisto-

Espiard S et al. PKA in Adrenocortical Tumors  …  Horm Metab Res 2014; 46: 869–875

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Review 871

872 Review PRKAR2B

Alterations of PRKAR2B have been observed in cortisol secreting adenomas. A complete loss of R2B protein has been observed by Western blot and immunohistochemistry in a subset of cortisolsecreting adenomas. This type of tumor lacking R2B represents a significant subgroup, likely to be half of these cortisol secreting adenomas [46, 47]. The mechanisms of R2B loss are unclear. No mutation of PRKAR2B has been reported in adrenal tumors so far. The transcriptome of cortisol secreting adenomas differs dramatically from the transcriptome of nonsecreting adrenocortical adenomas [48]. However, no clear loss of expression of R2B is observed in these tumors. It is likely that R2B loss is due to protein degradation. It is tempting to speculate that catalytic subunit activation by mutation (see below) might alter the stability of the regulatory subunits playing a role in R2B loss. It has been shown that the inhibition of the regulatory subunit type 2 by R2-selective cAMP analogue inhibits proliferation and induces apoptosis in adrenocortical mouse Y-1 cells; while the inhibition of the regulatory subunit type 2 by R1-selective cAMP analogue stimulates cell proliferation. In mice, RIIβ is mainly expressed in the brain and in the white and brown adipose tissues [49]. Male RIIβ null mice (RIIβ–/– mice) are lean, insulin sensitive and have an increase of their lifespan [49, 50], but no abnormal adrenal phenotype have been reported. PRKAR2B inactivation in these transgenic animal lead to increased PKA activity in fat tissue.

Alterations of the Catalytic Subunits of PKA in Adrenocortical Tumors

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PRKACA

The use of next generation sequencing has been recently very powerful to identify new genes of adrenocortical tumors [51, 52]. Using exome sequencing Felix Beuschlein and collaborators identified somatic activating mutations of the catalytic alpha subunit of PKA in a subset of cortisol secreting adrenocor▶  Fig. 2a) [11]. Shortly after 3 additional studies tical adenomas ( ● have reported using rather similar next generation sequencing approaches the same mutation of the PRKACA gene [30, 53, 54]. The PRKACA mutations observed in these 4 studies are somatic mutations found only on the tumor DNA. The c.617A > G/p. L206R hotspot mutation was found in heterozygote state in more than 40 % of the cortisol producing adrenocortical adenoFig. 2  Alterations of the catalytic subunits in adrenocortical tumors. a PRKACA mutations (yellow lightning) lead to constitutive activation of the catalytic Cα protein with loss of its interaction with the regulatory subunits. b Duplication of Cβ has been described in a case of CNC and is associated with increase of PKA activity. (Color figure available online only).

Espiard S et al. PKA in Adrenocortical Tumors  …  Horm Metab Res 2014; 46: 869–875

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chemisty, an increased nuclear accumulation of β-catenin in nodules from PPNAD have been observed [39]. PKA can phosphorylate glycogen synthase kinase-3β (GSK3β) that stabilizes the protein of β-catenin. Transcriptome studies in PPNAD have demonstrated overexpression of genes involved in the Wnt pathway such as WISP2, β-catenin (CTNNB1), and GSK3β [31]. Somatic mutations of CTNNB1 have also been found in macronodules of PPNAD, suggesting that the occurrence of this secondary event in the background of PRKAR1A inactivation increases tumor development [39, 40]. PKA may affect Wnt pathway through micro-RNA regulation [41]. Various mouse models have been developed to investigate the in vivo effects of PRKARIA deficiency. The complete knock-out mice (PRKAR1A–/–) die during embryonic development [36]. Therefore transgenic mice carrying an inducible antisense-construct of PRKARIA exon 2 under the control of a tetracycline responsive promoter has been initially developed [42]. In this model, animals develop tumors that share some similarities with the tumors observed in Carney Complex. Heterozygous PRKAR1A + /– mice can live, and the development of sarcoma and lymphoma as well as extracardiac tumors are observed. In these models a high incidence of thyroid lesions is observed and some adrenal lesions reminiscent of PPNAD. Most of the animals show a mesenchymal and epithelial hyperplasia in a variety of tissues, that causes spindle cell schwanoma and squamous papilloma tumors. The formation of tumors in these transgenic mice is associated with altered PKA activity [43]. Latter mice model allowing tissue-specific inactivation of PRKARIA have been added to target the adrenal cortex and investigate the role of RIα loss in the initiation and development of PPNAD; mice lacking PRKARIA specifically in the adrenal cortex (AdKO) have been developed by crossing a PRKARIA floxed mice with the akr1b7-Cre mice line. In this model, PRKARIA deficiency induces adrenal cortex hyperactivity and bilateral hyperplasia as observed in PPNAD. A zonation defect characterized by the appearance of a fetal adrenal zone is observed in these mice [44]. Most adult AdKO adrenal glands express high levels of CYP17 (cytochrome P450 17, coding 17α-hydroxylase). The PRKARIA transcripts are decreased but there is an increase in RIIβ and Cα by post-transcription regulation, with enhanced PKA activity. In this model, increased proliferation and resistance to apoptosis are both observed. This is at least in part explained by activation of the mTOR pathway as shown by an increased BAD phosphorylation that correlates with increased cell survival [45].

Review 873

Reference

Cao Y et al. [30] Goh G et al. [53] Beuschlein F et al. [11] Sato M et al. [54] Total

Cortisol

Cortisol

Other unilateral

secreting

secreting ACA

adrenocortical

ACA

with overt CS

tumor

65.5 % (57/87) 23.6 % (13/55) 22.2 % (22/99) 52.3 % (34/65) 41.2 % (126/306)

NA 35.7 % (10/28) 37.2 % (22/59) 58.2 % (32/55) 45 % (64/142)

0 % (0/3) 1 0 % (0/8) 2 0 % (0/40) 3 – –

NA: Not available 1

 Other unilateral adrenocortical tumors investigated: 3 adrenocortical oncocytomas

2

 Other unilateral adrenocortical tumors investigated: 8 adrenocortical carcinomas

3

 Other unilateral adrenocortical tumors investigated: 20 aldosterone-producing

Conclusion

adenomas and 20 inactive adenomas

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▶  Table 1). One other insertion, the c.595_596insCAC/ mas (ACA) ( ● L199_Cys200insTrp, has been found in one patient with cortisol producing ACA [11]. Clinical correlations have been established. The p.L206R hotspot is significantly associated with younger age at the diagnosis, smaller tumor, and over Cushing’s syndrome [11, 53, 54]. Indeed, this mutation is mainly found in patient with clinical overt Cushing’s syndrome [11, 53, 54]. PRKACA mutations are observed in patients with a more severe endocrine phenotype: lesser suppression of serum cortisol level after overnight dexamethasone suppression, higher urinary cortisol and higher midnight cortisol levels [11]. The hotspot L206R is located in a highly conserved active-site cleft of the catalytic subunits to which the regulatory subunits binds. Cell culture studies have demonstrated that the 2 mutants are constitutively hyperactive, do not respond to cAMP stimulation, and are resistant to inhibition by the regulatory subunits R1α [53, 54] and R2β [11]. L206R PRKACA protein has a dominant effect over the wild-type subunit [11]. The mutated protein can still be inhibited by the PKA inhibitor PKI [30]. Resistance to regulatory subunits inhibition is explained by a loss of interaction between the L206R mutant catalytic subunit and the regulatory subunit R1α, demonstrated by co-immunoprecipitation studies [53, 54]. The activation of the PKA enzymatic activity and a higher expression of steroidogenic enzymes have been demonstrated in adrenal tissue from mutated patients [11, 53]. Interestingly, constitutive activation of PKA in cells transfected with the L206R mutant, can be suppressed by PRKACA inhibitors (H89 and KT5720), but not by the competitive inhibitor of cAMP binding for the R subunit Rp-cAMPS [30, 54]. Beyond these 2 mutations in ACAs, germline duplications of a region in chromosome 19p including PRKACA in 5 patients with cortisol secreting adrenal hyperplasia has also been shown by CGH study [11]. A duplication was inherited and beard by a mother and her son who have mild insidious Cushing’s syndrome diagnosed after the third decade and caused by bilateral macronodular hyperplasia. The 3 others patients were young boys with severe disease and micronodular or macronodular bilateral adrenal hyperplasia [11]. Germline duplication of PRKACA appears to be also a new etiology of bilateral adrenal hyperplasia, associated preferentially with early severe Cushing’s syndrome.

PRKACB

Recently the first case of genetic alteration of the catalytic subu▶  Fig. 2b). A germline triplication of nit beta has been described ( ●

Genetic alterations of the subunits of PKA, mainly R1A and Cα, are involved in the tumor of the adrenal cortex. Most patients with Carney Complex or isolated PPNADs harbor heterozygous germline inactivating mutations of PRKAR1A, which can be also detected as a somatic alteration in some sporadic endocrine tumors. Most of PRKAR1A mutations result in the total loss of protein suggesting that PRKAR1A might function as a tumor suppressor gene. Due to the recent development of exome sequencing in adrenal tumors, activating somatic mutations of PRKACA have been recently reported in cortisol secreting tumors. These are likely to act as oncogene. Interestingly, both PRKAR1A inactivation and PRKACA activation lead to increased PKA activity. In keeping with the effect of ACTH/cAMP on steroidogenesis, these alterations are observed in cortisol secreting tumors responsible for marked Cushing’s syndrome. Similarly, in agreement with the effect of cAMP on cellular differentiation, these alterations are observed in benign slowly growing tumors. Targeting the cAMP/PKA pathway should clearly be a candidate approach to treat cortisol secreting tumors and cortisol excess.

Conflict of Interest

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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.

References

1 Gallo-Payet N, Payet MD. Mechanism of action of ACTH: beyond cAMP. Microsc Res Tech 2003; 61: 275–287 2 de Joussineau C, Sahut-Barnola I, Levy I, Saloustros E, Val P, Stratakis CA, Martinez A. The cAMP pathway and the control of adrenocortical development and growth. Mol Cell Endocrinol 2012; 351: 28–36 3 Rosenberg D, Groussin L, Bertagna X, Bertherat J. cAMP pathway alterations from the cell surface to the nucleus in adrenocortical tumors. Endocr Res 2002; 28: 765–775 4 Taylor SS, Zhang P, Steichen JM, Keshwani MM, Kornev AP. PKA: lessons learned after twenty years. Biochim Biophys Acta 2013; 1834: 1271–1278 5 Rosenberg D, Groussin L, Jullian E, Perlemoine K, Bertagna X, Bertherat J. Role of the PKA-regulated transcription factor CREB in development and tumorigenesis of endocrine tissues. Ann N Y Acad Sci 2002; 968: 65–74 6 Lania A, Mantovani G, Spada A. cAMP pathway and pituitary tumorigenesis. Ann Endocrinol (Paris) 2012; 73: 73–75 7 Vezzosi D, Bertherat J, Groussin L. Pathogenesis of benign adrenocortical tumors. Best Pract Res Clin Endocrinol Metab 2010; 24: 893–905 8 Almeida MQ, Stratakis CA. How does cAMP/protein kinase A signaling lead to tumors in the adrenal cortex and other tissues? Mol Cell Endocrinol 2011; 336: 162–168

Espiard S et al. PKA in Adrenocortical Tumors  …  Horm Metab Res 2014; 46: 869–875

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chromosome 1p31.1, including PRKACB, has been found in a 19-years-old woman with acromegaly, pigmented spots and myxomas but without Cushing’s syndrome [12]. This triplication comes with an increase of the catalytic subunit Cβ expression and PKA activation in lymphocyte. The in vivo study, in mice carrying a transgene for human PRKACB, has shown an increase of growth hormone level in these mice. No PRKACB mutations nor other 1p31 amplifications were found in 132 additional patients with Carney complex and PRKAR1A mutated or wild-type [12]. No PRKACB mutation has been described so far in the different whole exome sequencing performed in adrenocortical adenomas.

Table 1  Frequency of L206R hotspot and L199_Cys200insTrp mutations in ACA.

9 Riou JP, Evain D, Perrin F, Saez JM. Adenosine 3□,5□-cyclic monophosphate dependent protein kinase in human adrenocortical tumors. J Clin Endocrinol Metab 1977; 44: 413–419 10 Kirschner LS, Carney JA, Pack SD, Taymans SE, Giatzakis C, Cho YS, ChoChung YS, Stratakis CA. Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat Genet 2000; 26: 89–92 11 Beuschlein F, Fassnacht M, Assie G, Calebiro D, Stratakis CA, Osswald A, Ronchi CL, Wieland T, Sbiera S, Faucz FR, Schaak K, Schmittfull A, Schwarzmayr T, Barreau O, Vezzosi D, Rizk-Rabin M, Zabel U, Szarek E, Salpea P, Forlino A, Vetro A, Zuffardi O, Kisker C, Diener S, Meitinger T, Lohse MJ, Reincke M, Bertherat J, Strom TM, Allolio B. Constitutive activation of PKA catalytic subunit in adrenal Cushing’s syndrome. N Engl J Med 2014; 370: 1019–1028 12 Forlino A, Vetro A, Garavelli L, Ciccone R, London E, Stratakis CA, Zuffardi O. PRKACB and Carney complex. N Engl J Med 2014; 370: 1065–1067 13 Carney JA, Gordon H, Carpenter PC, Shenoy BV, Go VL. The complex of myxomas, spotty pigmentation, and endocrine overactivity. Medicine (Baltimore) 1985; 64: 270–283 14 Rothenbuhler A, Stratakis CA. Clinical and molecular genetics of Carney complex. Best Pract Res Clin Endocrinol Metab 2010; 24: 389–399 15 Espiard S, Bertherat J. Carney complex. Front Horm Res 2013; 41: 50–62 16 Groussin L, Jullian E, Perlemoine K, Louvel A, Leheup B, Luton JP, Bertagna X, Bertherat J. Mutations of the PRKAR1A gene in Cushing’s syndrome due to sporadic primary pigmented nodular adrenocortical disease. J Clin Endocrinol Metab 2002; 87: 4324–4329 17 Carney JA, Libe R, Bertherat J, Young WF. Primary Pigmented Nodular Adrenocortical Disease: The Original 4 Cases Revisited After 30 Years for Follow-up, New Investigations, and Molecular Genetic Findings. Am J Surg Pathol 2014, doi:10.1097/PAS.0000000000000220 18 Bertherat J, Horvath A, Groussin L, Grabar S, Boikos S, Cazabat L, Libe R, Rene-Corail F, Stergiopoulos S, Bourdeau I, Bei T, Clauser E, Calender A, Kirschner LS, Bertagna X, Carney JA, Stratakis CA. Mutations in regulatory subunit type 1A of cyclic adenosine 5□-monophosphate-dependent protein kinase (PRKAR1A): phenotype analysis in 353 patients and 80 different genotypes. J Clin Endocrinol Metab 2009; 94: 2085–2091 19 Horvath A, Bertherat J, Groussin L, Guillaud-Bataille M, Tsang K, Cazabat L, Libe R, Remmers E, Rene-Corail F, Faucz FR, Clauser E, Calender A, Bertagna X, Carney JA, Stratakis CA. Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update. Hum Mutat 2010; 31: 369–379 20 Groussin L, Kirschner LS, Vincent-Dejean C, Perlemoine K, Jullian E, Delemer B, Zacharieva S, Pignatelli D, Carney JA, Luton JP, Bertagna X, Stratakis CA, Bertherat J. Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD. Am J Hum Genet 2002; 71: 1433–1442 21 Greene EL, Horvath AD, Nesterova M, Giatzakis C, Bossis I, Stratakis CA. In vitro functional studies of naturally occurring pathogenic PRKAR1A mutations that are not subject to nonsense mRNA decay. Hum Mutat 2008; 29: 633–639 22 Meoli E, Bossis I, Cazabat L, Mavrakis M, Horvath A, Stergiopoulos S, Shiferaw ML, Fumey G, Perlemoine K, Muchow M, Robinson-White A, Weinberg F, Nesterova M, Patronas Y, Groussin L, Bertherat J, Stratakis CA. Protein kinase A effects of an expressed PRKAR1A mutation associated with aggressive tumors. Cancer Res 2008; 68: 3133–3141 23 Blyth M, Huang S, Maloney V, Crolla JA, Karen Temple I. A 2.3Mb deletion of 17q24.2-q24.3 associated with □Carney Complex plus’. Eur J Med Genet 2008; 51: 672–678 24 Horvath A, Bossis I, Giatzakis C, Levine E, Weinberg F, Meoli E, RobinsonWhite A, Siegel J, Soni P, Groussin L, Matyakhina L, Verma S, Remmers E, Nesterova M, Carney JA, Bertherat J, Stratakis CA. Large deletions of the PRKAR1A gene in Carney complex. Clin Cancer Res 2008; 14: 388–395 25 Bruystens JG, Wu J, Fortezzo A, Kornev AP, Blumenthal DK, Taylor SS. PKA RIalpha homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease. Structure 2014; 22: 59–69 26 Groussin L, Horvath A, Jullian E, Boikos S, Rene-Corail F, Lefebvre H, Cephise-Velayoudom FL, Vantyghem MC, Chanson P, Conte-Devolx B, Lucas M, Gentil A, Malchoff CD, Bertagna X, Stratakis CA, Bertherat J. A PRKAR1A mutation associated with primary pigmented nodular adrenocortical disease in 12 kindreds. J Clin Endocrinol Metab 2006; 91: 1943–1949

27 Poukoulidou T, Maiter D, Bertherat J, Beauloye V. A rare case of familial Cushing’s syndrome with a common presentation of weight gain due to a mutation of the PRKAR1A gene causing isolated primary pigmented nodular adrenocortical disease. J Pediatr Endocrinol Metab 2014 [Epub ahead of print] 28 Pereira AM, Hes FJ, Horvath A, Woortman S, Greene E, Bimpaki E, Alatsatianos A, Boikos S, Smit JW, Romijn JA, Nesterova M, Stratakis CA. Association of the M1V PRKAR1A mutation with primary pigmented nodular adrenocortical disease in two large families. J Clin Endocrinol Metab 2010; 95: 338–342 29 Bertherat J, Groussin L, Sandrini F, Matyakhina L, Bei T, Stergiopoulos S, Papageorgiou T, Bourdeau I, Kirschner LS, Vincent-Dejean C, Perlemoine K, Gicquel C, Bertagna X, Stratakis CA. Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expression and activity. Cancer Res 2003; 63: 5308–5319 30 Cao Y, He M, Gao Z, Peng Y, Li Y, Li L, Zhou W, Li X, Zhong X, Lei Y, Su T, Wang H, Jiang Y, Yang L, Wei W, Yang X, Jiang X, Liu L, He J, Ye J, Wei Q, Li Y, Wang W, Wang J, Ning G. Activating hotspot L205R mutation in PRKACA and adrenal Cushing’s syndrome. Science 2014; 344: 913–917 31 Horvath A, Mathyakina L, Vong Q, Baxendale V, Pang AL, Chan WY, Stratakis CA. Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation. J Clin Endocrinol Metab 2006; 91: 584–596 32 Ragazzon B, Cazabat L, Rizk-Rabin M, Assie G, Groussin L, Fierrard H, Perlemoine K, Martinez A, Bertherat J. Inactivation of the Carney complex gene 1 (protein kinase A regulatory subunit 1A) inhibits SMAD3 expression and TGF beta-stimulated apoptosis in adrenocortical cells. Cancer Res 2009; 69: 7278–7284 33 Bouizar Z, Ragazzon B, Viou L, Hortane M, Bertherat J, Rizk-Rabin M. 8ClcAMP modifies the balance between PKAR1 and PKAR2 and modulates the cell cycle, growth and apoptosis in human adrenocortical H295R cells. J Mol Endocrinol 2010; 44: 331–347 34 Cazabat L, Ragazzon B, Varin A, Potier-Cartereau M, Vandier C, Vezzosi D, Risk-Rabin M, Guellich A, Schittl J, Lechene P, Richter W, Nikolaev VO, Zhang J, Bertherat J, Vandecasteele G. Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters. Hum Mol Genet 2014; 23: 1163–1174 35 Robinson-White A, Hundley TR, Shiferaw M, Bertherat J, Sandrini F, Stratakis CA. Protein kinase-A activity in PRKAR1A-mutant cells, and regulation of mitogen-activated protein kinases ERK1/2. Hum Mol Genet 2003; 12: 1475–1484 36 Amieux PS, Howe DG, Knickerbocker H, Lee DC, Su T, Laszlo GS, Idzerda RL, McKnight GS. Increased basal cAMP-dependent protein kinase activity inhibits the formation of mesoderm-derived structures in the developing mouse embryo. J Biol Chem 2002; 277: 27294–27304 37 Nadella KS, Saji M, Jacob NK, Pavel E, Ringel MD, Kirschner LS. Regulation of actin function by protein kinase A-mediated phosphorylation of Limk1. EMBO Rep 2009; 10: 599–605 38 Mavrakis M, Lippincott-Schwartz J, Stratakis CA, Bossis I. Depletion of type IA regulatory subunit (RIalpha) of protein kinase A (PKA) in mammalian cells and tissues activates mTOR and causes autophagic deficiency. Hum Mol Genet 2006; 15: 2962–2971 39 Gaujoux S, Tissier F, Groussin L, Libe R, Ragazzon B, Launay P, Audebourg A, Dousset B, Bertagna X, Bertherat J. Wnt/beta-catenin and 3′,5′-cyclic adenosine 5′-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors. J Clin Endocrinol Metab 2008; 93: 4135–4140 40 Tadjine M, Lampron A, Ouadi L, Horvath A, Stratakis CA, Bourdeau I. Detection of somatic beta-catenin mutations in primary pigmented nodular adrenocortical disease. Clin Endocrinol (Oxf) 2008; 69: 367–373 41 Iliopoulos D, Bimpaki EI, Nesterova M, Stratakis CA. MicroRNA signature of primary pigmented nodular adrenocortical disease: clinical correlations and regulation of Wnt signaling. Cancer Res 2009; 69: 3278–3282 42 Griffin KJ, Kirschner LS, Matyakhina L, Stergiopoulos S, Robinson-White A, Lenherr S, Weinberg FD, Claflin E, Meoli E, Cho-Chung YS, Stratakis CA. Down-regulation of regulatory subunit type 1A of protein kinase A leads to endocrine and other tumors. Cancer Res 2004; 64: 8811–8815 43 Kirschner LS, Kusewitt DF, Matyakhina L, Towns WH 2nd, Carney JA, Westphal H, Stratakis CA. A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues. Cancer Res 2005; 65: 4506–4514

Espiard S et al. PKA in Adrenocortical Tumors  …  Horm Metab Res 2014; 46: 869–875

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874 Review

44 Sahut-Barnola I, de Joussineau C, Val P, Lambert-Langlais S, Damon C, Lefrancois-Martinez AM, Pointud JC, Marceau G, Sapin V, Tissier F, Ragazzon B, Bertherat J, Kirschner LS, Stratakis CA, Martinez A. Cushing’s syndrome and fetal features resurgence in adrenal cortex-specific Prkar1a knockout mice. PLoS Genet 2010; 6: e1000980 45 de Joussineau C, Sahut-Barnola I, Tissier F, Dumontet T, Drelon C, BatisseLignier M, Tauveron I, Pointud JC, Lefrancois-Martinez AM, Stratakis CA, Bertherat J, Val P, Martinez A. mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD). Hum Mol Genet 2014 [Epub ahead of print] 46 Vincent-Dejean C, Cazabat L, Groussin L, Perlemoine K, Fumey G, Tissier F, Bertagna X, Bertherat J. Identification of a clinically homogenous subgroup of benign cortisol-secreting adrenocortical tumors characterized by alterations of the protein kinase A (PKA) subunits and high PKA activity. Eur J Endocrinol 2008; 158: 829–839 47 Mantovani G, Lania AG, Bondioni S, Peverelli E, Pedroni C, Ferrero S, Pellegrini C, Vicentini L, Arnaldi G, Bosari S, Beck-Peccoz P, Spada A. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: relationship with cell proliferation. Exp Cell Res 2008; 314: 123–130 48 Wilmot Roussel H, Vezzosi D, Rizk-Rabin M, Barreau O, Ragazzon B, Rene-Corail F, de Reynies A, Bertherat J, Assie G. Identification of gene expression profiles associated with cortisol secretion in adrenocortical adenomas. J Clin Endocrinol Metab 2013; 98: E1109–E1121 49 Cummings DE, Brandon EP, Planas JV, Motamed K, Idzerda RL, McKnight GS. Genetically lean mice result from targeted disruption of the RII beta subunit of protein kinase A. Nature 1996; 382: 622–626

50 Enns LC, Morton JF, Treuting PR, Emond MJ, Wolf NS, Dai DF, McKnight GS, Rabinovitch PS, Ladiges WC. Disruption of protein kinase A in mice enhances healthy aging. PLoS One 2009; 4: e5963 51 Assie G, Libe R, Espiard S, Rizk-Rabin M, Guimier A, Luscap W, Barreau O, Lefevre L, Sibony M, Guignat L, Rodriguez S, Perlemoine K, Rene-Corail F, Letourneur F, Trabulsi B, Poussier A, Chabbert-Buffet N, Borson-Chazot F, Groussin L, Bertagna X, Stratakis CA, Ragazzon B, Bertherat J. ARMC5 mutations in macronodular adrenal hyperplasia with Cushing’s syndrome. N Engl J Med 2013; 369: 2105–2114 52 Assie G, Letouze E, Fassnacht M, Jouinot A, Luscap W, Barreau O, Omeiri H, Rodriguez S, Perlemoine K, Rene-Corail F, Elarouci N, Sbiera S, Kroiss M, Allolio B, Waldmann J, Quinkler M, Mannelli M, Mantero F, Papathomas T, De Krijger R, Tabarin A, Kerlan V, Baudin E, Tissier F, Dousset B, Groussin L, Amar L, Clauser E, Bertagna X, Ragazzon B, Beuschlein F, Libe R, Reynies A, Bertherat J. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet 2014; 46: 607–612 53 Goh G, Scholl UI, Healy JM, Choi M, Prasad ML, Nelson-Williams C, Kuntsman JW, Korah R, Suttorp AC, Dietrich D, Haase M, Willenberg HS, Stalberg P, Hellman P, Akerstrom G, Bjorklund P, Carling T, Lifton RP. Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors. Nat Genet 2014; 46: 613–617 54 Sato Y, Maekawa S, Ishii R, Sanada M, Morikawa T, Shiraishi Y, Yoshida K, Nagata Y, Sato-Otsubo A, Yoshizato T, Suzuki H, Shiozawa Y, Kataoka K, Kon A, Aoki K, Chiba K, Tanaka H, Kume H, Miyano S, Fukayama M, Nureki O, Homma Y, Ogawa S. Recurrent somatic mutations underlie corticotropin-independent Cushing’s syndrome. Science 2014; 344: 917–920

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