Molecular genetics of adrenocortical tumor formation and potential pharmacologic targets

Abstract

Abnormalities in the cAMP/PKA signaling pathway have been linked to the formation of benign adrenal tumors, as well as a possible predisposition to adrenocortical cancer. Mutations in the G-protein coupled receptor are associated with McCune-Albright syndrome and ACTH-independent macronodular adrenal hyperplasia, while defects in cAMP-dependent protein kinase A can lead to the development of Carney’s complex, as well as primary pigmented nodular adrenocortical disease (PPNAD), and micronodular adrenocortical hyperplasia (MAH). Defects in phosphodiesterases, which regulate cAMP levels, have also been demonstrated in PPNAD and MAH. The Wnt signaling pathway, which is involved in oncogenesis in a variety of tumors, has also been implicated in adrenocortical tumorigenesis. MicroRNA profiling has added to the our understanding of the signaling pathways involved in tumor formation in the adrenal cortex. Will this all lead to the development of specific targets for pharmacologic therapies? In this article, we review the molecular genetics of adrenocortical tumors and refer to potential targets for pharmacologic therapy.

Introduction

Adrenocorticotropic hormone (ACTH) is perhaps the most important ligand for the activation of cyclic adenosine monophosphate (cAMP) signaling in adrenocortical cells. The ACTH receptor (MC2R) is a G-protein coupled receptor (GPCR); after ACTH binding to its receptor the Gsα subunit of the heterotrimeric G proteins is acctivated. The newly dissociated Gsα subunit protein interacts with adenylate cyclase to produce cAMP. Phosphodiesterases (PDEs) catalyze the breakdown of cAMP and play a critical role in the regulation of cytosolic levels of cAMP. cAMP dependent protein kinase (PKA), which is a heterotetramer that consists of two regulatory subunits, each of which is bound to a catalytic subunit, is activated by cAMP binding to the regulatory subunits^1,2. The activated catalytic subunits of PKA then phosphorylate targets in the cytoplasm and the nucleus, including cAMP response element-binding (CREB) protein, thereby activating DNA transcription of cAMP-responsive element containing genes³. Abnormalities in any of the steps on this pathway can lead to alterations in cell activation and proliferation.

The Wnt (wingless-type) signaling pathway, which regulates cell proliferation and differentiation, is considered to be essential to adrenocortical development and function⁴. The binding of Wnt ligand to the Frizzled receptor inactivates glycogen synthase kinase 3β (GSK3β), and results in an accumulation of β-catenin. Nuclear translocation of β-catenin results in transcription of target genes, the end result of which is cell proliferation. The Wnt pathway is the “final common pathway” for several signaling pathways, including the cAMP/PKA pathway. In particular, in steroidogenic cells, cAMP/PKA interacts with GSK3β⁵, and its target transcription factor, CREB, is essential for myogenic gene expression induced by Wnt⁶.

G-protein coupled receptor abnormalities

The first association of adrenal tumors with abnormalities in PKA signaling was observed in McCune-Albright syndrome, a disease caused by an activating mutation in the Gsα subunit (the GNAS gene) of the GPCR. The presence of this somatic mutation leads to unregulated cAMP production, thereby resulting in autonomous hyperfunction of affected tissues, as well as unregulated cell growth. This, in turn, leads to continuous stimulation of adenylate cyclase and PKA activation, as well as a number of manifestations, most notably fibrous dysplasia, café au lait skin pigmentation and autonomous endocrine dysfunction⁷. When present, adrenal pathology in this syndrome consists of bilateral macronodular adrenocortical hyperplasia with overproduction of cortisol^8,9

Cortisol production in ACTH-independent macronodular adrenal hyperplasia (AIMAH, also known as massive macronodular adrenocortical disease) can be the result of abnormally expressed GPCRs, which are regulated by ligands other than ACTH in this disorder^10,11. In a screening study by Mircescu, et al.¹², 20 patients with adrenal Cushing’s syndrome were studied for the presence of abnormal GPCRs. All 6 patients with ACTH-independent macronodular adrenal hyperplasia were found to have at least one abnormal GPCR. In these patients, hormones such as gastric inhibitory polypeptide, vasopressin, or catecholamines are able to stimulate their aberrantly expressed GPCRs, which leads to activation of the cAMP/PKA signaling system. This results in unregulated cortisol production, thereby leading to the development of Cushing’s syndrome. Interestingly, 3 out of 13 patients with unilateral adenomas were also found to have abnormal GPCRs. This data suggests possible new pharmacologic therapies specifically targeted to the abnormal receptor.

Abnormalities of PKA regulation

In contrast to over-expression of the GPCRs or activating GNAS mutations, Carney complex (CNC) is the result of inactivating mutations of the PRKAR1A gene, which codes for the type 1α regulatory subunit of PKA^13,14. CNC is characterized by multiple endocrine neoplasias, including skin tumors, myxomas, schwannomas, and endocrine neoplasms, including primary pigmented nodular adrenocortical disease (PPNAD) in the majority of patients^15,16. Germline mutations in the PRKAR1A gene are inherited in an autosomal dominant fashion and result most of the time (but not alays) in the complete lack of normal PRKAR1A protein in affected tissues. Loss of this tumor suppressor leads to increased responsiveness to cAMP-stimulated activity in CNC tumors while basal PKA activity is normal or decreased. While the majority of patients with PRKAR1A mutations have functionally null mutations¹³, variants with mutations leading to abnormal PRKAR1A proteins have also been described¹⁷. In a study evaluating CNC patients by Bertherat, et al.¹⁸, the clinical phenotype and genotype was described. Those patients with null mutations typically had less severe disease compared with those patients with mutant PRKAR1A protein. In 12% of patients, isolated PPNAD was the only manifestation.

Somatic mutations in the PRKAR1A gene or loss of the 17q22–24 region (that harbors the gene) were also found in 5 out of 22 patients with sporadic adrenal adenomas, and in 8 of 15 patient with adrenocortical carcinomas in another study by Bertherat, et al.¹⁹. These patients had similar clinical characteristics as patients with PPNAD, including elevated cortisol levels and paradoxical response to glucocorticoid suppression. In addition, adenomas with loss of 17q region had increased PKA activity in response to cAMP as compared with those adenomas without mutations. This research expanded the role of the PRKAR1A gene as a tumor suppressor.

Mutations in phosphodiesterases, the enzymes that break down cAMP, have also been associated with adrenal tumors. In micronodular adrenocortical hyperplasia (MAH), a disease that shares many similarities with PPNAD (including ACTH-independent hypercortisolism) small nodules form in both adrenal glands, although unlike in PPNAD, the nodules are not pigmented. Since these patients did not have any mutations in PRKAR1A gene, Horvath, et al.²⁰ performed a genome-wide genotyping, and discovered a mutation in the 2q31–35 region, which codes for phosphodiesterase 11A (PDE11A). This inactivating mutation results in elevated cAMP levels, and therefore increased PKA activity. Mutations in the gene encoding PDE11A were also found to be present in a small percentage of the normal population, suggesting that the gene may also contribute to predisposition to adrenal hyperplasia and adenomas²¹. A second genetic locus coding for phosphodiesterase 8B (PDE8B) was more recently found to be involved in isolated PPNAD, MAH and other adrenal tumors²².

The role of the Wnt signaling pathway and miRNAs in adrenal tumorigenesis

Activation of Wnt signaling and subsequent β-catenin accumulation has been linked to human cancers. Mutations in genes along this pathway have been associated with colon cancer²³, gastric cancer²⁴, and hepatocellular carcinoma²⁵, and also occur in adrenocortical tumors. Mutations in the Wnt pathway leading to excessive accumulation of β-catenin were found in 38% of adrenocortical adenomas, and 85% of adrenocortical carcinomas. The majority of mutations were activating somatic mutations of the β-catenin gene, and were more common in non-functional adrenocortical adenomas²⁶. Moreover, over-expression of genes in the Wnt signaling pathway, including WNT1-inducible signaling pathway protein 2 (WISP2) in AIMAH²⁷ have been demonstrated, while in PPNAD, over-expression of six genes involved in the Wnt signaling pathway, including GSK3β, WISP2, and β-catenin have been established²⁸.

MicroRNAs (miRNAs) are short, noncoding, single-stranded RNA sequences which regulate gene expression. MiRNAs act at the post-transcriptional level, via binding to the 3’ untranslated region of mRNA, and regulate gene expression through repression of translation or by degradation of mRNA. They play a critical role in the regulation of cell proliferation, apoptosis, differentiation, and growth.

Due to their important role in cell proliferation, miRNAs have been linked to oncogenesis, both as oncogenes and tumor suppressors. One of the first studies to link miRNAs to tumorigenesis was by Calin, et al.²⁹. Chromosomal analysis was performed in patients with B-cell chronic lymphocytic leukemia to evaluate for gene deletions on chromosome 13q14, a region known to be deleted in the majority of CLL patients. Two small, noncoding RNAs, miR15 and miR16, were found to be deleted in 68% of CLL patients, while their expression is up-regulated in normal B cells. This data suggested that miR15 and miR16 act as tumor suppressors, and their deletion results in unregulated cell proliferation.

Synthesis of adrenal hormones such as cortisol is tightly regulated, and recent research by Riester, et al.³⁰ demonstrated the important role of miRNA as modulators of the glucocorticoid receptor. Expression of miRNA in the adrenal glands of mice was studied under basal conditions, and during ACTH stimulation. The expression of the glucocorticoid receptor was significantly increased after 10 minutes, followed by up-regulation of miR96, miR101a, miR142-3p, and miR433, and subsequent down-regulation of the glucocorticoid nuclear receptor by 60 minutes following ACTH stimulation. Thus, in response to stress, the glucocorticoid nuclear receptor is able to immediately turn on transcription of genes to result in synthesis of steroids, and is quickly inhibited by miRNAs after the stress reaction is completed.

MiRNAs were recently linked to adrenal tumors via the Wnt signaling pathway in both PPNAD and MMAD. MiR-449 was found to be highly down-regulated in PPNAD, while its target gene, WISP2, was found to be highly expressed compared to normal adrenal tissue. When PKA activity was inhibited in PPNAD cells, miR-449 expression was increased, while WISP2 expression decreased, thus demonstrating that PKA activates the Wnt pathway through miR-449 inhibition in PPNAD cells. Of clinical relevance, down-regulation of let-7b, a miRNA associated with lung and other cancers, was correlated with higher midnight cortisol levels, and thus with worsening disease severity³¹. With regards to AIMAH, a number of miRNAs were found to be differentially expressed in AIMAH tissue compared to normal adrenal tissue. Most notably, miR-200b was the highest down-regulated miRNA in AIMAH, and it was found to inhibit MATR3, a nuclear protein which regulates transcription. Specifically, MATR3 is degraded by PKA in normal tissues, and suggests an additional link to the cAMP/PKA signaling pathway in AIMAH, as well as a role for miRNAs in adrenal tumor formation. Interestingly, over-expression of miR-130a and miR-382 in AIMAH was positively correlated with higher midnight cortisol levels, as in PPNAD³².

Pharmacologic targets

The elucidation of the cAMP/PKA pathway has allowed for multiple potential targets for pharmacotherapy. One of the most well-studied inhibitors of the pathway is mifepristone (RU-486), a glucocorticoid receptor (GR) antagonist. It was first used to treat glucocorticoid excess in a patient with Cushing’s syndrome due to ectopic ACTH production³³. Mifepristone has high affinity for both the GR and the progesterone receptor (PR), and acts as a GR and PR antagonist in vivo³⁴, but does not bind to the mineralocorticoid or estrogen receptors. It has been used in the treatment of Cushing’s disease or ectopic ACTH-producing tumors that are not amenable to surgery, as well as in adrenocortical carcinoma to treat hypercortisolism.

RU-486 was studied in vitro in PPNAD. In a study evaluating one of the hallmarks of PPNAD, the paradoxical increase in cortisol production in response to dexamethasone, Louiset, et al.³⁵ cultured PPNAD cells along with various modulators of the cAMP/PKA pathway, and exposed them to RU-486. Mifepristone was found to block the paradoxical dexamethasone-induced increase in cortisol levels, as well as basal cortisol production.

Another compound that has been widely used in in vitro studies on the cAMP/PKA pathway is H89, a potent and selective inhibitor of PKA. In a study evaluating steroid production in the human placenta, the inhibition of PKA by H89 resulted in decreased progesterone synthesis in the mitochondria³⁶. H89 has also been evaluated for its clinical utility, as a cardioprotective agent. Myocardial ischemia is characterized, in part, by cAMP accumulation and activation of PKA. H89 has been demonstrated to reduce postischemic contractile recovery as well as reducing the size of infarct in rat heart isolates³⁷. In PPNAD cell cultures, miR-449 is down-regulated, while WISP2 expression is up-regulated. H89 treatment increased miR-449, as well as decreasing WISP2 expression, restoring normal cell cycle regulation³¹.

Given the important role of miRNA in regulation of adrenal cell signaling, inhibitors of miRNA have also been studied. Recent research has shown that let-7, a miRNA which functions as a tumor suppressor, is deficient in a number of tumors, including non-small-cell lung cancer. Replacement of let-7 miRNA resulted in decreased tumor size when injected intratumorally in two mouse lung cancer models, as well as reduced tumor burden in established lung tumors when given intranasally³⁸. Let-7 was also able to prevent growth of lung tumors³⁹. In the previously referenced study, Iliopoulos, et al.,³¹ also evaluated the effects of inhibition of miR-449 on WISP2 expression. Inhibition by as-miR-449 resulted in up-regulation of its target gene, WISP2, thus counteracting its tumor suppressor role. These studies suggest miRNA has a potential role as a therapeutic target for adrenal tumors.

The Wnt pathway, downstream from cAMP and PKA, has also been the focus of pharmacologic intervention. Quercetin, a flavonoid with known anti-carcinogenic properties, has been shown to inhibit the Wnt/β-catenin pathway. In a study by Park, et al.⁴⁰, the effects of Quercetin on colon cancer cells with genetic mutations resulting in constitutively activated β-catenin was evaluated. Binding of β-catenin to its transcription factor was inhibited by Quercetin, along with binding of the transcription factor to its DNA-binding sites. In addition, Quercetin reduced nuclear levels of both β-catenin and its transcription factor. In addition to Quercetin, there are a number of inhibitors in the pathway currently under investigation, including inhibitors at the Wnt receptor, inhibitors that target cytosolic signaling, and those that target nuclear signaling. Currently, there are at least two inhibitors of the Wnt pathway in Phase I trials, and offer the potential for targeted therapy for a number of tumors with known Wnt signaling defects. There are no known trials of these molecules in adrenocortical tumors, although it is clear that this would be worth exploring.

Conclusion

Mutations in the cAMP/PKA signaling pathway are present in the majority of benign cortisol-producing tumors of the adrenal cortex. The discovery of the role of the cAMP/PKA pathway, as well as Wnt signaling and miRNAs, and their importance in tumorigenesis, have lead to exciting new developments for potential pharmacologic targets. More studies are needed to elucidate the role of genetic variants in the formation of adrenal incidentalomas. In the future, specifically targeted pharmacologic therapy may replace surgical intervention as the treatment modality of choice for benign adrenal tumors.