Genetics and epigenetics of adrenocortical tumors

Abstract

Adrenocortical tumors are common neoplasms. Most are benign, nonfunctional and clinically irrelevant. However, adrenocortical carcinoma is a rare disease with a dismal prognosis and no effective treatment apart from surgical resection. The molecular genetics of adrenocortical tumors remain poorly understood. For decades, molecular studies relied on a small number of samples and were directed to candidate-genes. This approach, based on the elucidation of the genetics of rare genetic syndromes in which adrenocortical tumors are a manifestation, has led to the discovery of major dysfunctional molecular pathways in adrenocortical tumors, such as the IGF pathway, the Wnt pathway and TP53. However, with the advent of high-throughput methodologies and the organization of international consortiums to obtain a larger number of samples and high-quality clinical data, this paradigm is rapidly changing. In the last decade, genome-wide expression profile studies, microRNA profiling and methylation profiling allowed the identification of subgroups of tumors with distinct genetic markers, molecular pathways activation patterns and clinical behavior. As a consequence, molecular classification of tumors has proven to be superior to traditional histological and clinical methods in prognosis prediction. In addition, this knowledge has also allowed the proposal of molecular-targeted approaches to provide better treatment options for advanced disease. This review aims to summarize the most relevant data on the rapidly evolving field of genetics of adrenal disorders.

Keywords: Adenoma; Adrenocortical; Carcinoma; Epigenetics; Genetics; Hyperplasia.

Figure 1. The cAMP signaling pathway in the adrenal cortex

A) ACTH binds to its specific seven-domain transmembrane receptors (MC2R), leading to activation the Ga_s protein, which will activate adenyl cyclase (AC) and, therefore, cAMP production. After binding of cAMP to PKA regulatory subunits, the catalytic subunits are released and activated. The free catalytic subunit (C) then phosphorylates CREB at serine 133, which is translocated to the nucleus and activates the transcription of target genes. cAMP is inactivated by phosphodiesterases, allowing the PKA tetramer to reassembled, terminating its activity. B) Anomalous activation of cAMP pathway in adrenocortical disease. At cell membrane level: 1. activating mutations of protein Ga_s lead to constitutive activation; 2. expression of “illicit” G protein-coupled receptors cause pathway activation in response to unusual ligands. Inactivating mutations of PKA regulatory subunit PRKAR1A or LOH at the gene locus lead to constitutive catalytic subunits activation. Inactivating mutations of phosphodiesteraes (PDE) lead to an increase in cytoplasmatic cAMP levels, augmenting PKA activity.

Figure 1. The cAMP signaling pathway in the adrenal cortex

A) ACTH binds to its specific seven-domain transmembrane receptors (MC2R), leading to activation the Ga_s protein, which will activate adenyl cyclase (AC) and, therefore, cAMP production. After binding of cAMP to PKA regulatory subunits, the catalytic subunits are released and activated. The free catalytic subunit (C) then phosphorylates CREB at serine 133, which is translocated to the nucleus and activates the transcription of target genes. cAMP is inactivated by phosphodiesterases, allowing the PKA tetramer to reassembled, terminating its activity. B) Anomalous activation of cAMP pathway in adrenocortical disease. At cell membrane level: 1. activating mutations of protein Ga_s lead to constitutive activation; 2. expression of “illicit” G protein-coupled receptors cause pathway activation in response to unusual ligands. Inactivating mutations of PKA regulatory subunit PRKAR1A or LOH at the gene locus lead to constitutive catalytic subunits activation. Inactivating mutations of phosphodiesteraes (PDE) lead to an increase in cytoplasmatic cAMP levels, augmenting PKA activity.

Figure 2. Physiology of aldosterone secretion in the normal adrenal cortex

A) In the absence of stimulus, the activity of the ATP1A1 Na⁺/K⁺ATPase and the KCNJ5 potassium channel create an electrical gradient between the inner surface and the outer surface of the plasma membrane. The electrical gradient (polarization) closes the L-type and T-type voltage-gated calcium channels. In addition, the activity of the ATP2B3 calcium pump keeps the cytosolic levels of calcium very low. B) The activation of the angiotensin receptor (ATR) by angiotensin 2 (AT2) inhibits the activity of the ATP1A1 Na⁺/K⁺ ATPase and decreases the K⁺ permeability of the KCNJ5 channel. The resulting depolarization of the plasma membrane will open the voltage-gated calcium channel, causing an inward calcium flow, elevating its cytosolic levels. As a result, a cytosolic calcium-sensitive protein kinase (CAMK) will be activated, promoting the phosphorilation of transcription factors such as CREB, NURR1, NGFIB and ATF1, which in turn will promote CYP11B2 (aldosterone synthase) transcription. C) Molecular mechanisms beyond aldosterone overproduction in aldosterone-producing adenomas. A permanent increase in cytosolic calcium levels causes constitutive activation of CAMK and downstream pathways, leading to aldosterone overproduction. The increased cytosolic calcium may be the result of a permanent depolarization of plasma membrane caused by somatic KCNJ5 mutations (which will result in loss of to selectivity of the channel to K ions, allowing an influx of Na⁺) or by inactivating ATP1A1 Na⁺/K⁺ ATPase mutations. In addition, high cytosolic calcium levels may be also resultant of inactivating mutations of the ATP2B3 Ca⁺² ATPase.

Figure 2. Physiology of aldosterone secretion in the normal adrenal cortex

A) In the absence of stimulus, the activity of the ATP1A1 Na⁺/K⁺ATPase and the KCNJ5 potassium channel create an electrical gradient between the inner surface and the outer surface of the plasma membrane. The electrical gradient (polarization) closes the L-type and T-type voltage-gated calcium channels. In addition, the activity of the ATP2B3 calcium pump keeps the cytosolic levels of calcium very low. B) The activation of the angiotensin receptor (ATR) by angiotensin 2 (AT2) inhibits the activity of the ATP1A1 Na⁺/K⁺ ATPase and decreases the K⁺ permeability of the KCNJ5 channel. The resulting depolarization of the plasma membrane will open the voltage-gated calcium channel, causing an inward calcium flow, elevating its cytosolic levels. As a result, a cytosolic calcium-sensitive protein kinase (CAMK) will be activated, promoting the phosphorilation of transcription factors such as CREB, NURR1, NGFIB and ATF1, which in turn will promote CYP11B2 (aldosterone synthase) transcription. C) Molecular mechanisms beyond aldosterone overproduction in aldosterone-producing adenomas. A permanent increase in cytosolic calcium levels causes constitutive activation of CAMK and downstream pathways, leading to aldosterone overproduction. The increased cytosolic calcium may be the result of a permanent depolarization of plasma membrane caused by somatic KCNJ5 mutations (which will result in loss of to selectivity of the channel to K ions, allowing an influx of Na⁺) or by inactivating ATP1A1 Na⁺/K⁺ ATPase mutations. In addition, high cytosolic calcium levels may be also resultant of inactivating mutations of the ATP2B3 Ca⁺² ATPase.

Figure 2. Physiology of aldosterone secretion in the normal adrenal cortex

A) In the absence of stimulus, the activity of the ATP1A1 Na⁺/K⁺ATPase and the KCNJ5 potassium channel create an electrical gradient between the inner surface and the outer surface of the plasma membrane. The electrical gradient (polarization) closes the L-type and T-type voltage-gated calcium channels. In addition, the activity of the ATP2B3 calcium pump keeps the cytosolic levels of calcium very low. B) The activation of the angiotensin receptor (ATR) by angiotensin 2 (AT2) inhibits the activity of the ATP1A1 Na⁺/K⁺ ATPase and decreases the K⁺ permeability of the KCNJ5 channel. The resulting depolarization of the plasma membrane will open the voltage-gated calcium channel, causing an inward calcium flow, elevating its cytosolic levels. As a result, a cytosolic calcium-sensitive protein kinase (CAMK) will be activated, promoting the phosphorilation of transcription factors such as CREB, NURR1, NGFIB and ATF1, which in turn will promote CYP11B2 (aldosterone synthase) transcription. C) Molecular mechanisms beyond aldosterone overproduction in aldosterone-producing adenomas. A permanent increase in cytosolic calcium levels causes constitutive activation of CAMK and downstream pathways, leading to aldosterone overproduction. The increased cytosolic calcium may be the result of a permanent depolarization of plasma membrane caused by somatic KCNJ5 mutations (which will result in loss of to selectivity of the channel to K ions, allowing an influx of Na⁺) or by inactivating ATP1A1 Na⁺/K⁺ ATPase mutations. In addition, high cytosolic calcium levels may be also resultant of inactivating mutations of the ATP2B3 Ca⁺² ATPase.

Figure 3

Molecular mechanism beyond IGF2 overexpression in ACC. IGF2 is located in chromosome 11p12, downstream to the cell cycle regulator CDKN1C and upstream to the H19 transcription regulator. In normal cells, IGF2 is imprinted on the maternal allele and CDKN1C and H19 are imprinted on the paternal allele. In ACC, the maternal copy of these genes is lost and the paternal copy gets duplicated, resulting in biallelic IGF2 expression and in transcriptional silencing of CDKN1C and H19.

Figure 4. Wnt signaling pathway in normal and cancer cells

A) In the absence of the ligand, cytosolic β-catenin is continuosly phosphorilated by the «destruction complex». This phosphorilation targets β-catenin to proteasomal (PR) degradation. B) After binding of Wnt to Frizzled and LRP5/6, GSK3 and CK1 phosphorilate LRP5/6, which in turn recruits axin. The ultimate consequence is the dissociation of the «destruction complex» and accumulation of unphosphorilated β-catenin on the cytosol which is translocated to the nucleus and activates the transcription of target genes. C) One of the mechanisms by which the Wnt pathway is constitutively activated in cancer cells is the presence of an activating somatic mutation on CTNNB1 gene. As a result of an abnormal phosphorilation site, the mutant β-catenin is no longer phosphorilated by the «destruction complex», being accumulated at the cytosol and translocated to the nucleus, where it activates the trancription of target genes.

Figure 5

Tyrosine-kinase coupled receptors (TKs) that are abnormally activated in ACC and downstream signaling pathways. The effects of pharmacological inhibition of some of these molecules have been evaluated in pre-clinical studies and clinical trials. The pharmacological compounds and specific targets are indicated.

Figure 6

Molecular findings in the whole spectrum of nodular adrenocortical disease. Some alterations are shared by benign disease (hyperplasias and ACA) and ACC and are thought to be early events in the tumorigenesis process. Alterations that are exclusive of ACC are thought to be late events.