Phosphodiesterases (PDEs) are enzymes that regulate the intracellular levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate, and, consequently, exhibit a central role in multiple cellular functions. The pharmacological exploitation of the ability of PDEs to regulate specific pathways has led to the discovery of drugs with selective action against specific PDE isoforms. Considerable attention has been given to the development of selective PDE inhibitors, especially after the therapeutic success of PDE5 inhibitors in the treatment of erectile dysfunction. Several associations between PDE genes and genetic diseases have been described, and more recently PDE11A and PDE8B have been implicated in predisposition to tumor formation. This review focuses on the possible function of PDEs in a variety of tumors, primarily in endocrine glands, both in tumor predisposition and as potential therapeutic targets.

The second messenger, cAMP, is one of the most important regulatory signals for control of steroidogenesis. This review focuses on current knowledge about regulation of cyclic nucleotides by phosphodiesterases (PDEs) in steroidogenic tissues. The first PDE known to directly regulate steroidogenesis was PDE2, the cGMP-stimulated PDE. PDE2 mediates ANP/cGMP-induced decreases in aldosterone production. Recently, the PDE8 family has been shown to control steroidogenesis in two tissues. Specifically, PDE8A regulates testosterone production by itself and in concert with additional IBMX-sensitive PDEs. PDE8B modulates basal corticosterone synthesis via acute and chronic mechanisms. In addition to cAMP-dependent pathways, cGMP signaling also can promote steroidogenesis, and PDE5 modulates this process. Finally, PDE mutations may lead to several human diseases characterized by abnormal steroid levels.

The cAMP-protein kinase A pathway plays a central role in the development and physiology of endocrine tissues. cAMP mediates the intracellular effects of numerous peptide hormones. Various cellular and molecular alterations of the cAMP-signaling pathway have been observed in endocrine diseases. Phosphodiesterases (PDEs) are key regulatory enzymes of intracellular cAMP levels. Indeed, PDEs are the only known mechanism for inactivation of cAMP by catalysis to 5'-AMP. It has been suggested that disruption of PDEs could also have a role in the pathogenesis of many endocrine diseases. This review summarizes the most recent advances concerning the role of the PDEs in the physiopathology of endocrine diseases. The potential significance of this knowledge can be easily envisaged by the development of drugs targeting specific PDEs.

The first highly potent and selective PDE8 inhibitors are disclosed. The initial tetrahydroisoquinoline hit was transformed into a nipecotic amide series in order to address a reactive metabolite issue. Reduction of lipophilicity to address metabolic liabilities uncovered an interesting diastereomer-dependent trend in turnover by human microsomes.

Objective: To review current knowledge on the involvement of cAMP and interacting signaling pathways in predisposition to tumor formation in primary pigmented nodular adrenocortical disease (PPNAD), a type of bilateral adrenal hyperplasia (BAH) related to the multiple endocrine neoplasia Carney complex (CNC), and also in isolated PPNAD and other BAHs.Methods: We review the pertinent literature.Results: Almost a decade ago, we discovered that PPNAD and CNC are caused by PRKAR1A mutations. PRKAR1A encodes the protein kinase A (PKA) regulatory subunit type 1A, an important regulator of cyclic adenosine monophosphate (cAMP) signaling in most cells. Recently, we described PKA or PRKAR1A abnormalities in a variety of other BAHs; in some of these cases, mutations in additional genes of the cAMP signaling pathway, the phosphodiesterases (PDEs), were identified. Transcriptomic analyses of human lesions or animal models showed that abnormal cAMP/PKA signaling in adrenals, and also in other tissues, such as the bone, lead to proliferation of tissue-specific pluripotential cells through activation of the Wnt signaling.Conclusions: Recent findings indicate the relevance of cAMP signaling in the pathogenesis of adrenocortical disease, and point to the Wnt signaling pathway as a potential important mediator of tumorigenesis related to increased cAMP and/or PKA signaling.

Familial pituitary adenomas can occur in MEN1 and Carney complex, as well as in the recently characterized familial isolated pituitary adenoma (FIPA) syndrome. FIPA is an autosomal dominant disease with incomplete penetrance, characterized by early-onset disease, often aggressive tumor growth and a predominance of somatotroph and lactotroph adenomas. In 20% of FIPA families, heterozygous mutations have been described in the aryl hydrocarbon receptor interacting (AIP) gene, whereas in other families the causative gene(s) are unknown. It has been suggested that AIP is a tumor suppressor gene and although experimental data support this hypothesis, the exact molecular mechanism by which its disruption leads to tumorigenesis is unclear. Here we discuss the clinical, genetic and molecular features of patients with FIPA.

Somatically acquired, activating mutations of GNAS, the gene encoding the stimulatory G-protein Gsalpha subunit, have been identified in kidney, thyroid, pituitary, leydig cell, adrenocortical and, more recently, in colorectal tumours, suggesting that mutations such as R201C may be oncogenic in these tissues. To study the role of GNAS in intestinal tumourigenesis, we placed GNAS R201C under the control of the A33-antigen promoter (Gpa33), which is almost exclusively expressed in the intestines. The GNAS R201C mutation has been shown to result in the constitutive activation of Gsalpha and adenylate cyclase and to lead to the autonomous synthesis of cyclic adenosine monophosphate (cAMP). Gpa33(tm1(GnasR201C)Wtsi/+) mice showed significantly elevated cAMP levels and a compensatory upregulation of cAMP-specific phosphodiesterases in the intestinal epithelium. GNAS R201C alone was not sufficient to induce tumourigenesis by 12 months, but there was a significant increase in adenoma formation when Gpa33(tm1(GnasR201C)Wtsi/+) mice were bred onto an Apc(Min/+) background. GNAS R201C expression was associated with elevated expression of Wnt and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) pathway target genes, increased phosphorylation of ERK1/2 MAPK and increased immunostaining for the proliferation marker Ki67. Furthermore, the effects of GNAS R201C on the Wnt pathway were additive to the inactivation of Apc. Our data strongly suggest that activating mutations of GNAS cooperate with inactivation of APC and are likely to contribute to colorectal tumourigenesis.

Familial pituitary adenomas occurr in the classical syndromes of MEN1 and Carney Complex as well as in Familial Isolated Pituitary Adenomas (FIPA), an autosomal dominant disease with incomplete penetrance. In some families and also rarely in sporadic tumours germline mutations of a gene located on chromosome 11q13 known as the aryl hydrocarbon receptor interacting protein have been found. This article discusses the AIP mutations in these groups and the different molecular interactions of AIP that may play a role in pituitary tumour formation.

Carney complex (CNC) is an inherited tumor predisposition associated with pituitary tumors, including GH-producing pituitary adenomas and rare reports of prolactinomas. This disease is caused by mutations in PRKAR1A, which encodes the type 1A regulatory subunit of the cAMP-dependent protein kinase, PKA. Loss of PRKAR1A causes enhanced PKA signaling, which leads to pituitary tumorigenesis. Mutations in the gene have not been detected in sporadic pituitary tumors, but there is some data to suggest that non-genomic mechanisms may cause loss of protein expression. Unlike CNC patients, mice heterozygous for Prkar1a mutations do not develop pituitary tumors, although complete knockout of the gene in the Pit1 lineage of the pituitary produces GH-secreting pituitary adenomas. These data indicate that complete loss of Prkar1a/PRKAR1A is able to cause pituitary tumors in mice and men. The pattern of tumors is likely related to the signaling pathways employed in specific pituitary cell types.

BACKGROUND: Human phosphodiesterase (PDE) type 8B (PDE8B) is located at 5q14.1 and is known as the PDE with the highest affinity to cAMP. We recently described a family with bilateral micronodular adrenocortical disease that was apparently caused by an inactivating PDE8B mutation (H305P). As a result of a genome-wide study, a strong association between six polymorphic variants in the PDE8B promoter and serum levels of the thyroid-stimulating hormone (TSH) has been recently reported. Despite an extended analysis of the regions surrounding 5q14.1, no other potential genetic variants that could be responsible for the associated TSH levels were found. METHODS: In this study, we genotyped by polymerase chain reaction the described six polymorphic variants in the PDE8B promoter in the family with micronodular adrenocortical disease and inactivating PDE8B mutation and analyzed their correlation with individual TSH values in the family members. RESULTS: We observed complete segregation between the reported association and individual TSH values in the family we studied. Haplotype analysis showed that the haplotype associated with the high TSH levels is different from the one that segregated with H305P, suggesting that the mutation most probably has arisen on an allele independent of the high TSH-associated allele. CONCLUSIONS: The proposed mechanism by which PDE8B may influence TSH levels is through control of cAMP signaling. Our analysis revealed separate segregation of an inactivating PDE8B allele from the high-TSH-allele and showed low TSH levels in persons who carry an inactivating PDE8B allele. These data suggest that, indeed, PDE8B may be involved in regulation of TSH levels.

BACKGROUND Carney complex (CNC) is a multiple endocrine neoplasia syndrome due to inactivating mutations in the PRKAR1A gene that codes for type Iα regulatory (RIα) subunit of protein kinase A. Most PRKAR1A mutations are subject to nonsense mRNA decay (NMD) and, thus, lead to haploinsufficiency. METHODS AND SETTING Patient phenotyping for CNC features and DNA, RNA, protein, and transfection studies were carried out at a research center. RESULTS We describe in unrelated kindreds with CNC four naturally occurring PRKAR1A mutations (1055del4, 1067del4ins5, 1076delTTins13, and 1142del4) that are predicted to escape NMD because they are located in the last coding exon of the gene. The phenotype of CNC was not different from that in other patients with the condition, although the number of patients was small. Each of the mutations caused a frameshift that led to a new stop codon into the 3' untranslated open reading frame, predicting an elongated protein that, however, was absent in patient-derived cells. After site-directed mutagenesis, in vitro transcription, and cell-free translation experiments, the expected size mutant proteins were present. However, when the mutant constructs were transfected in adrenal (NCI-295), testicular (N-TERA), and embryonic (HEK293) cells and despite the presence of the mutant mRNA, Western blot analysis indicated that there were no longer proteins. The subsequent application of proteasome inhibitors to cells transfected with the mutant constructs led to the detection of the aberrant proteins, although a compound that affects protein folding had no effect. The wild-type protein was also decreased in both patient-derived cells and/or tissues as well as in the in vitro systems used in this study. CONCLUSIONS This was the first demonstration of proteasomal degradation of RIα protein variants leading to PRKAR1A haploinsufficiency and CNC, adding protein surveillance to NMD in the cellular mechanisms overseeing RIα synthesis. In agreement with the molecular data, CNC patients bearing PRKAR1A defects that extend the open reading frame did not have a different phenotype, although this has to be confirmed in a larger number of patients.

PURPOSE: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in approximately 70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A-mutation-negative individuals with Carney complex. EXPERIMENTAL DESIGN: To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2. RESULTS: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma. CONCLUSIONS: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31-2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31-2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

BACKGROUND Carney complex (CNC) is a familial multiple neoplasia syndrome frequently associated with primary pigmented nodular adrenocortical disease (PPNAD), a bilateral form of micronodular adrenal hyperplasia that leads to Cushing's syndrome (CS). Germline PRKAR1A mutations cause CNC and only rarely isolated PPNAD. PATIENTS AND METHODS PRKAR1A mutation analysis in two large families with CS and no other CNC manifestations demonstrated a M1V germline mutation; a total of 21 asymptomatic individuals were screened, and mutation carriers were evaluated for CNC. The mutation was expressed in vitro and functionally tested for its effects on protein kinase A function. RESULTS Presymptomatic testing identified five first-degree relatives who were M1V carriers and who were all diagnosed with subclinical, mild CS at ages ranging from 20-56 yr. There were no other signs of CNC. In a cell-free system, we detected a shorter compared with the wild-type type 1alpha regulatory subunit of protein kinase A (PRKAR1A) protein (43 kDa). This was not identified in cell lines from the patients or in transfection experiments in HEK293 cells that showed no detectable PRKAR1A protein from the M1V-bearing constructs. In these cells, the mutant mRNA was expressed in a 1:1 ratio. CONCLUSION In two large families, the M1V PRKAR1A mutation resulted in a PPNAD-only phenotype with significant variability both in terms of age of onset and clinical severity. Expression studies showed a unique effect of this sequence change. This study has implications for genetic counseling of carriers of this PRKAR1A mutation and patients with CNC and PPNAD and for the study of PRKAR1A-related tumorigenesis.

Inactivating germline mutations in phosphodiesterase 11A (PDE11A) have been implicated in adrenal tumor susceptibility. PDE11A is highly expressed in endocrine steroidogenic tissues, especially the testis, and mice with inactivated Pde11a exhibit male infertility, a known testicular germ cell tumor (TGCT) risk factor. We sequenced the PDE11A gene-coding region in 95 patients with TGCT from 64 unrelated kindreds. We identified 8 nonsynonymous substitutions in 20 patients from 15 families: four (R52T, F258Y, G291R, and V820M) were newly recognized, three (R804H, R867G, and M878V) were functional variants previously implicated in adrenal tumor predisposition, and one (Y727C) was a known polymorphism. We compared the frequency of these variants in our patients to unrelated controls that had been screened and found negative for any endocrine diseases: only the two previously reported variants, R804H and R867G, known to be frequent in general population, were detected in these controls. The frequency of all PDE11A-gene variants (combined) was significantly higher among patients with TGCT (P = 0.0002), present in 19% of the families of our cohort. Most variants were detected in the general population, but functional studies showed that all these mutations reduced PDE activity, and that PDE11A protein expression was decreased (or absent) in TGCT samples from carriers. This is the first demonstration of the involvement of a PDE gene in TGCT, although the cyclic AMP signaling pathway has been investigated extensively in reproductive organ function and their diseases. In conclusion, we report that PDE11A-inactivating sequence variants may modify the risk of familial and bilateral TGCT.

Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1 alpha Delta 184-236 (R1 alpha Delta 6)]. We compared in vitro R1 alpha Delta 6 with wild-type (wt) R1 alpha. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1 alpha and mutant (mt) R1 alpha; we observed by confocal microscopy R1 alpha tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C alpha). Introduction of the R1 alpha Delta 6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1 alpha protein in wt-R1 alpha- and R1 alpha Delta 6-transfected cells but the former also exhibited discrete aggregates of R1 alpha that bound C alpha; these were absent in R1 alpha Delta 6-transfected cells and did not bind C alpha at baseline or in response to cyclic AMP. Other changes induced by R1 alpha Delta 6 included decreased nuclear C alpha. We conclude that R1 alpha Delta 6 leads to increased PKA activity through the mt-R1 alpha decreased binding to C alpha and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis.

Several types of adrenocortical tumors that lead to Cushing syndrome may be caused by aberrant cyclic AMP (cAMP) signaling. We recently identified patients with micronodular adrenocortical hyperplasia who were carriers of inactivating mutations in the 2q-located phosphodiesterase 11A (PDE11A) gene. We now studied the frequency of two missense substitutions, R804H and R867G, in conserved regions of the enzyme in several sets of normal controls, including 745 individuals enrolled in a longitudinal cohort study, the New York Cancer Project. In the latter, we also screened for the presence of the previously identified PDE11A nonsense mutations. R804H and R867G were frequent among patients with adrenocortical tumors; although statistical significance was not reached, these variants affected significantly enzymatic function in vitro with variable increases in cAMP and/or cyclic guanosine 3',5'-monophosphate levels in HeLa and HEK293 cells. Adrenocortical tissues carrying the R804H mutation showed 2q allelic losses and higher cyclic nucleotide levels and cAMP-responsive element binding protein phosphorylation. We conclude that missense mutations of the PDE11A gene that affect enzymatic activity in vitro are present in the general population; protein-truncating PDE11A mutations may also contribute to a predisposition to other tumors, in addition to their association with adrenocortical hyperplasia. We speculate that PDE11A genetic defects may be associated with adrenal pathology in a wider than previously suspected clinical spectrum that includes asymptomatic individuals.(Cancer Res 2006; 66(24): 11571-5).

CONTEXT Most tumors in Carney complex (CNC) are benign, including primary pigmented nodular adrenocortical disease (PPNAD), the main endocrine tumor in CNC. Adrenocortical cancer (AC) has never been observed in the syndrome. Herein, we describe a large Azorean family with CNC caused by a point mutation in the PRKAR1A gene coding for type 1-α (RIα) regulatory subunit of the cAMP-dependent protein kinase A, in which the index patient presented with AC. OBJECTIVE We studied the genotype-phenotype correlation in CNC. DESIGN AND SETTING We reported on case series and in vitro testing of the PRKAR1A mutation in a tertiary care referral center. PATIENTS Twenty-two members of a family were investigated for Cushing syndrome and other CNC components; their DNA was sequenced for PRKAR1A mutations. RESULTS Cushing syndrome due to PPNAD occurred in four patients, including the proposita who presented with AC and three who had Cushing syndrome and/or PPNAD. Lentigines were found in six additional patients who did not have PPNAD. A base substitution (c.439A>G/p.S147G) in PRKAR1A was identified in the proposita, in the three others with PPNAD, in the proposita's twin daughters who had lentigines but no evidence of hypercortisolism, and in five other family members, including one without lentigines or evidence of hypercortisolism. Unlike in other RIα defects, loss of heterozygosity was not observed in AC. The S147G mutation was compared to other expressed PRKAR1A mutations; it led to decreased cAMP and catalytic subunit binding by RIα and increased protein kinase A activity in vitro. CONCLUSIONS In a large family with CNC, one amino acid substitution caused a spectrum of adrenal disease that ranged from lack of manifestations to cancer. PPNAD and AC were the only manifestations of CNC in these patients, in addition to lentigines. These data have implications for counseling patients with CNC and are significant in documenting the first case of AC in the context of PPNAD.

CONTEXT Primary pigmented nodular adrenocortical disease, associated with Carney complex, is caused by mutations in PRKAR1A (mt-PRKAR1A), a gene that codes for the regulatory subunit type 1alpha (RIalpha) of cAMP-dependent protein kinase (PKA). PRKAR1A inactivation is associated with dysregulated PKA activity that is thought to result in tumorigenesis. mt-PRKAR1A-bearing lymphocytes from Carney complex patients exhibit enhanced cell proliferation associated with increased expression of the MAPK ERK1/2 pathway. OBJECTIVE The objective of the study was to determine how PKA and its subunits and ERK1/2 and their molecular partners change in the presence of PRKAR1A mutations in adrenocortical tissue. DESIGN PKA activity and subunit expression, ERK1/2, other immunoassays, and immunohistochemistry on adrenocortical samples from patients with germline normal or mt-PRKAR1A were analyzed. RESULTS Increased cAMP-stimulated total kinase activity was associated with mt-PRKAR1A. PKA subunit expression analysis in mt-PRKAR1A tissues, by quantitative mRNA assay and immunoblotting, showed a 2.4-fold (P = 0.02) and 1.8-fold (P = 0.09) decrease in RIalpha's message and protein, respectively, and increases in other PKA subunits. Immunoassays showed 2-fold (P = 0.03) and 6-fold (P = 0.03) decreases in baseline ERK1/2, with corresponding increases in phosphorylated (p) ERK1/2 in mt-PRKAR1A samples. B-raf kinase, p-MEK1/2, and p-c-Myc, but not p-Akt/protein kinase B, were significantly increased. Immunohistochemistry studies supported these data. CONCLUSIONS mt-PRKAR1A causes increased total cAMP-stimulated kinase activity, likely the result of up-regulation of other PKA subunits caused by down-regulation of RIalpha, as seen in human lymphocytes and mouse animal models. These changes, associated with enhanced MAPK activity, may be, in part, responsible for the proliferative signals that result in primary pigmented nodular adrenocortical disease.

BACKGROUND: Genetic aberrations in various components of cAMP signalling pathway predispose to endocrine tumors. Growing evidence has shown that mutations in the phosphodiesterases (PDEs) are involved in the predisposition to adrenocortical neoplastic conditions. OBJECTIVE: Screen for genetic variations in PDE8Bamong patients with different types of adrenocortical tumors. DESIGN AND SUBJECTS: Case-control study followed by functional analyses. 216 unrelated patients with different types of adrenocortical tumors and 192 healthy control individuals. METHODS: BI-DIRECTIONAL SANGER SEQUENCING, IN VITRO CELL LINE TRANSFECTION, IN SILICO MODELLING.: RESULTS: Nine different PDE8B sequence changes, 6 novel and 3 previously reported, were identified in our patients and controls. Two of the variations, seen only in the patient group, showed significant potential to impair protein function, both in vitro and in silico. CONCLUSION: PDE8B is another gene in which variations may contribute to predisposition of adrenocortical tumors.