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Cardiac myxomas are rare tumors that usually occur as sporadic lesions or,more rarely, in the familial form,mostly in the context of Carney complex (CNC). The molecular basis for the development of cardiac myxomas is unclear. However, somatic activating mutations in the GNAS1 gene (the gsp oncogene) are detected in the myocardium ofMcCune-Albright syndrome patients while germ-line mutations in the PRKAR1A gene are associated with CNC and familial myxomas. We investigated the presence of activating missense mutations in the GNAS1 gene as well as of inactivating mutations in PRKAR1A in 29 sporadically occurring cardiac myxomas. No gsp and no PRKAR1A mutations were found by direct sequencing of PCR products amplified from tumoral DNA. This is the first study including a large series of sporadic, isolated cardiac myxomas and showing that these cardiac neoplasms do not share the same mutations found in familial forms.

Background: The "complex of myxomas, spotty skin pigmentation, and endocrine overactivity," or "Carney complex" (CNC), is caused by inactivating mutations of the regulatory subunit type 1A of the cAMP-dependent protein kinase (PRKAR1A) gene and as yet unknown defect(s) in other gene(s). Delineation of a genotype-phenotype correlation for CNC patients is essential for understanding PRKAR1A function and providing counseling and preventive care., Methods: A transatlantic consortium studied the molecular genotype and clinical phenotype of 353 patients (221 females and 132 males, age 34 +/- 19 yr) who carried a germline PRKAR1A mutation or were diagnosed with CNC and/or primary pigmented nodular adrenocortical disease., Results: A total of 258 patients (73%) carried 80 different PRKAR1A mutations; 114 (62%) of the index cases had a PRKAR1A mutation. Most PRKAR1A mutations (82%) led to lack of detectable mutant protein (nonexpressed mutations) because of nonsense mRNA mediated decay. Patients with a PRKAR1A mutation were more likely to have pigmented skin lesions, myxomas, and thyroid and gonadal tumors; they also presented earlier with these tumors. Primary pigmented nodular adrenocortical disease occurred earlier, was more frequent in females, and was the only manifestation of CNC with a gender predilection. Mutations located in exons were more often associated with acromegaly, myxomas, lentigines, and schwannomas, whereas the frequent c.491-492delTG mutation was commonly associated with lentigines, cardiac myxomas, and thyroid tumors. Overall, nonexpressed PRKAR1A mutations were associated with less severe disease., Conclusion: CNC is genetically and clinically heterogeneous. Certain tumors are more frequent, with specific mutations providing some genotype-phenotype correlation for PRKAR1A mutations.

Context: The GHRH-protein kinase A (PKA) signalling pathway is essential for cell proliferation and GH synthesis/secretion in somatotrophs. An inactivating mutation of PRKAR1A is one of the causes of somatotrophinoma in Carney complex (CNC). The basal PKA activity of somatotroph adenoma cells from CNC has not been evaluated because of a limited amount of available tissue., Objective: This study examined how the PRKAR1A mutation affects the PKA signalling pathway in a human somatotrophinoma with a PRKAR1A mutation., Design and Setting: Somatotrophinoma cells from a 40-year-old male patient with CNC were used. The patient had a novel somatic heterozygous germline frameshift mutation (227delT) in PRKAR1A leading to a premature stop codon. The tumour showed loss of heterozygosity (LOH) at 17q23-24. Primary cultured adenoma cells were subjected to electrophysiological experiments to evaluate PKA signalling in individual cells., Results: GHRH did not increase the nonselective cation current or the voltage-gated calcium current in these adenoma cells, in contrast to nonadenomatous somatotroph cells in which these currents increase through the PKA pathway. Application of a PKA inhibitor inhibited the basal currents in these adenoma cells, results that were not observed in nonadenomatous somatotrophs. These data indicate that the basal currents are already increased and cannot be further increased by GHRH., Conclusions: The results demonstrate that PKA is activated at the basal state in these adenoma cells. The data also show that both the nonselective cation current and the voltage-gated calcium current, vital regulators of GH secretion downstream of PKA, are maximally increased in these cells. These maximally increased currents probably account for the excessive GH secretion.

The genesis of pituitary tumours is still under debate. Although these neoplasias are monoclonal in origin, mutations of GNAS1, the gene encoding the alpha subunit of the stimulatory G-protein, Gs, are the only mutational changes unequivocally associated with growth hormone (GH)-secreting adenomas. However, despite the growth advantage that this oncogene has been demonstrated to confer in vitro, patients carrying this mutation have a similar clinical and biochemical phenotype to those who do not carry it. This discrepancy is due to the occurrence of events able to counteract the biological effect of the mutation. Consistent with a potential role of the cyclic adenosine monophosphate pathway in the proliferation of somatotrophs, germline mutations of the gene encoding the type 1alpha regulatory subunit of protein kinase A (PRKAR1A) have been found in patients with the Carney complex, a syndrome including GH-secreting adenomas, whereas alterations in the expression levels of this subunit are frequently observed in sporadic adenomas., (Copyright 2009 S. Karger AG, Basel.)

Ectopic thymic tissue within the thyroid gland is rare. Patients with a complex of myxomas, spotty skin pigmentation, and endocrine overactivity, collectively known as Carney complex (CNC), have a predisposition towards the development of thyroid abnormalities, but there are no reports of thymic defects in CNC. We present the case of a 12-year-old boy with CNC and a growing thyroid nodule. The patient had the c.682 C > T (Arg228X) pathogenic PRKAR1A mutation. Hemithyroidectomy for a Hürthle cell adenoma led to the confirmation of distinct intrathyroidal ectopic thymic tissue. Thymic abnormalities have not been previously reported in CNC.

Cardiac myxomas are the most common primary tumors of the heart, although little is known about their etiology. Mutations of the protein kinase A regulatory subunit gene PRKAR1A cause inherited myxomas in the setting of the Carney complex tumor syndrome, providing a possible window for understanding their pathogenesis. We recently reported that cardiac-specific knockout of this gene causes myxomatous changes in the heart, although the mice die during gestation from cardiac failure. In this review, we discuss these findings and place them in the larger understanding of how protein kinase A dysregulation might affect cardiac function and cause myxomagenesis.

The study involved 63 patients with an echocardiographic, surgical and histopathologic diagnosis of cardiac myxoma who were seen over a period of 20 years. Tumor recurrence or relapse was documented in five of these patients (7.9%), 3 of whom had a confirmed diagnosis of Carney complex, while one other patient had a probable diagnosis. Genetic studies demonstrated abnormalities in the PRKAR1A gene on chromosome 17 in 2 patients and their immediate family. In 11 of the 58 patients who did not experience relapse of the myxoma, genetic studies failed to show any abnormality. In conclusion, the possible presence of the Carney complex should be investigated in patients with multiple myxomas or with a cardiac myxoma whose location is atypical.

Objective: Toxic thyroid adenoma (TA) is a common cause of hyperthyroidism. Mutations in the TSH receptor (TSHR) gene, and less frequently in the adenylate cyclase-stimulating G alpha protein (GNAS) gene, are well established causes of TA in Europe. However, genetic causes of TA remain unknown in a small percentage of cases. We report the first study to investigate mutations in TSHR, GNAS, protein kinase, cAMP-dependent, regulatory, type I alpha (PRKAR1A) and RAS genes, in a large series of TA from Galicia, an iodine-deficient region in NW Spain., Design and Methods: Eighty-five TA samples were obtained surgically from 77 hyperthyroid patients, operated on for treatment of non-autoimmune toxic nodular goitre. After DNA extraction, all coding exons of TSHR, GNAS and PRKAR1A genes, and exons 2 and 3 of HRAS, KRAS and NRAS were amplified by PCR and sequenced. Previously unreported mutants were cloned in expression vectors and their basal constitutive activities were determined by quantification of cAMP response element (CRE)-luciferase activity in CO7 cells transfected with wild-type and mutant plasmids., Results: TSHR gene mutations were found in 52 (61.2%) samples, GNAS gene mutations in 4 (4.71%) samples and no PRKAR1A or RAS mutations were found. Only three previously unreported mutations were found, two affecting the TSHR, A623F and I635V, and one affecting the G-protein alpha-subunit (Gsalpha), L203P. All mutant proteins showed higher CRE-luciferase activity than their wild-type counterparts., Conclusions: TA in a hyperthyroid population living in Galicia, a Spanish iodine-deficient region, harbours elevated frequencies of TSHR and GNAS mutations activating the cAMP pathway. However, the genetic cause of TA was undetermined in 34% of the TA samples.

Objective: Primary pigmented nodular adrenocortical disease (PPNAD) is the main endocrine manifestation of Carney complex, a multiple neoplasia syndrome caused by PRKAR1A gene mutations. The presence of PRKAR1A loss of heterozygosity (LOH) in adrenocortical tumorigenesis remains controversial. The aim of the present study is to investigate the presence of PRKAR1A LOH in adrenocortical cells in a patient with Carney complex., Methods: The LOH was investigated using a PRKAR1A informative intragenic marker by GeneScan software analysis in DNA obtained from laser-captured microdissected cells of several adrenal nodules., Patients: A young adult male patient with Carney complex and his family were studied., Results: A novel heterozygous mutation (p. Y21X) was identified at PRKAR1A in blood DNA of the male proband and his relatives. No PRKAR1A LOH was evidenced in the laser-captured microdissected cells from PPNAD tissue by different methodologies., Conclusion: We identified a new PRKAR1A nonsense mutation and in addition we did not evidence PRKAR1A LOH in laser-captured nodules cells, suggesting that adrenocortical tumorigenesis in PPNAD may occurs apart from the second hit.

Signaling events leading to Schwann cell tumor initiation have been extensively characterized in the context of neurofibromatosis (NF). Similar tumors are also observed in patients with the endocrine neoplasia syndrome Carney complex, which results from inactivating mutations in PRKAR1A. Loss of PRKAR1A causes enhanced protein kinase A activity, although the pathways leading to tumorigenesis are not well characterized. Tissue-specific ablation of Prkar1a in neural crest precursor cells (TEC3KO mice) causes schwannomas with nearly 80% penetrance by 10 months. These heterogeneous neoplasms were clinically characterized as genetically engineered mouse schwannomas, grades II and III. At the molecular level, analysis of the tumors revealed almost complete loss of both NF proteins, despite the fact that transcript levels were increased, implying posttranscriptional regulation. Although Erk and Akt signaling are typically enhanced in NF-associated tumors, we observed no activation of either of these pathways in TEC3KO tumors. Furthermore, the small G proteins Ras, Rac1, and RhoA are all known to be involved with NF signaling. In TEC3KO tumors, all three molecules showed modest increases in total protein, but only Rac1 showed significant activation. These data suggest that dysregulated protein kinase A activation causes tumorigenesis through pathways that overlap but are distinct from those described in NF tumorigenesis.

Carney complex is an autosomal dominantly inherited disease characterized by skin pigmentation, myxoma, primary pigmented nodular adrenocortical disease (PPNAD), and acromegaly. However, only a few incidences of PPNAD combined with acromegaly are observed in patients. The type 1alpha regulatory subunit of cAMP-dependent protein kinase (PRKAR1A) has been identified in patients as a causative gene for Carney complex by a positional cloning approach. Here, we report a female patient diagnosed with Cushing's syndrome and a GH-producing pituitary adenoma without otherwise evident acromegaly that could be diagnosed only by specialized endocrinological tests. Based on family history of acromegaly (mother and sister) and the fact that the combination of both diseases is very rare, genetic diagnosis involving Carney complex was considered to be appropriate. The 10 exons and flanking regions of PRKAR1A were screened for mutations by direct DNA sequencing. The patient and her mother and sister were found to have the same, novel frameshift mutation resulting from a single base deletion in exon 6 coding cAMP-binding domain A, denoted c.597delC in PRKAR1A. This single base deletion generated an immature stop codon at the sixth codon (p.Phe200LeufsX6). Even family members with the same mutation can show distinct phenotypes, suggesting that Carney complex is a multifactorial disorder comprising various genetic and environmental factors. Genetic diagnosis makes it possible to prepare more effective therapeutic strategies for patients and gene carriers and to avoid unnecessary tests for non-carriers in the family of the patient.

Background: Carney complex is an autosomal dominant endocrine disorder associated with skin involvement., Objective: To describe the dermatological signs of patients diagnosed with Carney complex (CNC) or primary pigmented adrenocortical nodular disease (PPNAD)., Methods: We conducted a prospective, single-center descriptive study of inpatients and outpatients at a university hospital endocrinology department. Sixteen patients from 14 families diagnosed with CNC or PPNAD were prospectively included in the study between September 2003 and March 2006. Data collected were age at enrollment; sex; Fitzpatrick skin phototype; the presence, location, and density of classic CNC skin lesions--lentigines, freckles, blue nevi, cutaneous myxoma--and other non-disease-specific skin lesions. Histopathologic analysis was carried out in cases in which the lesions were thought to be degenerative or to confirm the diagnosis. Patients were systematically assessed for endocrine and visceral involvement and genotyped for the PRKAR1A gene., Results: Twelve patients had lentiginosis (75%), 7 patients had blue nevi (43%), and 5 patients had cutaneous myxoma (31%). Patients could be classified into 3 groups based on skin signs: patients with no prominent skin lesions (n = 3), patients with skin lesions that could not be directly linked to CNC (n = 4), and patients with cutaneous lesions suggestive of CNC (n = 9). We found a correlation between dermatological and endocrine signs in 3 groups of patients: patients with few lesions, patients with an intermediate phenotype, and patients with both many endocrine signs and dermatological signs., Limitations: The classification proposed in our study should be validated on more patients., Conclusions: Skin manifestations are heterogeneous in patients with CNC, and skin phenotype seems to be correlated with endocrine phenotype.

Context: Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia syndrome (OMIM 160980). About 70% of cases are familiar; most have mutations of the PRKAR1A gene on chromosome 17q22-24. There is little phenotype-genotype correlation known to date., Objective: To study the genotype-phenotype correlation in a family with newly diagnosed CNC and three generations of subjects bearing the same PRKAR1A mutation. The proband was diagnosed with hepatocellular carcinoma, a tumour that appears to be associated with CNC., Design: The study consisted of clinical and genetic analysis of a total of 10 individuals belonging to a large Italian family., Patients: The index case was referred for PRKAR1A gene mutation analysis because he met the diagnostic criteria for a clinical diagnosis of CNC., Results: The PRKAR1A-inactivating mutation c.502 +1G > A in the intron 5 splice-donor site was detected after bidirectional sequencing of germline DNA. The mutation causes a frameshift in the transcribed sequence and a nonsense mRNA that was shown to be degraded; this leads to PRKAR1A haploinsufficiency in all tissues. All available relatives were screened first by DNA testing and, if the latter was positive, by clinical, biochemical and imaging means., Conclusions: A novel PRKAR1A mutation with an apparently low penetrance and variable expression is reported; the same mutation is also associated with a hepatocellular carcinoma. This is the first time a PRKAR1A mutation is reported in individuals who were diagnosed with CNC after retrospective family screening and following the identification of a proband; the finding has implications for genetic counselling on PRKAR1A and/or CNC.

Most adrenocortical tumors are benign; adrenocortical carcinomas are rare but their prognosis is poor and few therapeutic options are available. In most adrenocortical tumors, the morphological approach provides enough elements to establish the differential diagnosis between a benign and a malignant tumor but in few cases, it is insufficient. Moreover, morphology is limited for predicting prognosis of adrenocortical carcinomas. These observations led to development of other approaches, in particular immunohistochemical and genetic approaches. The comprehension of the genetic syndromes associated with adrenocortical tumors led to progress in the identification of genetic abnormalities involved in sporadic adrenocortical tumorigenesis. Thus, in sporadic adrenocortical tumorigenesis, IGF-II overexpression and cyclin E overproduction have been associated with 11p15 alterations which are observed in Bethwith-Wiedemann syndrome and TP53 inactivating mutations and 17p13 locus abnormalities which are observed in Li-Fraumeni syndrome. Activation of the Wnt/ss-catenin signaling pathway which is observed in familial adenomatous polyposis has been found in adrenocortical adenomas and carcinomas associated to mutations of CTNNB1, the gene coding ss-catenin, suggesting a central role for this pathway in adrenocortical tumorigenesis. These genetics findings already have had repercussions for patients via the development of molecular markers for diagnosis and prognosis; in the future they should be helpful in the development of new therapeutics.

Background: The Wnt/beta-catenin and cAMP signaling pathways play an important role in adrenal cortex tumorigenesis. Somatic activating mutations of the beta-catenin gene (CTNNB1) are the most frequent genetic defects identified both in adrenocortical adenomas (ACAs) and adrenocortical cancers (ACCs). PRKAR1A mutations leading to cAMP pathway dysregulation are observed in primary pigmented nodular adrenocortical diseases (PPNADs) and some sporadic ACAs., Objective: The objective of the investigation was to study Wnt/beta-catenin dysregulation in adrenocortical tumors (ACTs) with cAMP pathway genetic alteration and search for secondary CTNNB1 somatic mutations in heterogeneous tumors., Patients and Methods: Nine PPNADs, including five with macronodules, three ACAs with PRKAR1A somatic mutations, and one heterogeneous tumor with ACC developed within an ACA, were studied by immunohistochemistry and DNA sequencing., Results: beta-Catenin accumulation was observed in all PPNADs, ACAs with PRKAR1A mutations, and the ACC component of the heterogeneous tumor. CTNNB1 somatic activating mutations were found in the macronodule of two of the five macronodular PPNADs, in one ACA with a PRKAR1A somatic mutation, and in the malignant part of the heterogeneous ACT., Conclusions: The Wnt/beta-catenin pathway is activated in PPNADs and ACAs with PRKAR1A mutations, suggesting a cross talk between the cAMP and Wnt/beta-catenin pathways in ACT development. In addition, the occurrence as an additional hit of a CTNNB1 somatic mutation is associated with larger or more aggressive ACTs. This underlines the importance of the Wnt/beta-catenin pathway in adrenal cortex tumorigenesis and the importance of genetic accumulation in the progression of ACTs.

Background: Primary pigmented nodular adrenocortical disease (PPNAD) leads to Cushing syndrome (CS) and is often associated with Carney complex (CNC). Genetic alterations of the type 1-alpha regulatory subunit of cAMP-dependent protein kinase A (PRKAR1A) and phosphodiesterase 11A4 (PDE11A) genes have been found in PPNAD. Recent studies have demonstrated that beta-catenin mutations are frequent in adrenocortical adenomas and carcinomas and that the Wnt-signalling pathway is involved in PPNAD tumorigenesis. We hypothesized that adrenocortical adenomas that form in the context of PPNAD may harbour beta-catenin mutations., Methods: We studied 18 patients with CS secondary to PPNAD who were screened for germline PRKAR1A and PDE11A mutations. Tumor DNA was extracted from pigmented adrenocortical adenoma and nodular adrenal hyperplasia. Mutation analysis of exons 3 and 5 of beta-catenin was performed using polymerase chain reaction and direct sequencing. Sections from formalin-fixed, paraffin-embedded tumour samples were studied by immunohistochemistry with an antibody against beta-catenin., Results: Nine patients were carrying germline PRKAR1A mutations and one patient had a PDE11A mutation. We found somatic beta-catenin mutations in 2 of 18 patients (11%). In both cases, the mutations occurred in relatively large adenomas that had formed in the background of PPNAD. Tumor DNA analysis revealed a heterozygous ACC-to-GCC missense mutation in codon 41 (T41A) and a TCT-to-CCT missense mutation in codon 45 (S45P) of exon 3 of the beta-catenin gene that was confirmed at the cDNA level. There were no alterations in the DNA of PPNAD-adjacent tissues and lymphocytes from the patients, indicating somatic events. Immunohistochemistry showed nuclear accumulation of beta-catenin in more than 90% of cells in adenomatous tissue whereas no nuclear immunoreactivity was detected in adjacent PPNAD nodular cells. Nuclear translocation of beta-catenin protein in the PPNAD adenoma suggests activation of the Wnt-beta-catenin pathway in PPNAD., Conclusions: We report, for the first time, beta-catenin mutations in adenomas associated with PPNAD, further implicating Wnt-beta-catenin signalling in tumorigenesis linked to bilateral adrenal hyperplasias.

Mutations of the PRKAR1A gene are an important cause of Carney complex (CC). The PRKAR1A gene encodes the type 1A regulatory subunit of cAMP-dependent protein kinase A. We have identified one mutation of PRKAR1A (553delG) in three members of the same family affected by CC. This mutation was not identified in six unaffected family members, 12 patients with sporadic cardiac myxoma and 100 non-related healthy individuals. The novel mutation (553delG) is predicted to produce a frameshift leading to a premature stop codon. RNA analysis in the index patient showed normal size transcripts in RT-PCR amplicons of several exons, but an overall tendency to lower amounts of transcripts in relation to GAPDH controls. In Western blot analyses only full-length protein was present without any evidence of truncated product. These data suggest that the mutant allele might be a null allele due to degradation of the mutant mRNA via nonsense-mediated decay.

Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.

Patients presenting with primary pigmented nodular adrenocortical disease (PPNAD), Carney complex (CNC), or sporadic tumors were previously found to carry germline mutations in the human type Ialpha regulatory subunit (RIalpha) of adenosine 3',5'-cyclic monophosphate (cyclic AMP [cAMP])-dependent protein kinase (PKA; PRKAR1A). Although about 90% of disease-causing PRKAR1A mutations lead to premature stop codon generation and subsequent degradation of the mutant message by nonsense-mediated mRNA decay (NMD), here we describe seven PRKAR1A mutations whose mRNAs do not seem to undergo NMD and instead result in an expressed mutant RIalpha protein. The expressed mutations (p.Ser9Asn, p.Glu60_Lys116del [Delta-exon 3], p.Arg74Cys, p.Arg146Ser, p.Asp183Tyr, p.Ala213Asp, and p.Gly289Trp) were spread over all the functional RIalpha domains, and all of them exhibited increased PKA activity, which we attribute to decreased binding to cAMP and/or the catalytic subunit. Our data further corroborate the previous finding that altered PRKAR1A function, not only haploinsufficiency, is enough to elevate PKA activity which is apparently associated with tumorigenesis in tissues affected by CNC. In some cases, as with the Delta-exon 3 mutation, we may even conclude that the presence of a mutant PRKAR1A protein may be more harmful than allelic loss.

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.

Dysregulation of protein kinase A (PKA) activity, caused by loss of function mutations in PRKAR1A, is known to induce tumor formation in the inherited tumor syndrome Carney complex (CNC) and is also associated with sporadic tumors of the thyroid and adrenal. We have previously shown that Prkar1a(+/-) mice develop schwannomas reminiscent of those seen in CNC and that similar tumors are observed in tissue-specific knockouts (KO) of Prkar1a targeted to the neural crest. Within these tumors, we have previously described the presence of epithelial islands, although the nature of these structures was unclear. In this article, we report that these epithelial structures are derived from KO cells originating in the neural crest. Analysis of the mesenchymal marker vimentin revealed that this protein was markedly down-regulated not only from the epithelial islands, but also from the tumor as a whole, consistent with mesenchymal-to-epithelial transition (MET). In vitro, Prkar1a null primary mouse embryonic fibroblasts, which display constitutive PKA signaling, also showed evidence for MET, with a loss of vimentin and up-regulation of the epithelial marker E-cadherin. Reduction of vimentin protein occurred at the posttranslational level and was rescued by proteasomal inhibition. Finally, this down-regulation of vimentin was recapitulated in the adrenal nodules of CNC patients, confirming an unexpected and previously unrecognized role for PKA in MET.

Background: Protein kinase A signaling has long been known to play an important role in cardiac function. Dysregulation of the protein kinase A system, caused by mutation of the protein kinase A regulatory subunit gene PRKAR1A, causes the inherited tumor syndrome Carney complex, which includes cardiac myxomas as one of its cardinal features. Mouse models of this genetic defect have been unsatisfactory because homozygote null animals die early in development and heterozygotes do not exhibit a cardiac phenotype., Methods and Results: To study the cardiac-specific effects resulting from complete loss of Prkar1a, we used cre-lox technology to generate mice lacking this protein specifically in cardiomyocytes. Conditional knockout mice died at day 11.5 to 12.5 of embryogenesis with thin-walled, dilated hearts. These hearts showed elevated protein kinase A activity and decreased cardiomyocyte proliferation before demise. Analysis of the expression of transcription factors required for cardiogenesis revealed downregulation of key cardiac transcription factors such as the serum response factor, Gata4, and Nkx2-5. Although heart wall thickness was reduced overall, specific areas exhibited morphological changes consistent with myxomatous degeneration in the walls of knockout hearts., Conclusions: Loss of Prkar1a from the heart causes a failure of proper myocardial development with subsequent cardiac failure and embryonic demise. These changes appear to be due to suppression of cardiac-specific transcription by increased protein kinase A activity. These biochemical changes lead to myxoma-like changes, indicating that these mice may be a good model with which to study the formation of these tumors.

Carney complex (CNC) is an inherited neoplasia syndrome characterized by spotty skin pigmentation, myxomas, endocrine tumors, and schwannomas. Among the endocrine tumors that comprise the syndrome, GH-producing pituitary tumors are seen in approximately 10% of patients, although biochemical abnormalities of the GH axis are much more common. To explore the role of loss of the CNC gene PRKAR1A on pituitary tumorigenesis, we produced a tissue-specific knockout (KO) of this gene in the mouse. For these studies, we generated a mouse line expressing the cre recombinase in pituitary cells using the rat GHRH receptor promoter. These mice were then crossed with Prkar1a conditional null animals to produce tissue-specific KOs. Although prolactinomas were observed in KO and control mice, the KO mice exhibited a significantly increased frequency of pituitary tumors compared with wild-type or conventional Prkar1a(+/-) mice. Characterization of the tumors demonstrated they were composed of cells of the Pit1 lineage that stained for GH, prolactin, and TSH. At the biochemical level, levels of GH in the serum of KO animals were markedly elevated compared with controls, regardless of the presence of a frank tumor. These data indicate that complete loss of Prkar1a is sufficient to allow the formation of pituitary tumors and abnormalities of the GH axis, in close analogy to human patients with CNC.

Carney complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, and schwannomas. Some patients with the complex also develop myxoid bone tumors termed osteochondromyxomas. To study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a(+/-) mice develop bone tumors with high frequency, although these lesions have not yet been characterized, either from human patients or from mice. Bone tumors from Prkar1a(+/-) mice were heterogeneous, including elements of myxomatous, cartilaginous, and bony differentiation that effaced the normal bone architecture. Immunohistochemical analysis identified an osteoblastic origin for the abnormal cells associated with islands of bone. To better understand these cells at the biochemical level, we isolated primary cultures of tumoral bone and compared them with cultures of bone from wild-type animals. The tumor cells exhibited the expected decrease in Prkar1a protein and exhibited increased PKA activity. At the phenotypic level, we observed that tumor cells behaved as incompletely differentiated osteoblasts and were able to form tumors in immunocompromised mice. Examination of gene expression revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. Tumor cells exhibited enhanced growth in response to PKA-stimulating agents, suggesting that tumorigenesis in osteoblast precursor cells is driven by effects directly mediated by the dysregulation of PKA.

Context: Inactivating mutations of PRKAR1A, the regulatory subunit type 1A (RIalpha) of protein kinase A (PKA), are associated with tumor formation., Objective: Our objective was to evaluate the role of PKA isozymes on proliferation and cell cycle., Methods: A cell line with RIalpha haploinsufficiency due to an inactivating PRKAR1A mutation (IVS2+1 G-->A) was transfected with constructs encoding PKA subunits. Genetics, PKA subunit mRNA and protein expression and proliferation, aneuploidy, and cell cycle status were assessed. To identify factors that mediate PKA-associated cell cycle changes, we studied E2F and cyclins expression in transfected cells and E2F's role by small interfering RNA; we also assessed cAMP levels and baseline and stimulated cAMP signaling in transfected cells., Results: Introduction of PKA subunits led to changes in proliferation and cell cycle: a decrease in aneuploidy and G(2)/M for the PRKAR1A-transfected cells and an increase in S phase and aneuploidy for cells transfected with PRKAR2B, a known PRKAR1A mutant (RIalphaP), and the PKA catalytic subunit. There were alterations in cAMP levels, PKA subunit expression, cyclins, and E2F factors; E2F1 was shown to possibly mediate PKA effects on cell cycle by small interfering RNA studies. cAMP levels and constitutive and stimulated cAMP signaling were altered in transfected cells., Conclusion: This is the first immortalized cell line with a naturally occurring PRKAR1A-inactivating mutation that is associated in vivo with tumor formation. PKA isozyme balance is critical for the control of cAMP signaling and related cell cycle and proliferation changes. Finally, E2F1 may be a factor that mediates dysregulated PKA's effects on the cell cycle.

The three-component nanoparticle of this investigation consisted of an anti-type I regulatory subunit alpha of the cyclic AMP-dependent protein kinase A (RIalpha) antisense phosphorodiamidate morpholino (MORF) oligomer, a tat peptide and the anti-HER2 Herceptin antibody each biotinylated and each linked via streptavidin and tested in SUM190 (HER2+), SUM149 (HER2-) and SK-BR-3 (HER2+) cells in culture, using both radioactivity and fluorescent labels on the antisense and control sense MORF. Within the nanoparticle, the antibody provides specific binding to the target cells, the tat improves cellular delivery and the MORF provides the specific retention of the radioactivity in the target cell nucleus. The results show that within the nanoparticle, the Herceptin was still able to bind to its determinant; that the MORF escaped entrapment with its mRNA-binding ability preserved and that the tat maintained its carrier function. Fluorescence microscopy showed evidence of antisense MORF internalization, separation from Herceptin and migration to the nucleus. In conclusion, streptavidin appears to provide an easy means of mixing and matching components to improve the tumor-specific targeting, cell membrane transport, pharmacokinetics and other properties of antisense and other oligomers. Combining the three components of this investigation with streptavidin apparently did not interfere with the properties of each component in cell culture and significantly improved delivery.

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.

The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n=16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n=5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

We report the molecular and cytogenetic characterization of a novel variant of acute promyelocytic leukemia (APL). The bone marrow showed 88% hypergranular promyelocytes, and the karyotype was 47,XY,+22 [5]/46,XY[30]. Fluorescence in situ hybridization (FISH) indicated disruption and deletion of the 5'-end of the RARA gene. Treatment with all-trans retinoic acid, idarubicin, and arsenic trioxide induced cytogenetic complete remission without morphologic evidence of residual leukemia. The diagnostic marrow was negative for PML-RARA transcripts by reverse transcription-polymerase chain reaction (RT-PCR), but an atypical product was observed. Sequencing showed partial homology to the PRKAR1A gene, encoding the regulatory subunit type I-alpha of cyclic adenosine monophosphate-dependent protein kinase. RT-PCR using specific primers for PRKAR1A and RARA amplified 2 transcript splice variants of a PRKAR1A-RARA fusion gene, and PRKAR1A and RARA FISH probes confirmed the fusion. This novel PRKAR1A-RARA gene rearrangement is the fifth variant APL in which the RARA partner gene has been identified and the second known rearrangement of PRKAR1A in a malignant disease. This trial was registered at www.actr.org.au with the Australian Clinical Trials Registry as number 12605000070639.

Pigmented epithelioid melanocytoma (PEM) is a recently described entity comprising most cases previously described as "animal-type melanoma" and epithelioid blue nevus (EBN) occurring in patients with the multiple neoplasia syndrome Carney complex (CNC). Mutations of the protein kinase A regulatory subunit type 1alpha (R1alpha) (coded by the PRKAR1A gene) are found in more than half of CNC patients. In this study, we investigated whether PEM and EBN are related at the molecular level, and whether changes in the PRKAR1A gene status and the expression of the R1alpha protein may be involved in the pathogenesis of PEM and other melanocytic lesions. Histologic analysis of hematoxylin and eosin-stained sections and immunohistochemistry (IHC) with R1alpha antibody were performed on 34 sporadic PEMs, 8 CNC-associated PEMs from patients with known PRKAR1A mutations, 297 benign and malignant melanocytic tumors (127 conventional sections of 10 compound nevi, 10 Spitz nevi, 5 deep-penetrating nevi, 5 blue nevi, 6 cellular blue nevi, 2 malignant blue nevi, 3 lentigo maligna, and 86 melanomas of various types); in addition, 170 tissue microarray sections consisting of 35 benign nevi, 60 primary melanomas, and 75 metastatic melanomas, and 5 equine dermal melanomas, were examined. Histologic diagnoses were based on preexisting pathologic reports and were confirmed for this study. DNA studies [loss of heterozygosity (LOH) for the 17q22-24 locus and the PRKAR1A gene sequencing] were performed on 60 melanomas and 7 PEMs. IHC showed that R1alpha was expressed in all but one core from tissue microarrays (169/170), and in all 127 melanocytic lesions evaluated in conventional sections. By contrast, R1alpha was not expressed in the 8 EBN from patients with CNC and PRKAR1A mutations. Expression of R1alpha was lost in 28 of 34 PEMs (82%). R1alpha was expressed in the 5 equine melanomas studied. DNA studies correlated with IHC findings: there were no PRKAR1A mutations in any of the melanomas studied and the rate of LOH for 17q22-24 was less than 7%; 5 of the 7 PEMs showed extensive 17q22-24 LOH but no PRKAR1A mutations. The results support the concept that PEM is a distinct melanocytic tumor occurring in a sporadic setting and in the context of CNC. They also suggest that PEM differs from melanomas in equine melanotic disease, further arguing that the term animal-type melanoma may be a misnomer for this group of lesions. Loss of expression of R1alpha offers a useful diagnostic test that helps to distinguish PEM from lesions that mimic it histologically.

Primary pigmented nodular adrenocortical disease (PPNAD) is a form of bilateral adrenocortical hyperplasia that is often associated with corticotrophin (ACTH)-independent Cushing's syndrome (CS) and is characterized by small to normal-sized adrenal glands containing multiple small cortical pigmented nodules (1,2). PPNAD may occur in an isolated form or associated with a multiple neoplasia syndrome, the complex of spotty skin pigmentation, myxomas, and endocrine overactivity, or Carney complex, in which Cushing's syndrome is the most common endocrine manifestation (3). Molecular studies have led to the identification of several genes, defects in which may predispose PPNAD formation; all of these molecules play important role for the cAMP signaling pathway. This review intends to present the most recent knowledge of the pathology and molecular genetics of the benign bilateral adrenocortical lesions, as well as to discuss the modern tools for diagnostics and treatment of this condition.

The Carney complex (CNC) is a dominantly inherited syndrome characterized by spotty skin pigmentation, endocrine overactivity and myxomas. Skin pigmentation anomalies include lentigines and blue naevi. The most common endocrine gland manifestations are acromegaly, thyroid and testicular tumors, and adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). PPNAD, a rare cause of Cushing's syndrome, is due to primary bilateral adrenal defect that can be also observed in some patients without other CNC manifestations or familial history of the disease. Myxomas can be observed in the heart, skin and breast. Cardiac myxomas can develop in any cardiac chamber and may be multiple. One of the putative CNC genes located on 17q22-24, (PRKAR1A), has been identified to encode the regulatory subunit (R1A) of protein kinase A. Heterozygous inactivating mutations of PRKAR1A were reported initially in 45 to 65% of CNC index cases, and may be present in about 80% of the CNC families presenting mainly with Cushing's syndrome. PRKAR1A is a key component of the cAMP signaling pathway that has been implicated in endocrine tumorigenesis and could, at least partly, function as a tumor suppressor gene. Genetic analysis should be proposed to all CNC index cases. Patients with CNC or with a genetic predisposition to CNC should have regular screening for manifestations of the disease. Clinical work-up for all the manifestations of CNC should be performed at least once a year in all patients and should start in infancy. Cardiac myxomas require surgical removal. Treatment of the other manifestations of CNC should be discussed and may include follow-up, surgery, or medical treatment depending on the location of the tumor, its size, the existence of clinical signs of tumor mass or hormonal excess, and the suspicion of malignancy. Bilateral adrenalectomy is the most common treatment for Cushing's syndrome due to PPNAD.

First described in the mid 80's, Carney Complex (CNC) is a rare, dominantly heritable disorder with features overlapping those of McCune-Albright syndrome (MAS) and other multiple endocrine neoplasia (MEN) syndromes like MEN type 1 (MEN 1). Pituitary tumors have been described in a number of patients with CNC; they present with elevated growth hormone (GH) levels and mild hyperprolactinemia. However, most patients with CNC have mild hypersomatomammotropinemia starting in adolescence; this is similar to the situation in MAS patients: in both disorders, pituitary hyperplasia appears to precede tumor development. Familial pituitary tumor syndromes such as CNC provide an important insight into the genetics and molecular pathology of pituitary and other endocrine tumors. Our understanding of these conditions is expanding rapidly due to the identification of the causative genes and the availability of murine disease models. The present report reviews the clinical findings related to pituitary tumor development among patients with CNC and provides an update on murine models of the complex.

Primary Pigmented Nodular Adrenocortical Disease (PPNAD) is a rare primary bilateral adrenal defect causing corticotropin-independent Cushing's syndrome. It occurs mainly in children and young adults. Macroscopic appearance of the adrenals is characteristic with small pigmented micronodules observed in the cortex. PPNAD is most often diagnosed in patients with Carney complex (CNC), but it can also be observed in patients without other manifestations or familial history (isolated PPNAD). The CNC is an autosomal dominant multiple neoplasia syndrome characterized by the association of myxoma, spotty skin pigmentation and endocrine overactivity. One of the putative CNC genes has been identified as the gene of the regulatory R1A subunit of protein kinase A (PRKAR1A), located at 17q22-24. Germline heterozygous inactivating mutations of PRKAR1A have been reported in about 45% of patients with CNC, and up to 80% of CNC patients with Cushing's syndrome due to PPNAD. Interestingly, such inactivating germline PRKAR1A mutations have also been found in patients with isolated PPNAD. The hot spot PRKAR1A mutation termed c.709[-7-2]del6 predisposes mostly to isolated PPNAD, and is the first clear genotype/phenotype correlation described for this gene. Somatic inactivating mutations of PRKAR1A have been observed in macronodules of PPNAD and in sporadic cortisol secreting adrenal adenomas. Isolated PPNAD is a genetic heterogenous disease, and recently inactivating mutations of the gene of the phosphodiesterase 11A4 (PDE11A4) located at 2q31-2q35 have been identified in patients without PRKAR1A mutations. Interestingly, both PRKAR1A and PDE11A gene products control the cAMP signaling pathway, which can be altered at various levels in endocrine tumors.