PRKAR1A Research Articles

Heterogeneous Clinical Manifestations of Cushing’s Syndrome in a Family with Primary Pigmented Nodular Adrenocortical Disease

Purpose: To evaluate the clinical manifestations of Cushing’s Syndrome (CS) and associated genetic mutation in patients with Primary Pigmented Nodular Adrenocortical Disease (PPNAD). Methods: Seven family members were screened for mutations of the PRKAR1A gene. Gene mutation screening used genomic DNA (from peripheral blood leukocytes and, in some cases, adrenal gland tissue) and subsequent DNA sequencing. The five patients showing genetic mutation were assessed clinically for baseline cortisol and adrenocorticotropic hormone and adrenal imaging studies (abdominal computed tomography and adrenal scintigraphy). Low-dose and high-low dexamethasone suppression tests were performed in these five patients. Results: PRKAR1A gene mutation was detected in five of the seven family members. Four of the five gene mutation-positive patients presented with overt CS due to pathology-proven PPNAD. After unilateral adrenalectomy in these four patients, overt CS persisted for two patients and CS symptoms abated for the other two. The remission period of CS was >11 years in one instance (case III-2) and continues for >12 years in the other (case II-4), with no observable disease of the contralateral non-resected adrenal gland. For each of the two remission patients, one adrenal gland had been larger (by abdominal computed tomography) and had had stronger function (by 131I-6β- iodomethyl-19-norcholesterol scintigraphy) than the other. In one (case III-2) of the two remission patients, one adrenal displayed focal PPNAD while the other displayed diffuse PPNAD. The clinical manifestations of CS did not resolve after unilateral adrenalectomy in case II-2 and III-7. The fifth gene mutation-positive patient (case III-4) is still in the latent stage of CS. Conclusions: The clinical manifestations of CS and adrenal image features in patients with PPNAD are heterogeneous. Detailed adrenal imaging of these patients is necessary to assist with the decision to perform unilateral adrenalectomy.

Carney Complex

ABSTRACT The complex of myxomas, spotty skin pigmentation, and endocrine over activity or Carney complex (CNC) (MIM no. 160980) is an autosomal dominant disorder that was described in 1985 by Carney. The diagnosis of CNC is made if two of the main manifestations of the syndrome are present, these need to be confirmed by histology, biochemical testing, or imaging. Alternatively, the diagnosis is made when one of the criteria is present and the patient is a carrier of a known inactivating mutation of the PRKAR1A gene. Most cases of CNC are caused by inactivating mutations in the gene encoding one of the subunits of the protein kinase A (PKA) tetrameric enzyme, namely regulatory subunit type1 alpha (PRKAR1A), located at 17q22-24. Endocrine, dermatologic, and cardiac anomalies are the main manifestations of CNC. Skin abnormalities are present in almost 77% of the CNC patients. Variety of endocrine gland tumors are observed in CNC patients, namely growth hormone secreting pituitary adenoma (acromegaly), thyroid adenomas or carcinomas, testicular tumors (large cell calcifying sertoli cell tumors), and ovarian cyst. Cardiac myxoma is the most common primary tumor affecting the heart, accounting for nearly half of cardiac neoplasms. Approximately, 30-60% of CNC patients will develop cardiac myxoma, usually at much younger ages than the sporadic tumors. A high degree of suspicion, complete evaluation, genetic counseling is important aspect of management of Carney's disease. Once confirmed, surgical removal remains the mainstay of treatment. How to cite this article Majumdar G, Agarwal SK, Pande S, Chandra B. Carney Complex. World J Endoc Surg 2014;6(1):1-6.

Malignant Melanotic Schwannian Tumor

Melanotic schwannomas (MSs), variably associated with the Carney complex, are rare tumors that usually involve spinal nerve roots but may occur in other locations. Clinicopathologic evaluation poorly predicts the behavior of MS. Fewer than 200 cases have been reported. We report a series of 40 well-characterized MSs, one of the largest series to date. The tumors were comprehensively evaluated, and clinical follow-up was obtained. Immunohistochemistry for S100 protein, Melan-A, HMB45, tyrosinase, glial fibrillary acidic protein (GFAP), EMA, SMARCB1, Ki-67 antigen, ASMTL, and the Carney complex-associated PRKAR1A gene product was performed using commercially available antibodies and the Ventana Ultraview detection system. Gene microarray study was conducted on formalin-fixed, paraffin-embedded blocks from 10 MSs and the results compared with previous data from melanoma and schwannoma. Differentially expressed genes were selected at >3-fold and P<0.001. The Fisher exact test was used for statistical analysis. The tumors occurred in 18 male and 22 female patients (mean age 41 y; range, 11 to 84 y) and involved the paravertebral nerve roots (N=31), mediastinum (N=3), sacrum, cauda equina, para-aortic region, fifth cranial nerve, buttock, and cerebellum (N=1 each). Two patients had known Carney complex, and 1 patient also had a cutaneous myxoma, suggestive of Carney complex. The tumors expressed S100 protein (21/25, 84%), Melan-A (23/25, 92%), HMB45 (25/25, 100%), tyrosinase (25/25, 100%), GFAP (0/24, 0%), EMA (0/9, 0%), SMARCB1 (retained in 25/25, 100%), and ASMTL (5/19, 26%); PRKAR1A expression was lost in 7/20 cases (35%). Ki-67-labeling index was <5% in 23/25 cases (92%) and 5% to 10% in 2/25 cases (8%). Gene expression profiling showed significant differences between MS, melanoma, and conventional schwannoma. Clinical follow-up (26/40, 65%; mean 55 mo; range, 1 to 300 mo) showed local recurrences in 9/26 (35%) and metastases in 11/26 (44%) patients. Fourteen patients were alive without disease, 5 were alive with disease, and 7 had died of disease. Only a mitotic rate >2/10 HPF correlated with metastases (P=0.008). The clinicopathologic features of tumors with and without psammoma bodies were identical. We conclude that MSs are distinctive malignant tumors, rather than benign neoplasms with occasionally unpredictable behavior, and propose their reclassification as "malignant melanotic schwannian tumors." Loss of PRKAR1A expression suggests a link to Carney complex, even when this history is absent.

A novel PRKAR1A gene mutation in a young patient with cardiac myxoma

Myxomas are the most common clinically detected primary cardiac tumours, located predominantly in the left atrium. They occur sporadically in 90% of cases, especially women with mean age of 50 years. About 7% of cardiac myxomas occur in a setting wherein these tumors occur at atypical or multiple sites in young patients with extra-cardiac manifestations as well. This is designated as Carney’s complex, which may be familial or sporadic. Genetic studies have confirmed that these multi-systemic manifestations are a result of mutations in the gene located at 17q22-24 coding protein kinase A, regulatory subunit 1-α (PRKAR-1-α). We report a case of recurrent (tumours detected at the ages of 12 and 20 years), multifocal myxomas with associated facial pigmentations and mucocutaneous angiomyxomas in a 20-year-old male. Screening for mutations in our patient by Sanger sequencing on whole genome amplified DNA revealed a heterozygous deletion at c.353_356delTACC as confirmed using forward and reverse primers. This has not been reported in PRKAR-1-α mutation database. Such a mutation was not seen in the immediate family members, indicating the sporadic nature of occurrence.

Deletions of the PRKAR1A Locus at 17q24.2-q24.3 in Carney Complex: Genotype-Phenotype Correlations and Implications for Genetic Testing

Carney complex (CNC) is a multiple neoplasia syndrome caused by PRKAR1A-inactivating mutations. One-third of the patients, however, have no detectable PRKAR1A coding sequence defects. Small deletions of the gene were previously reported in few patients, but large deletions of the chromosomal PRKAR1A locus have not been studied systematically in a large cohort of patients with CNC. A tertiary care referral center was the setting for analysis of an international cohort of patients with CNC. Methods included genome-wide array analysis followed by fluorescent in situ hybridization, mRNA, and other studies as well as a retrospective analysis of clinical information and phenotype-genotype correlation. We detected 17q24.2-q24.3 deletions of varying size that included the PRKAR1A gene in 11 CNC patients (of 51 tested). Quantitative PCR showed that these patients had significantly lower PRKAR1A mRNA levels. Phenotype varied but was generally severe and included manifestations that are not commonly associated with CNC, presumably due to haploinsufficiency of other genes in addition to PRKAR1A. A significant number (21.6%) of patients with CNC that are negative in currently available testing may have PRKAR1A haploinsufficiency due to genomic defects that are not detected by Sanger sequencing. Array-based studies are necessary for diagnostic confirmation of these defects and should be done in patients with unusual and severe phenotypes who are PRKAR1A mutation-negative.

Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion

Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters

Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.

Abstract 3854: Novel hematopoietic neoplasms in prkar2a- deficient mice.

Abstract Background: Histiocytic sarcoma (HS) is an aggressive hematological neoplasm that responds poorly to therapy. The molecular etiology and pathology of this disease remain unclear, hampering the development of an effective therapy. Therefore, a need for more, and more realistic, animal models remains. Lymphoproliferative disorders have been reported in mice deficient for the prkar1a gene coding for the regulatory subunit type 1A of protein kinase A (PKA), but nothing is known about the role of type II PKA regulatory subunits in hematologic malignancies. Methods: Mice deficient for the Prkar1a and Prkar2a alleles were previously reported (Kirschner et al, 2005 και Burton et al, 1997) and were kept on a mixed genetic background (C57BL/129Sv). Mice were crossed to create prkar2a+/- and prkar2a-/-. Mice were phenotyped at the ages of 3-6-9-12-18 months or when they exhibited signs of advanced disease. Tissues were collected for histological and molecular analysis. Results: Unexpectedly, mice deficient for the prkar2a allele(s) developed lymphomas, with significant higher incidence compared to the prkar1a+/- mice [8/13 (61.5%) of the prkar2a+/- and 5/8 (62.5%) of the prkar2a-/- vs. 3/21(14.2%) of the prkar1a+/-, Fisher's exact test p=0.02]. Histology studies of sections stained with H&amp;E revealed a variety of pathologic changes in lymphoid and non-lymphoid tissues. Lesions characteristic of histiocytic sarcoma (HS) were found in spleen and abdominal lymph nodes, sometimes associated with leukemic infiltrates in lung and bone marrow (BM). Abnormal megakaryopoiesis in spleen and BM was associated with cells actively phagocytosing red cells, others with a pseudo Gaucher-like appearance and increased immature forms. Erythropoiesis was reduced in BM and spleen. Vascular thrombi were associated with schistocytosis. Accumulations of mostly mature plasma cells and some plasmablasts were present in the splenic red pulp together with large, active germinal centers. Cells with characteristics of diffuse large B cell lymphoma often occupied expanded splenic follicles and lymph nodes. Kidneys exhibited degenerative changes in glomeruli and tubules. Median age at presentation was 18 months (range 16-25 months). FACS analysis of BM cells revealed a decrease in late pro-B cells in the prkar2a deficient mice compared to the wild-type, indicating a developmental block at this stage of early B cell differentiation. Interestingly, Southern blot analysis of these B cell lymphomas showed that in most cases they were monoclonal with either one or both IgH alleles rearranged. Conclusions: Our data indicate that PRKAR2a may be involved in the development of malignant lesions of the hematologic lineage. The presented mouse model can be used to gain insights into the molecular and cellular origins of this rare neoplasm and provide a preclinical model in which to test novel therapeutic strategies. Citation Format: Emmanouil Saloustros, Paraskevi Salpea, Lina Gugglioti, Kittman Tsang, Anelia Horvath, Maria Nesterova, Chen-Feng Qi, Herbert C. Morse III, Constantine A. Stratakis. Novel hematopoietic neoplasms in prkar2a- deficient mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3854. doi:10.1158/1538-7445.AM2013-3854

Abstract 2736: Genetic deletion of the prkar1a in the endocrine pancreas promotes neuroendocrine carcinogenesis.

Abstract Background: Carney complex (CNC) is a rare disease inherited as an autosomal dominant trait, associated with various tumors, and caused most frequently by inactivation of the PRKAR1A gene. Recently a possible association of pancreatic neoplasms with CNC was reported in 9 out of 354 patients (2.5%), suggesting the potential role of PRKAR1A as a tumor suppressor gene in the pancreas. Methods: Prkar1afl/fl and pdx-1-CRE mice were previously reported (Kirschner et al, 2005; Lammert et al, 2001) and were kept in a mixed genetic background (C57Bl/6 x FVB). Pdx1 is a transcription factor expressed in beta-insulin producing cells and in some a-glucagon producing cells of the mouse adult pancreatic islet. Both prkar1a alleles were conditionally deleted during development by generating pdx1-Cre; prkar1af/f mice (Δ–prkar1a). Mice were phenotyped at the age of 2, 4 and 6 months and tissues were collected for histological and molecular analysis. Results: Mice developed endocrine or mixed endocrine/acinar cell carcinomas with 100% penetration by the age of 4-5 months. Malignant behavior of the tumors was defined by stromal invasion and regional lymph node metastasis. Histologically most tumors exhibited an organoid pattern as seen in the islet-cell tumors. Some tumors exhibited in addition foci suggestive of acinar cell differentiation. The primary neuroendocrine nature of the tumor cells was confirmed by immunohistochemical (IHC) staining for chromogranin and by electron microscopy, which revealed the characteristic neuroendocrine granules. Focal acinar differentiation was further supported by the finding of scattered chymotrypsin-positive cells by immunofluorescence (IF). Although the Δ-prkar1a mice developed hypoglycemia after overnight fasting, insulin and glucagon levels in the plasma didn't differ. The negative IHC staining for the most common produced peptides (insulin, c-peptide, glucagon, gastrin, somatostatin) suggested that these tumors are non-functional. The recombination that was confirmed by the detection of the mutated allele for the prkar1a in the tumor DNA, but neither in the germline of the KO animals nor in the islets DNA of the WT mice, resulted in increased PKA activity and lack of protein expression in the tumor compared to the normal pancreatic tissue. The latter finding is suggestive of the oncogenic effect of PRKAR1A inactivation. IF staining showed that the tumor cells were composed of a pdx1+/insulin- cell-population. We hypothesized that the recently identified multipotent pdx1+/insulin- cell in the adult pancreas, gives rise to endocrine, or mixed endocrine/acinar pancreatic malignancies upon PKA manipulation. Conclusions: Loss of prkar1a function predisposes the endocrine pancreas to invasive cancers. This supports the role of prkar1a as a tumor suppressor gene in the pancreas and provides valuable tools to evaluate novel therapeutics in endocrine pancreas malignancies. Citation Format: Emmanouil Saloustros, Maria Tsokos, Paraskevi Salpea, Matthew Starost, Sisi Liu, Li Guang, Maria Nesterova, Eva Szarek, Mehboob A. Hussain, Constantine A. Stratakis. Genetic deletion of the prkar1a in the endocrine pancreas promotes neuroendocrine carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2736. doi:10.1158/1538-7445.AM2013-2736

Novel mutations of the PRKAR1A gene in patients with acrodysostosis

Acrodysostosis is characterized by a peripheral dysostosis that is accompanied by short stature, midface hypoplasia, and developmental delay. Recently, it was shown that heterozygous point mutations in the PRKAR1A gene cause acrodysostosis with hormone resistance. By mutational analysis of the PRKAR1A gene we detected four different mutations (p.Arg368Stop, p.Ala213Thr, p.Tyr373Cys, and p.Arg335Cys) in four of seven affected patients with acrodysostosis. The combination of clinical results, endocrinological parameters and in silico mutation analysis gives evidence to suppose a pathogenic effect of each mutation. This assumption is supported by the de novo origin of these mutations. Apart from typical radiological abnormalities of the hand bones, elevated thyroid stimulating hormone and parathyroid hormone values as well as short stature are the most common findings. Less frequent features are characteristic facial dysmorphisms, sensorineural hearing loss and mild intellectual disability. These results lead to the conclusion that mutations of PKRAR1A are the major molecular cause for acrodysostosis with endocrinological abnormalities. In addition, in our cohort of 44 patients affected with brachydactyly type E (BDE) we detected only one sequence variant of PRKAR1A (p.Asp227Asn) with an unclear effect on protein function. Thus, we conclude that PRKAR1A mutations may play no major role in the pathogenesis of BDE.

The complex of myxomas, spotty skin pigmentation and endocrine overactivity (Carney complex): imaging findings with clinical and pathological correlation

The complex of myxomas, spotty skin pigmentation and endocrine overactivity, or Carney complex (CNC), is a familial multiple endocrine neoplasia and lentiginosis syndrome. CNC is inherited in an autosomal dominant manner and is genetically heterogeneous. Its features overlap those of McCune-Albright syndrome and other multiple endocrine neoplasia (MEN) syndromes. Spotty skin pigmentation is the major clinical manifestation of the syndrome, followed by multicentric heart myxomas, which occur at a young age and are the lethal component of the disease. Myxomas may also occur on the skin (eyelid, external ear canal and nipple) and the breast. Breast myxomas, when present, are multiple and bilateral among female CNC patients, an entity which is also described as “breast-myxomatosis” and is a characteristic feature of the syndrome. Affected CNC patients often have tumours of two or more endocrine glands, including primary pigmented nodular adrenocortical disease (PPNAD), an adrenocorticotropin hormone (ACTH)-independent cause of Cushing’s syndrome, growth hormone (GH)-secreting and prolactin (PRL)-secreting pituitary adenomas, thyroid adenomas or carcinomas, testicular neoplasms (large-cell calcifying Sertoli cell tumours [LCCSCT]) and ovarian lesions (cysts and cancinomas). Additional infrequent but characteristic manifestations of CNC are psammomatous melanotic schwannomas (PMS), breast ductal adenomas (DAs) with tubular features, and osteochondromyxomas or “Carney bone tumour”. Teaching Points • Almost 60 % of the known CNC kindreds have a germline inactivating mutations in the PRKAR1A gene. • Spotty skin pigmentation is the major clinical manifestation of CNC, followed by heart myxomas. • Indicative imaging signs of PPNAD are contour abnormality and hypodense spots within the gland. • Two breast tumours may present in CNC: myxoid fibroadenomas (breast myxomatosis) and ductal adenomas. • Additional findings of CNC are psammomatous melanotic schwannomas (PMSs) and osteochondromyxomas.

Epithelioid and Fusiform Blue Nevus of Chronically Sun-damaged Skin, an Entity Distinct From the Epithelioid Blue Nevus of the Carney Complex

Epithelioid blue nevus (EBN) was first described in patients with Carney complex (CNC) and subsequently shown to also occur sporadically. Over 50% of patients with CNC harbor mutations in the gene PRKAR1A, which codes for protein kinase A regulatory subunit 1α (R1α) involved in the signaling pathway regulating melanogenesis and melanocytic proliferation. Immunohistochemical expression of R1α has been shown to be absent in the majority of pigmented epithelioid melanocytomas and all CNC-associated EBNs but present in melanomas and other melanocytic nevi. We have observed several examples of EBN occurring in chronically sun-damaged (CSD) skin with a predominance of epithelioid morphology but also containing a component of fusiform and conventional blue nevus cells, which we have termed epithelioid and fusiform blue nevus of CSD skin. Several of these cases demonstrated notable pleomorphism and nuclear atypia with rare mitotic activity raising concern for the possibility of melanoma; however, the clinical outcomes, detailed histologic review, and molecular results were most consistent with a benign melanocytic neoplasm. We report our clinical, histopathologic, immunohistochemistry, and fluorescence in situ hybridization experience with this distinct entity of epithelioid and fusiform blue nevus and demonstrate that it is a unique subtype of blue nevus occurring on CSD skin with a higher frequency of an associated conventional blue nevus component compared with EBN and without association with CNC or loss of R1α expression typically found in pigmented epithelioid melanocytoma and CNC-associated EBN. We also postulate that the epithelioid pattern may represent a subclone of the conventional blue nevus component induced by chronic UV damage.

Acrodysostosis syndromes

Acrodysostosis (ADO) refers to a heterogeneous group of rare skeletal dysplasia that share characteristic features including severe brachydactyly, facial dysostosis and nasal hypoplasia. The literature describing acrodysostosis cases has been confusing because some reported patients may have had other phenotypically related diseases presenting with Albright Hereditary Osteodystrophy (AHO) such as pseudohypoparathyroidism type 1a (PHP1a) or pseudopseudohypoparathyroidism (PPHP). A question has been whether patients display or not abnormal mineral metabolism associated with resistance to PTH and/or resistance to other hormones that bind G-protein coupled receptors (GPCR) linked to Gsα, as observed in PHP1a. The recent identification in patients affected with acrodysostosis of defects in two genes, PRKAR1A and PDE4D, both important players in the GPCR-Gsα-cAMP-PKA signaling, has helped clarify some issues regarding the heterogeneity of acrodysostosis, in particular the presence of hormonal resistance. Two different genetic and phenotypic syndromes are now identified, both with a similar bone dysplasia: ADOHR, due to PRKAR1A defects, and ADOP4 (our denomination), due to PDE4D defects. The existence of GPCR-hormone resistance is typical of the ADOHR syndrome. We review here the PRKAR1A and PDE4D gene defects and phenotypes identified in acrodysostosis syndromes, and discuss them in view of phenotypically related diseases caused by defects in the same signaling pathway.

Rac1 is required for Prkar1a-mediated Nf2 suppression in Schwann cell tumors

Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including Neurofibromatosis Types 1 (NF1) and 2 (NF2), Familial Schwannomatosis (FS) and Carney Complex (CNC). Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to play a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout of the CNC gene Prkar1a, which encodes the type 1A regulatory subunit of Protein Kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double knockout of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation.. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicates that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA.

Gastrointestinal stromal tumor: Our experience

Gastrointestinal stromal tumor: Our experience

A new PRKAR1A gene mutation in the carney complex syndrome: A case report

A new PRKAR1A gene mutation in the carney complex syndrome: A case report

Identification of a Recurrent Mutation in the PRKAR1A Gene in Patients Affected with Chondrodysplasia and Hormone Resistance

Identification of a Recurrent Mutation in the PRKAR1A Gene in Patients Affected with Chondrodysplasia and Hormone Resistance

Resection of Multiple Recurrent Cardiac Myxomas in an Adult Man With Carney's Complex

Cardiac myxomas have many manifestations, including an association with endocrine overreactivity, unusual testicular tumors, and manifestations in Carney's complex [1McCarthy P.M. Piehler J.M. Schaff H.V. et al.The significance of multiple, recurrent, and “complex” cardiac myxomas.J Thorac Cardiovasc Surg. 1986; 91: 389-396PubMed Google Scholar]. We recently treated a 40-year-old man who presented with an embolic stroke. He had a family history of cardiac myxoma and underwent resection of a right atrial myxoma 7 years earlier.Echocardiography revealed multiple intracardiac masses in the left atrium (Fig 1A , arrow), right atrium, and right ventricle (Fig 1B, arrow). An ultrasound examination of the scrotum showed multiple bilateral testicular calcifications (Figs 2A, 2B), thought to be calcifying Sertoli cell tumors. A computed tomography scan of the abdomen showed no abnormalities, and the 24-hour urine cortisol level was within the normal reference range. Genetic evaluation revealed a heterozygous mutation in the PRKAR1A gene.Fig 2View Large Image Figure ViewerDownload (PPT)The patient underwent excision of 4 cardiac myxomas (left atrium, right atrium, and right ventricle; Fig 3) using cardiopulmonary bypass and standard aortic and bicaval cannulation. His postoperative course was unremarkable, and he was dismissed on the fourth postoperative day.Fig 3View Large Image Figure ViewerDownload (PPT) Cardiac myxomas have many manifestations, including an association with endocrine overreactivity, unusual testicular tumors, and manifestations in Carney's complex [1McCarthy P.M. Piehler J.M. Schaff H.V. et al.The significance of multiple, recurrent, and “complex” cardiac myxomas.J Thorac Cardiovasc Surg. 1986; 91: 389-396PubMed Google Scholar]. We recently treated a 40-year-old man who presented with an embolic stroke. He had a family history of cardiac myxoma and underwent resection of a right atrial myxoma 7 years earlier. Echocardiography revealed multiple intracardiac masses in the left atrium (Fig 1A , arrow), right atrium, and right ventricle (Fig 1B, arrow). An ultrasound examination of the scrotum showed multiple bilateral testicular calcifications (Figs 2A, 2B), thought to be calcifying Sertoli cell tumors. A computed tomography scan of the abdomen showed no abnormalities, and the 24-hour urine cortisol level was within the normal reference range. Genetic evaluation revealed a heterozygous mutation in the PRKAR1A gene. The patient underwent excision of 4 cardiac myxomas (left atrium, right atrium, and right ventricle; Fig 3) using cardiopulmonary bypass and standard aortic and bicaval cannulation. His postoperative course was unremarkable, and he was dismissed on the fourth postoperative day.

Abstract 1360: A mouse model of double heterozygosity for protein kinase A regulatory subunits promotes osteoblastic differentiation of cAMP-induced bone tumors

Abstract Background: Carney Complex (CNC) is a multiple neoplasia syndrome inherited in an autosomal dominant manner and causing various endocrine and bone tumors. The PRKAR1A gene coding for the regulatory subunit type 1A of protein kinase A (PKA) is mutated in CNC patients, but tumorogenesis mechanisms are not well understood. Regulatory subunits suppress activity of catalytic subunits of PKA responsible for cAMP signaling and cell differentiation and maturation. Mice with a deleted prkar1a allele develop a variety of tumors overlapping those of CNC patients. Deletion of a catalytic subunit allele prkaca+/− on the prkar1a+/ background unexpectedly increased the numer and aggressiveness of bone tumors without development of schwannomas or thyroid lesions. Cells from prkar1a+/−prkaca+/ tumors have more type II PKA complexes, indicating that unbalanced expression of type II and type I regulatory subunits may also dysregulates PKA catalytic activity, potentially increasing tumorigenesis. Methods: To explore this hypothesis, single mice with single alleles of prkar1a, prkar2a and prkar2b were crossed to generate heterozygous mice for double heterozygotes prkar1a+/−/prkar2a+/− or prkar1a+/−/prkar2b+/− and double knockouts. The resulting mice of 3,6,9,12,18 month age were phenotyped and compared to prkar1a+/−. Tumor specimens were examined by histology, polarized microscopy and confocal Raman micro-spectroscopy. Results: Mice with both alleles prkar1a+/+ did not develop any tumors. Double heterozygotes developed bone tumors whose onset age was delayed to 9 months in prkar1a+/−/prkar2b+/− mice compared to 3-6 months in the others. However, the mean number of tumors per animal became similar for prkar1a+/− and double heterozygotes by 12 months. Histology of the bone tumors showed abnormal proliferation of a fibroblastoid-like cell with better osteogenic differentiation in the lesions from double heterozygous mice. Polarized microscopy and Raman micro-spectroscopy revealed that bone material growing in tumors was immature and poorly organized in prkar1a+/− mice (disorganized collagen and osteocytes and undermineralized matrix). Prkar2α+/− deletion on the prkar1α+/− background rescued local organization and mineralization but worsened global organization of cortical tumor bone. The prkar1a+/−/prkar2b+/− deletion rescued both global and local organization and mineralization, resulting in formation of mature bone. Conclusion: Prkar1a haploinsufficiency requires both prkar2a and prkar2b for tumorigenesis in most tissues except bone. In bone, however, prkar1a is the major tumor-suppressor gene. Unexpectedly, Prkar2b or prkar2a deficiencies in addition to prkar1a lead to better differentiation of bone tumors, indicating a compensating role for the type II regulatory subunits in bone tumirogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1360. doi:1538-7445.AM2012-1360

Abstract 954: COX-2 inhibition reduces bone tumor growth in animal models:A role for celecoxib treatment in cAMP/protein kinase A-induced tumors

Abstract Background: Carney Complex (CNC) is a multiple neoplasia syndrome characterized by skin lesions, endocrine and other tumors, including osteochondromyxomas. It is caused by inactivating mutations in the PRKAR1A gene encoding the regulatory type 1A subunit of cAMP-dependent protein kinase A (PKA). This subunit is the main suppressor of activity of catalytic subunit (PRCKACA) of PKA which regulates cell proliferation and differentiation. Loss of PRKAR1A increases PKA activity and causes tumors in CNC-affected tissues. Mice with deletion of one prkar1a allele (prkar1a+/−) develop various CNC-like tumors and multiple tail osteotic lesions. Further deletion of a prkaca allele on the prkar1a+/− background unexpectedly increases the number and severity of bone lesions and reduces their age of onset. Recent data indicate that increased PKA activity stimulates the inflammasome without inflammation in mouse and human cells. The released prostaglandin E2 (PGE2) further increases cAMP, which in turn activates PKA and Wnt signaling and promotes the growth of these bony tumors. These data led us to investigate celecoxib, which is a selective inhibitor of cyclooxygenase-2 (COX-2) and therefore of PGE2's release, in prkar1α+/−/prkacα+/− mice. Methods: Eleven out of twenty-two mice 6-12 months old were exposed to celecoxib-containing diet (1,500 mg/kg of NIH-31 diet). The remaining mice were fed the standard NIH-31 diet. After a six-week treatment, we looked at the number, the PKA and phosphodiesterase (PDE) activities, c AMP levels and histology of bony lesions. Sixteen and seven days before sacrifice, mice were injected with calcein which labels bone forming surfaces. From calcein fluorescence observed under microscope, the rate of apposition of cortical bone at periosteal side was measured. This rate reflects tumor growth rate because growing tumors remain encapsulated by cortical bone forming at periosteal side and resorbing at endosteal side. Results: Celecoxib reduced the rate of tumor growth by two-fold as measured by calcein labeling (p=0.016). Histological examination revealed decreased cellularity in the lesions of treated animals due to treatment effect on the tumor cells. Treatment reduced PKA enzymatic activity in the tail tumors as measured by high-performance liquid chromatography (4.0 vs 3.0 CMP/ug protein, p=0.07) whereas cAMP levels and total PDE activity did not change. The expression of all inflammasome-related genes (ets-1, caspase-1, il1b and cox2) was down-regulated by at least 70% in the bone tumors from treated animals compared to controls. Conclusion: COX-2 inhibition reduces PKA activity and bone tumor growth in prkar1α+/−/prkacα+/− mice. Since local tumor control is clinically meaningful for patients with cAMP-induced tumors, celecoxib may merit consideration as the first molecular targeted therapy in this setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 954. doi:1538-7445.AM2012-954