Paraskevi Salpea - Academia.edu (original) (raw)
Papers by Paraskevi Salpea
Diabetes Research and Clinical Practice, 2020
Diabetes Research and Clinical Practice, 2019
Diabetes Research and Clinical Practice, 2019
Endocrine-Related Cancer, 2016
Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predispos... more Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predisposition to pancreatic tumors; it is caused most frequently by the inactivation of the PRKAR1A gene, a regulator of the cyclic AMP (cAMP)-dependent kinase (PKA). The method used was to create null alleles of prkar1a in mouse cells expressing pdx1 (Δ-Prkar1a). We found that these mice developed endocrine or mixed endocrine/acinar cell carcinomas with 100% penetrance by the age of 4–5 months. Malignant behavior of the tumors was seen as evidenced by stromal invasion and metastasis to locoregional lymph nodes. Histologically, most tumors exhibited an organoid pattern as seen in the islet-cell tumors. Biochemically, the lesions exhibited high PKA activity, as one would expect from deleting prkar1a. The primary neuroendocrine nature of these tumor cells was confirmed by immunohistochemical staining and electron microscopy, the latter revealing the characteristic granules. Although the Δ-Prkar1a ...
Molecular and cellular endocrinology, Jan 4, 2016
Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like ... more Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1a grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkar1a and Prkaca (coding for Cα) haploinsufficiency lead to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSCs. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1α and Cα with 1,500 mg/kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene ...
Journal of Experimental & Clinical Cancer Research, 2015
Background: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunit... more Background: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIα, RIβ, RIIα, RIIβ) and four catalytic subunits (Cα, Cβ, Cγ, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIα) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIα and RIIβ, respectively) are also viable but have not been studied for their susceptibility to any tumors. Methods: Cohorts of Prkar1a +/− , Prkar2a +/− , Prkar2a −/− , Prkar2b +/− and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed. Results: Both Prkar2a −/− and Prkar2a +/− mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms. Conclusions: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIα's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms.
Endocrine-Related Cancer, 2015
Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney comple... more Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney complex (CNC) or isolated, leads to ACTH-independent Cushing's syndrome (CS). CNC and PPNAD are caused typically by inactivating mutations of PRKAR1A, a gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). Mice lacking Prkar1a, specifically in the adrenal cortex (AdKO) developed CS caused by bilateral adrenal hyperplasia (BAH), which is formed from the abnormal proliferation of fetal-like adrenocortical cells. Celecoxib is a cyclooxygenase 2 (COX2) inhibitor. In bone, Prkar1a inhibition is associated with COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of bone stromal cells. We hypothesized that COX2 inhibition may have an effect in PPNAD. In vitro treatment of human cell lines, including one from a patient with PPNAD, with celecoxib resulted in decreased cell viability. We then treated AdKO and control m...
Human molecular genetics, Jan 5, 2015
Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated... more Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated with bone lesions such as osteochondromyxomas (OMX). The most frequent cause for CNC is PRKAR1A deficiency; PRKAR1A codes for type-I regulatory subunit of protein kinase A (PKA). Prkar1a(+/-) mice developed OMX, fibrous dysplasia-like lesions (FDL), and other tumors. Tumor tissues in these animals had increased PKA activity due to an unregulated PKA catalytic subunit and increased PKA type II (PKA-II) activity mediated by the PRKAR2A and PRKAR2B subunits. To better understand the effect of altered PKA activity on bone we studied Prkar2a and Prkar2b knock out and heterozygous mice; none of these mice developed bone lesions. When Prkar2a(+/-) and Prkar2b(+/-) mice were used to generate Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) animals, bone lesions formed that looked like those of the Prkar1a(+/-) mice. However, better overall bone organization and mineralization and fewer FDL...
European journal of endocrinology / European Federation of Endocrine Societies, Jan 30, 2015
Carney Complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of... more Carney Complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of the skin and mucosa, cardiac, cutaneous and other myxomas, and multiple endocrine tumors. The disease is caused by inactivating mutations or large deletions of the PRKAR1A gene located at 17q22-24 coding for the regulatory subunit type I alpha of protein kinase A (PKA) gene. Most recently, components of the complex have been associated with defects of other PKA subunits, such as the catalytic subunits PRKACA (adrenal hyperplasia) and PRKACB (pigmented spots, myxomas, pituitary adenomas). In this report, we review CNC, its clinical features, diagnosis, treatment, and molecular etiology including PRKAR1A mutations and the newest on PRKACA and PRKACB defects especially as they pertain to adrenal tumors and Cushing's syndrome.
Cancer Research, 2013
Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Histiocyt... more Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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&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
Cancer Research, 2013
Background: Carney complex (CNC) is a rare disease inherited as an autosomal dominant trait, asso... more 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 didn9t 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
Endocrine Abstracts, 2015
European Journal of Endocrinology, 2015
ObjectiveWe have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) ... more ObjectiveWe have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) and Cushing's syndrome (CS) caused by constitutive activation of the catalytic subunit of protein kinase A (PRKACA). By doing new in-depth analysis of their cytogenetic abnormality, we attempted a better genotype–phenotype correlation of their PRKACA amplification.DesignThis study is a case series.MethodsMolecular cytogenetic, genomic, clinical, and histopathological analyses were performed in five patients with CS.ResultsReinvestigation of the defects of previously described patients by state-of-the-art molecular cytogenetics showed complex genomic rearrangements in the chromosome 19p13.2p13.12 locus, resulting in copy number gains encompassing the entire PRKACA gene; three patients (one sporadic case and two related cases) were observed with gains consistent with duplications, while two sporadic patients were observed with gains consistent with triplications. Although all five pati...
Cancer Research, 2014
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Corticotropin-independent C... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Corticotropin-independent Cushing's syndrome may be caused by tumors or hyperplasia of the adrenal cortex. Until now genetic alterations explain only a small fraction of cases. The observation that a subset of adrenal adenomas is characterized by abnormal PKA activity despite the absence of mutations in candidate genes suggested as yet unknown alterations in the cAMP/PKA signaling cascade in these tumors. The aim of this study was the analysis of the genetic basis of Cushing's syndrome in order to reveal the gene/s responsible for the disease. Exome sequencing was performed in ten cortisol-producing adenomas and recurrent mutations in candidate genes were evaluated in additional 171 patients with adrenocortical tumors. Genome-wide copy number analysis was performed in 35 patients with cortisol secreting bilateral hyperplasias. The effects of these genetic defects were studied both clinically and in vitro. Exome sequencing in 8/10 adenomas revealed somatic mutations in the PRKACA gene, which encodes the main catalytic subunit of cyclic AMP-dependent protein kinase (PKA) (c.617A>C in seven and c.595_596insCAC in one). Overall, PRKACA somatic mutations were identified in a total of 22/59 (37%) adenomas from patients with overt Cushing's syndrome while these mutations were not detectable in patients with subclinical hypercortisolism (n=40) or in other adrenal tumors (n=82). Among 35 patients with cortisol producing hyperplasias, 5 (with two patients as first degree relatives) carried germline copy number gain of the chromosome 19 region including the PRKACA gene. In vitro studies demonstrated impaired inhibition of the mutant PRKACA by the PKA regulatory subunit, while cells from patients with germline chromosomal gains showed increased protein levels; in both cases, PKA activity was increased. The present study shows that more than one third of cortisol-producing adenomas associated with overt Cushing syndrome harbor unique somatic mutations of the main cAMP-dependent kinase catalytic subunit, PRKACA resulting in constitutive PKA activation. While in these patients the mutation is present only in tumor cells, germline duplication of the PRKACA gene was identified in a group of patients with bilateral adrenal hyperplasias. This is the first report of genetic alterations of the catalytic subunit of PKA linked to human disease: Germline PRKACA duplications with bilateral adrenal hyperplasias and somatic PRKACA mutations with unilateral cortisol producing adrenal adenomas. Citation Format: Fabio R. Faucz, Felix Beuschlei, Martin Fassnacht, Guilaume Assie, Davide Calebiro, Constantine Stratakis, Andrea Osswald, Cristina L. Ronchi, Thomas Wieland, Silviu Sbiera, Katrin Schaak, Anett Schmittfull, Thomas Schwarzmayr, Olivia Barreau, Delphine Vezzosi, Marthe Rizk-Rabbin, Ulrike Zabel, Eva Szarek, Paraskevi Salpea, Antonella Forlino, Annalisa Vetro, Orsetta Zuffardi, Caroline Kisker, Susanne Diener, Thomas Meitinger, Martin J. Lohse, Martin Reincke, Jerome Bertherat, Tim M. Strom, Bruno Allolio. Constitutive activation of PRKACA in adrenal Cushing's syndrome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-182. doi:10.1158/1538-7445.AM2014-LB-182
New England Journal of Medicine, 2014
Molecular and Cellular Endocrinology, 2014
The Journal of Clinical Endocrinology & Metabolism, 2014
Background: Carney complex (CNC) is a multiple neoplasia syndrome caused by PRKAR1A-inactivating ... more Background: 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. Setting: A tertiary care referral center was the setting for analysis of an international cohort of patients with CNC. Methods: 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. Results: 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 ...
Diabetes Research and Clinical Practice, 2020
Diabetes Research and Clinical Practice, 2019
Diabetes Research and Clinical Practice, 2019
Endocrine-Related Cancer, 2016
Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predispos... more Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predisposition to pancreatic tumors; it is caused most frequently by the inactivation of the PRKAR1A gene, a regulator of the cyclic AMP (cAMP)-dependent kinase (PKA). The method used was to create null alleles of prkar1a in mouse cells expressing pdx1 (Δ-Prkar1a). We found that these mice developed endocrine or mixed endocrine/acinar cell carcinomas with 100% penetrance by the age of 4–5 months. Malignant behavior of the tumors was seen as evidenced by stromal invasion and metastasis to locoregional lymph nodes. Histologically, most tumors exhibited an organoid pattern as seen in the islet-cell tumors. Biochemically, the lesions exhibited high PKA activity, as one would expect from deleting prkar1a. The primary neuroendocrine nature of these tumor cells was confirmed by immunohistochemical staining and electron microscopy, the latter revealing the characteristic granules. Although the Δ-Prkar1a ...
Molecular and cellular endocrinology, Jan 4, 2016
Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like ... more Osteochondromyxomas (OMX) in the context of Carney complex (CNC) and fibrous dysplasia (FD)-like lesions (FDLL) in mice, as well as isolated myxomas in humans may be caused by inactivation of PRKAR1A, the gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). OMXs and FDLL in mice lacking Prkar1a grow from abnormal proliferation of adult bone stromal cells (aBSCs). Prkar1a and Prkaca (coding for Cα) haploinsufficiency lead to COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of aBSCs. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. We hypothesized that COX-2 inhibition may have an effect in FD and FDLL. In vitro treatment of a human cell line prepared from a FD patient with Celecoxib resulted in decreased PGE2 and cell proliferation. Treatment of mice haploinsufficient for R1α and Cα with 1,500 mg/kg Celecoxib led to decreased PGE2 and proliferation and increased apoptosis, with a corresponding gene ...
Journal of Experimental & Clinical Cancer Research, 2015
Background: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunit... more Background: Protein kinase A (PKA) is a holoenzyme that consists of a dimer of regulatory subunits and two inactive catalytic subunits that bind to the regulatory subunit dimer. Four regulatory subunits (RIα, RIβ, RIIα, RIIβ) and four catalytic subunits (Cα, Cβ, Cγ, Prkx) have been described in the human and mouse genomes. Previous studies showed that complete inactivation of the Prkar1a subunit (coding for RIα) in the germline leads to embryonic lethality, while Prkar1a-deficient mice are viable and develop schwannomas, thyroid, and bone neoplasms, and rarely lymphomas and sarcomas. Mice with inactivation of the Prkar2a and Prkar2b genes (coding for RIIα and RIIβ, respectively) are also viable but have not been studied for their susceptibility to any tumors. Methods: Cohorts of Prkar1a +/− , Prkar2a +/− , Prkar2a −/− , Prkar2b +/− and wild type (WT) mice have been observed between 5 and 25 months of age for the development of hematologic malignancies. Tissues were studied by immunohistochemistry; tumor-specific markers were also used as indicated. Cell sorting and protein studies were also performed. Results: Both Prkar2a −/− and Prkar2a +/− mice frequently developed hematopoietic neoplasms dominated by histiocytic sarcomas (HS) with rare diffuse large B cell lymphomas (DLBCL). Southern blot analysis confirmed that the tumors diagnosed histologically as DLBCL were clonal B cell neoplasms. Mice with other genotypes did not develop a significant number of similar neoplasms. Conclusions: Prkar2a deficiency predisposes to hematopoietic malignancies in vivo. RIIα's likely association with HS and DLBCL was hitherto unrecognized and may lead to better understanding of these rare neoplasms.
Endocrine-Related Cancer, 2015
Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney comple... more Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney complex (CNC) or isolated, leads to ACTH-independent Cushing's syndrome (CS). CNC and PPNAD are caused typically by inactivating mutations of PRKAR1A, a gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). Mice lacking Prkar1a, specifically in the adrenal cortex (AdKO) developed CS caused by bilateral adrenal hyperplasia (BAH), which is formed from the abnormal proliferation of fetal-like adrenocortical cells. Celecoxib is a cyclooxygenase 2 (COX2) inhibitor. In bone, Prkar1a inhibition is associated with COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of bone stromal cells. We hypothesized that COX2 inhibition may have an effect in PPNAD. In vitro treatment of human cell lines, including one from a patient with PPNAD, with celecoxib resulted in decreased cell viability. We then treated AdKO and control m...
Human molecular genetics, Jan 5, 2015
Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated... more Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated with bone lesions such as osteochondromyxomas (OMX). The most frequent cause for CNC is PRKAR1A deficiency; PRKAR1A codes for type-I regulatory subunit of protein kinase A (PKA). Prkar1a(+/-) mice developed OMX, fibrous dysplasia-like lesions (FDL), and other tumors. Tumor tissues in these animals had increased PKA activity due to an unregulated PKA catalytic subunit and increased PKA type II (PKA-II) activity mediated by the PRKAR2A and PRKAR2B subunits. To better understand the effect of altered PKA activity on bone we studied Prkar2a and Prkar2b knock out and heterozygous mice; none of these mice developed bone lesions. When Prkar2a(+/-) and Prkar2b(+/-) mice were used to generate Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) animals, bone lesions formed that looked like those of the Prkar1a(+/-) mice. However, better overall bone organization and mineralization and fewer FDL...
European journal of endocrinology / European Federation of Endocrine Societies, Jan 30, 2015
Carney Complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of... more Carney Complex (CNC) is a rare autosomal dominant syndrome, characterized by pigmented lesions of the skin and mucosa, cardiac, cutaneous and other myxomas, and multiple endocrine tumors. The disease is caused by inactivating mutations or large deletions of the PRKAR1A gene located at 17q22-24 coding for the regulatory subunit type I alpha of protein kinase A (PKA) gene. Most recently, components of the complex have been associated with defects of other PKA subunits, such as the catalytic subunits PRKACA (adrenal hyperplasia) and PRKACB (pigmented spots, myxomas, pituitary adenomas). In this report, we review CNC, its clinical features, diagnosis, treatment, and molecular etiology including PRKAR1A mutations and the newest on PRKACA and PRKACB defects especially as they pertain to adrenal tumors and Cushing's syndrome.
Cancer Research, 2013
Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Histiocyt... more Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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&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
Cancer Research, 2013
Background: Carney complex (CNC) is a rare disease inherited as an autosomal dominant trait, asso... more 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 didn9t 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
Endocrine Abstracts, 2015
European Journal of Endocrinology, 2015
ObjectiveWe have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) ... more ObjectiveWe have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) and Cushing's syndrome (CS) caused by constitutive activation of the catalytic subunit of protein kinase A (PRKACA). By doing new in-depth analysis of their cytogenetic abnormality, we attempted a better genotype–phenotype correlation of their PRKACA amplification.DesignThis study is a case series.MethodsMolecular cytogenetic, genomic, clinical, and histopathological analyses were performed in five patients with CS.ResultsReinvestigation of the defects of previously described patients by state-of-the-art molecular cytogenetics showed complex genomic rearrangements in the chromosome 19p13.2p13.12 locus, resulting in copy number gains encompassing the entire PRKACA gene; three patients (one sporadic case and two related cases) were observed with gains consistent with duplications, while two sporadic patients were observed with gains consistent with triplications. Although all five pati...
Cancer Research, 2014
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Corticotropin-independent C... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Corticotropin-independent Cushing's syndrome may be caused by tumors or hyperplasia of the adrenal cortex. Until now genetic alterations explain only a small fraction of cases. The observation that a subset of adrenal adenomas is characterized by abnormal PKA activity despite the absence of mutations in candidate genes suggested as yet unknown alterations in the cAMP/PKA signaling cascade in these tumors. The aim of this study was the analysis of the genetic basis of Cushing's syndrome in order to reveal the gene/s responsible for the disease. Exome sequencing was performed in ten cortisol-producing adenomas and recurrent mutations in candidate genes were evaluated in additional 171 patients with adrenocortical tumors. Genome-wide copy number analysis was performed in 35 patients with cortisol secreting bilateral hyperplasias. The effects of these genetic defects were studied both clinically and in vitro. Exome sequencing in 8/10 adenomas revealed somatic mutations in the PRKACA gene, which encodes the main catalytic subunit of cyclic AMP-dependent protein kinase (PKA) (c.617A>C in seven and c.595_596insCAC in one). Overall, PRKACA somatic mutations were identified in a total of 22/59 (37%) adenomas from patients with overt Cushing's syndrome while these mutations were not detectable in patients with subclinical hypercortisolism (n=40) or in other adrenal tumors (n=82). Among 35 patients with cortisol producing hyperplasias, 5 (with two patients as first degree relatives) carried germline copy number gain of the chromosome 19 region including the PRKACA gene. In vitro studies demonstrated impaired inhibition of the mutant PRKACA by the PKA regulatory subunit, while cells from patients with germline chromosomal gains showed increased protein levels; in both cases, PKA activity was increased. The present study shows that more than one third of cortisol-producing adenomas associated with overt Cushing syndrome harbor unique somatic mutations of the main cAMP-dependent kinase catalytic subunit, PRKACA resulting in constitutive PKA activation. While in these patients the mutation is present only in tumor cells, germline duplication of the PRKACA gene was identified in a group of patients with bilateral adrenal hyperplasias. This is the first report of genetic alterations of the catalytic subunit of PKA linked to human disease: Germline PRKACA duplications with bilateral adrenal hyperplasias and somatic PRKACA mutations with unilateral cortisol producing adrenal adenomas. Citation Format: Fabio R. Faucz, Felix Beuschlei, Martin Fassnacht, Guilaume Assie, Davide Calebiro, Constantine Stratakis, Andrea Osswald, Cristina L. Ronchi, Thomas Wieland, Silviu Sbiera, Katrin Schaak, Anett Schmittfull, Thomas Schwarzmayr, Olivia Barreau, Delphine Vezzosi, Marthe Rizk-Rabbin, Ulrike Zabel, Eva Szarek, Paraskevi Salpea, Antonella Forlino, Annalisa Vetro, Orsetta Zuffardi, Caroline Kisker, Susanne Diener, Thomas Meitinger, Martin J. Lohse, Martin Reincke, Jerome Bertherat, Tim M. Strom, Bruno Allolio. Constitutive activation of PRKACA in adrenal Cushing's syndrome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-182. doi:10.1158/1538-7445.AM2014-LB-182
New England Journal of Medicine, 2014
Molecular and Cellular Endocrinology, 2014
The Journal of Clinical Endocrinology & Metabolism, 2014
Background: Carney complex (CNC) is a multiple neoplasia syndrome caused by PRKAR1A-inactivating ... more Background: 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. Setting: A tertiary care referral center was the setting for analysis of an international cohort of patients with CNC. Methods: 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. Results: 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 ...