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Barreau O.,Institute National Of La Sante Et Of La Recherche Medicale Unite 1016 | Barreau O.,French National Center for Scientific Research | Barreau O.,University of Paris Descartes | Assie G.,Institute National Of La Sante Et Of La Recherche Medicale Unite 1016 | And 29 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2013

Purpose: DNA methylation is a mechanism for gene expression silencing in cancer. Limited information is available for adrenocortical tumors. Abnormal methylation at the IGF2/H19 locus is common in adrenocortical carcinomas. Our aim was to characterize the methylation in adrenocortical carcinomas at a whole-genome scale and to assess its clinical significance and its impact on gene expression. Experimental Design: Methylation patterns of CpG islands in promoter regions of 51 adrenocortical carcinomas and 84 adenomas were studied by the Infinium Human Methylation 27 Beadchip (Illumina, San Diego, CA). Methylation of 33 genes was studied by methylation-specific multiplex ligation-dependent probe amplification (MRC-Holland, Amsterdam, The Netherlands) in 15 carcinomas. Gene expression data were available for 87 tumors from a previous study (HG-U133Plus2.0 AffymetrixGeneChip; Affymetrix, Santa Clara, CA). Clinical information, including patient features and survival, were available for all tumors. Results: Methylation was higher in carcinomas than in adenomas (t test P = 3.1 × 10-9). Unsupervised clustering of DNA methylation profiles identified two groups of carcinomas, one with an elevated methylation level, evoking a CpG island methylator phenotype (CIMP). The subgroup of hypermethylated carcinomas was further divided in two subgroups, with different levels of methylation (CIMP-high and CIMP-low). This classification could be confirmed by methylation-specific multiplex ligation-dependent probe amplification. Hypermethylation was associated with a poor survival (Cox model P = 0.02). The transcriptome/methylation correlation showed 1741 genes (of 12,250) negatively correlated; among the top genes were H19 and other tumor suppressors (PLAGL-1, G0S2, and NDRG2). Conclusions: This genome-wide methylation analysis reveals the existence of hypermethylated adrenocortical carcinomas, with a poorer prognosis. Hypermethylation in these tumors is important for silencing specific tumor suppressor genes. Copyright © 2013 by The Endocrine Society.


Assie G.,French Institute of Health and Medical Research | Assie G.,Center for Rare Adrenal Diseases | Letouze E.,Programme Cartes dIdentite des Tumeurs CIT | Fassnacht M.,Ludwig Maximilians University of Munich | And 67 more authors.
Nature Genetics | Year: 2014

Adrenocortical carcinomas (ACCs) are aggressive cancers originating in the cortex of the adrenal gland. Despite overall poor prognosis, ACC outcome is heterogeneous. We performed exome sequencing and SNP array analysis of 45 ACCs and identified recurrent alterations in known driver genes (CTNNB1, TP53, CDKN2A, RB1 and MEN1) and in genes not previously reported in ACC (ZNRF3, DAXX, TERT and MED12), which we validated in an independent cohort of 77 ACCs. ZNRF3, encoding a cell surface E3 ubiquitin ligase, was the most frequently altered gene (21%) and is a potential new tumor suppressor gene related to the Î 2-catenin pathway. Our integrated genomic analyses further identified two distinct molecular subgroups with opposite outcome. The C1A group of ACCs with poor outcome displayed numerous mutations and DNA methylation alterations, whereas the C1B group of ACCs with good prognosis displayed specific deregulation of two microRNA clusters. Thus, aggressive and indolent ACCs correspond to two distinct molecular entities driven by different oncogenic alterations. © 2014 Nature America, Inc.


de Krijger R.E.,Erasmus Medical Center | de Krijger R.E.,Reinier Of Graaf Hospital | Bertherat J.,French Institute of Health and Medical Research | Bertherat J.,University of Paris Descartes | Bertherat J.,Center for Rare Adrenal Diseases
Hormones and Cancer | Year: 2016

For the clinician, despite its rarity, adrenocortical cancer is a heterogeneous tumor both in term of steroid excess and tumor evolution. For patient management, it is crucial to have an accurate vision of this heterogeneity, in order to use a correct tumor classification. Pathology is the best way to classify operated adrenocortical tumors: to recognize their adrenocortical nature and to differentiate benign from malignant tumors. Among malignant tumors pathology also aims at prognosis assessment. Although progress has being made for prognosis assessment, there is still a need for improvement. Recent studies have established the value of Ki67 for adrenocortical cancer (ACC) prognostication, aiming also at standardization to reduce variability. The use of genomics to study adrenocortical tumors gives a very new insight in their pathogenesis and molecular classification. Genomics studies of ACC give now a clear description of the mRNA (transcriptome) and miRNA expression profile, as well as chromosomal and methylation alterations. Exome sequencing also established firmly the list of the main ACC driver genes. Interestingly, genomics study of ACC also revealed subtypes of malignant tumors with different pattern of molecular alterations, associated with different outcome. This leads to a new vision of adrenocortical tumors classification based on molecular analysis. Interestingly, these molecular classifications meet also the results of pathological analysis. This opens new perspectives on the development and use of various molecular tools to classify, along with pathological analysis, ACC, and guides patient management at the area of precision medicine. © 2015, Springer Science+Business Media New York.


Yu B.,French Institute of Health and Medical Research | Yu B.,French National Center for Scientific Research | Yu B.,University of Paris Descartes | Ragazzon B.,French Institute of Health and Medical Research | And 9 more authors.
Hormone and Metabolic Research | Year: 2012

Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis. © Georg Thieme Verlag KG Stuttgart · New York.


Espiard S.,French Institute of Health and Medical Research | Espiard S.,French National Center for Scientific Research | Espiard S.,University of Paris Descartes | Ragazzon B.,French Institute of Health and Medical Research | And 6 more authors.
Hormone and Metabolic Research | Year: 2014

Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40% of cortisol-secreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders. © Georg Thieme Verlag K.G. Stuttgart.


Espiard S.,French Institute of Health and Medical Research | Espiard S.,French National Center for Scientific Research | Espiard S.,University of Paris Descartes | Bertherat J.,French Institute of Health and Medical Research | And 3 more authors.
Endocrinology and Metabolism Clinics of North America | Year: 2015

Advances in genomics accelerated greatly progress in the study of the genetics adrenocortical tumors. Bilateral nodular hyperplasias causing Cushing's syndrome are frequently caused by germline alterations leading to cAMP/PKA pathway activation (micronodular) and ARMC5 inactivation (macronodular). Somatic mutations of β-catenin and PRKACA are observed in non secreting or cortisol producing adenomas, respectively. Alterations of the β-catenin (CTNN1B, ZNFR3) or TP53 pathways are found in carcinomas. Mutations in cancers are more common in aggressive tumors and correlate with transcriptome or methylation profiles. Identification of these alterations helps to refine the molecular classification of these tumors and to develop molecular diagnostic tools. © 2015 Elsevier Inc.


Assie G.,French Institute of Health and Medical Research | Assie G.,French National Center for Scientific Research | Assie G.,University of Paris Descartes | Giordano T.J.,University of Michigan | And 4 more authors.
Molecular and Cellular Endocrinology | Year: 2012

Transcriptome studies of adrenocortical tumors have shown clear differences between adenomas and carcinomas and identified two subgroups of carcinomas with different prognoses. This review focuses on how transcriptomes have enriched our knowledge about genes previously identified by classical candidate gene approaches, uncovered novel genes relevant to adrenocortical tumor biology, helped to identify and understand specific pathway alterations, and advanced the overall translational relevance of this field of research. © 2011 Elsevier Ireland Ltd.


Rothenbuhler A.,U.S. National Institutes of Health | Horvath A.,U.S. National Institutes of Health | Libe R.,French National Center for Scientific Research | Libe R.,University of Paris Descartes | And 17 more authors.
Clinical Endocrinology | Year: 2012

Background Genetic aberrations in various components of cAMP signalling pathway predispose to endocrine tumours. Mutations in the phosphodiesterases (PDEs) are involved in the predisposition to adrenocortical neoplastic conditions. Objective To screen for genetic variations in PDE8B among patients with different types of adrenocortical tumours. Design and subjects This is a case-control study followed by functional analyses. Two hundred and sixteen unrelated patients with different types of adrenocortical tumours and 192 healthy control individuals participated in the study. Methods Bidirectional Sanger sequencing, in vitro cell line transfection and in silico modelling are used in this study. Results Nine different PDE8B sequence changes, six novel and three previously reported, were identified in our patients and controls. Two of the variations, seen only in the patient group, showed significant potential to impair protein function, both in vitro and in silico. Conclusion PDE8B is another PDE gene in which variations may contribute to predisposition of adrenocortical tumours. © 2012 Blackwell Publishing Ltd.


Johnsen I.K.,Ludwig Maximilians University of Munich | Hahner S.,University of Würzburg | Briere J.-J.,French Institute of Health and Medical Research | Briere J.-J.,University Paris Diderot | And 10 more authors.
Hormone and Metabolic Research | Year: 2010

Storage and tissue handling of surgical tumor specimen have been recognized as critical steps that can potentially affect reproducibility and comparability of molecular endpoints between laboratories. In the preparation of adrenal tumor tissue banking, three different protocols that simulate warm ischemia upon tumor removal (protocol I), thawing and refreezing cycles (protocol II), as well as storage of vital tumor samples (protocol III) were applied. For the first two protocols, samples were subdivided and either snap frozen or treated with a RNA preserving agent (RPA) while in protocol III different storage media were compared. Following these procedures, recovery and integrity of DNA, RNA, and protein by means of pulsed field electrophoresis, long-range PCR, real-time PCR, immunoblot, and immunohistochemistry (protocol I and II) as well as cell viability and steroidogenic capacity (protocol III) were investigated. While DNA integrity was not influenced by different treatment modalities, expression levels of adrenal marker genes were more affected in samples after snap freezing in comparison to RPA pretreatment. Moreover, storage at room temperature before and after freezing could be demonstrated to decrease the relative amount of protein phosphorylation (ERK) and enzymatic activity (succinate cytochrome c reductase) while overall protein levels were not significantly affected. Similarly, morphological or immunohistochemical evaluation was comparable between groups. For primary cell cultures generated after storage of tumor samples similar rates of viability were observable while steroid output varied between the groups. Overall, on the basis of the presented endpoints standardized operational procedures can be defined for a proposed European adrenal tumor biobank. © Georg Thieme Verlag KG Stuttgart - New York.


Basso F.,French Institute of Health and Medical Research | Rocchetti F.,French Institute of Health and Medical Research | Rodriguez S.,French Institute of Health and Medical Research | Nesterova M.,U.S. National Institutes of Health | And 6 more authors.
Hormone and Metabolic Research | Year: 2014

The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression. © Georg Thieme Verlag K.G. Stuttgart.

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