Marthe Rizk-Rabin

Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome

BACKGROUND Corticotropin-independent Cushings syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushings syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

ARMC5 Mutations in Macronodular Adrenal Hyperplasia with Cushing's Syndrome

BACKGROUND Corticotropin-independent macronodular adrenal hyperplasia may be an incidental finding or it may be identified during evaluation for Cushings syndrome. Reports of familial cases and the involvement of both adrenal glands suggest a genetic origin of this condition. METHODS We genotyped blood and tumor DNA obtained from 33 patients with corticotropin-independent macronodular adrenal hyperplasia (12 men and 21 women who were 30 to 73 years of age), using single-nucleotide polymorphism arrays, microsatellite markers, and whole-genome and Sanger sequencing. The effects of armadillo repeat containing 5 (ARMC5) inactivation and overexpression were tested in cell-culture models. RESULTS The most frequent somatic chromosome alteration was loss of heterozygosity at 16p (in 8 of 33 patients for whom data were available [24%]). The most frequent mutation identified by means of whole-genome sequencing was in ARMC5, located at 16p11.2. ARMC5 mutations were detected in tumors obtained from 18 of 33 patients (55%). In all cases, both alleles of ARMC5 carried mutations: one germline and the other somatic. In 4 patients with a germline ARMC5 mutation, different nodules from the affected adrenals harbored different secondary ARMC5 alterations. Transcriptome-based classification of corticotropin-independent macronodular adrenal hyperplasia indicated that ARMC5 mutations influenced gene expression, since all cases with mutations clustered together. ARMC5 inactivation decreased steroidogenesis in vitro, and its overexpression altered cell survival. CONCLUSIONS Some cases of corticotropin-independent macronodular adrenal hyperplasia appear to be genetic, most often with inactivating mutations of ARMC5, a putative tumor-suppressor gene. Genetic testing for this condition, which often has a long and insidious prediagnostic course, might result in earlier identification and better management. (Funded by Agence Nationale de la Recherche and others.).

Vitamin D–Resistant Rickets and Type 1 Diabetes in a Child With Compound Heterozygous Mutations of the Vitamin D Receptor (L263R and R391S): Dissociated Responses of the CYP‐24 and rel‐B Promoters to 1,25‐Dihydroxyvitamin D3

We report here the first association between vitamin D–resistant rickets, alopecia, and type 1 diabetes in a child with compound heterozygous mutations in the VDR gene. Transfection studies suggest dissociated effects of VDR gene mutations on the regulation of genes involved in vitamin D metabolism and dendritic cell maturation.

Inactivation of the Carney Complex Gene 1 (Protein Kinase A Regulatory Subunit 1A) Inhibits SMAD3 Expression and TGFβ-Stimulated Apoptosis in Adrenocortical Cells

The cyclic AMP signaling pathway can be altered at multiple levels in endocrine tumors. Its central component is the protein kinase A (PKA). Carney complex (CNC) is a hereditary multiple neoplasia syndrome resulting from inactivating mutations of the gene encoding the PKA type I alpha regulatory subunit (PRKAR1A). Primary pigmented nodular adrenocortical disease is the most frequent endocrine tumor of CNC. Transforming growth factor beta (TGFbeta) regulates adrenal cortex physiology and signals through SMAD2/3. We used an interference approach to test the effects of PRKAR1A inactivation on PKA and TGFbeta pathways and on apoptosis in adrenocortical cells. PRKAR1A silencing stimulates PKA activity and increases transcriptional activity of a PKA reporter construct and expression of the endogenous PKA target, NR4A2, under basal conditions or after forskolin stimulation. PRKAR1A inactivation also decreased SMAD3 mRNA and protein levels via PKA, altering the cellular response to TGFbeta. SMAD3 expression was also inhibited by adrenocorticorticotropic hormone in the mouse adrenal gland and by forskolin in H295R cells. TGFbeta stimulates apoptosis in H295R cells, and this effect was counteracted by PRKAR1A inactivation. PRKAR1A silencing decreased the percentage of apoptotic cells and the cleavage of apoptosis mediators [caspase-3, poly(ADP-ribose) polymerase, and lamin A/C]. Inactivating mutations of PRKAR1A observed in adrenocortical tumors alter SMAD3, leading to resistance to TGFbeta-induced apoptosis. This cross-talk between the PKA and the TGFbeta signaling pathways reveals a new mechanism of endocrine tumorigenesis.

Phosphodiesterase 11A (PDE11A) Gene Defects in Patients with ACTH-Independent Macronodular Adrenal Hyperplasia (AIMAH): Functional Variants May Contribute to Genetic Susceptibility of Bilateral Adrenal Tumors

CONTEXT Phosphodiesterases (PDEs) are key regulatory enzymes of intracellular cAMP levels. PDE11A function has been linked to predisposition to adrenocortical tumors. OBJECTIVE The aim of the study was to study the PDE11A gene in a large cohort of patients with ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in control subjects. DESIGN The PDE11A entire coding region was sequenced in 46 patients with AIMAH and 192 controls. Two variants found in AIMAH patients were transiently expressed in HEK 293 and adrenocortical H295R cells for further functional studies. RESULTS The frequency of all PDE11A variants was significantly higher among patients with AIMAH (28%) compared to controls (7.2%) (P = 5 × 10(-5)). Transfection of the two PDE11A variants found in AIMAH patients only (D609N or M878V) showed that cAMP levels were higher, after forskolin stimulation, in cells transfected with the PDE11A mutants, compared to cells transfected with the wild-type PDE11A in HEK 293 cells (P < 0.05). Moreover, transfection with mutants PDE11A increased transcriptional activity of a cAMP-response element reporter construct compared to wild-type PDE11A in HEK 293 cells (P < 0.0004 for D609N and P < 0.003 for M878V) and in the adrenocortical H295R cells (P < 0.05 for D609N and M878V). In addition, analysis of cAMP levels in intact living culture cells by fluorescence resonance energy transfer probes showed increased cAMP in forskolin-treated cells transfected with PDE11A variants compared with wild-type PDE11A (P < 0.05). CONCLUSION We conclude that PDE11A genetic variants may increase predisposition to AIMAH.

The testis anion transporter TAT1 (SLC26A8) physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator channel: a potential role during sperm capacitation

The Slc26 gene family encodes several conserved anion transporters implicated in human genetic disorders, including Pendred syndrome, diastrophic dysplasia and congenital chloride diarrhea. We previously characterized the TAT1 (testis anion transporter 1; SLC26A8) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse, deletion of Tat1 caused male sterility due to a lack of sperm motility, impaired sperm capacitation and structural defects of the flagella. Ca(2+), Cl(-) and HCO(3)(-) influxes trigger sperm capacitation events required for oocyte fertilization; these events include the intracellular rise of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)-dependent protein phosphorylation. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in mature sperm and has been shown to contribute to Cl(-) and HCO(3)(-) movements during capacitation. Furthermore, several members of the SLC26 family have been described to form complexes with CFTR, resulting in the reciprocal regulation of their activities. We show here that TAT1 and CFTR physically interact and that in Xenopus laevis oocytes and in CHO-K1 cells, TAT1 expression strongly stimulates CFTR activity. Consistent with this, we show that Tat1 inactivation in mouse sperm results in deregulation of the intracellular cAMP content, preventing the activation of PKA-dependent downstream phosphorylation cascades essential for sperm activation. These various results suggest that TAT1 and CFTR may form a molecular complex involved in the regulation of Cl(-) and HCO(3)(-) fluxes during sperm capacitation. In humans, mutations in CFTR and/or TAT1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm.

Silencing mutated β-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R

Context Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target. Objective Similar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ). Results Following dox treatment a profound reduction in β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group. Conclusion In summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target.

Identification of Gene Expression Profiles Associated With Cortisol Secretion in Adrenocortical Adenomas

CONTEXT The cortisol secretion of adrenocortical adenomas can be either subtle or overt. The mechanisms leading to the autonomous hypersecretion of cortisol are unknown. OBJECTIVE The objective of the study was to identify the gene expression profile associated with the autonomous and excessive cortisol secretion of adrenocortical adenomas. PATIENTS AND METHODS The transcriptome of 22 unilateral adrenocortical adenomas (5 nonsecreting, 6 subclinical cortisol producing, 11 cortisol producing) was studied and correlated with cortisol secretion. Phosphodiesterase 8B (PDE8B) expression was measured by Western blot. RESULTS Unsupervised clustering identified 2 groups of adenomas with a difference in secretion level (P = .008). Cluster 1 included only cortisol-producing adenomas (8 of 11), whereas cluster 2 was an admixture of the nonsecreting, the subclinical cortisol-secreting, and 3 of the 11 cortisol-secreting adenomas (Fisher exact, P = .002). This cluster was driven by genes related to cortisol secretion and to extracellular matrix. More than 3000 genes correlated with cortisol secretion. Among the positively correlated were the steroidogenic enzymes, genes involved in cholesterol metabolism, and glutathione S-transferases. Among the negatively correlated genes were genes related to transcripts translation and the transcription factor GATA-6. The PDE8B, which inactivates the protein kinase A pathway, unexpectedly showed the strongest positive correlation with cortisol secretion, confirmed by Western blot. The protein kinase A-activity to cAMP ratio was increased in adenomas with high PDE8B levels, suggesting counterregulation to limit downstream activation of the pathway. CONCLUSION The transcriptome of adrenocortical adenomas reveals a major association with cortisol secretion and identifies specific groups of genes implicated in steroid secretion, suggesting that cAMP signaling alterations might be frequent in cortisol-secreting adenomas.

IGF2 Promotes Growth of Adrenocortical Carcinoma Cells, but Its Overexpression Does Not Modify Phenotypic and Molecular Features of Adrenocortical Carcinoma

Insulin-like growth factor 2 (IGF2) overexpression is an important molecular marker of adrenocortical carcinoma (ACC), which is a rare but devastating endocrine cancer. It is not clear whether IGF2 overexpression modifies the biology and growth of this cancer, thus more studies are required before IGF2 can be considered as a major therapeutic target. We compared the phenotypical, clinical, biological, and molecular characteristics of ACC with or without the overexpression of IGF2, to address these issues. We also carried out a similar analysis in an ACC cell line (H295R) in which IGF2 expression was knocked down with si- or shRNA. We found no significant differences in the clinical, biological and molecular (transcriptomic) traits between IGF2-high and IGF2-low ACC. The absence of IGF2 overexpression had little influence on the activation of tyrosine kinase pathways both in tumors and in H295 cells that express low levels of IGF2. In IGF2-low tumors, other growth factors (FGF9, PDGFA) are more expressed than in IGF2-high tumors, suggesting that they play a compensatory role in tumor progression. In addition, IGF2 knock-down in H295R cells substantially impaired growth (>50% inhibition), blocked cells in G1 phase, and promoted apoptosis (>2-fold). Finally, analysis of the 11p15 locus showed a paternal uniparental disomy in both IGF2-high and IGF2-low tumors, but low IGF2 expression could be explained in most IGF2-low ACC by an additional epigenetic modification at the 11p15 locus. Altogether, these observations confirm the active role of IGF2 in adrenocortical tumor growth, but also suggest that other growth promoting pathways may be involved in a subset of ACC with low IGF2 expression, which creates opportunities for the use of other targeted therapies.

Differential Expression of Parathyroid Hormone–Related Protein in Adrenocortical Tumors: Autocrine/Paracrine Effects on the Growth and Signaling Pathways in H295R Cells

Adrenocortical tumors (ACT) are rare and heterogeneous, but their pathogenesis is unclear. The oncoprotein parathyroid hormone–related protein (PTHrP), found in many common tumors, can regulate their growth in an autocrine/paracrine fashion through the PTH-R1 receptor. Little is known about the role of PTHrP in ACT. We monitored the synthesis of PTHrP and PTH-R1 in a series of 25 ACT: 12 adrenocortical carcinomas (ACC) and 13 adrenocortical adenomas (ACA), and investigated the effects of PTHrP(1-34) on H295R cells derived from an ACC. PTH-R1 mRNA and proteins were detected by real-time PCR and Western blotting in all the ACT samples and in H295R cells. Their concentrations did not differ significantly from one ACT to another. PTHrP mRNA was assayed by quantitative real-time PCR. It was detected in 90% of ACC, and in 10% of ACA. There was a positive correlation with the prognostic factors, McFarlane stage and Weiss score. Tissue-specific PTHrP protein processing was shown by Western blotting. Immunohistochemical staining revealed numerous, dense foci of PTHrP-containing cells in ACC, but few positive cells in ACA or normal tissue. PTHrP stimulated the growth of H295R cells, whereas a specific anti-PTHrP antibody and a PTHrP-R1 antagonist both enhanced their apoptosis. PTHrP activated both adenylate cyclase/protein kinase A and the intracellular calcium/protein kinase C pathways via PTHrP-R1. The active synthesis of PTHrP is linked to poor prognosis in ACC, in which it may act as an autocrine/paracrine factor in tumor growth and malignancy. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2275–85)