Activation of cyclic AMP signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline PRKAR1A defects versus those due to somatic GNAS mutations.

CONTEXT: The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP or protein kinase pathway. PRKAR1A-inactivating mutations are responsible for primary pigmented nodular adrenocortical disease, whereas somatic GNAS activating mutations cause macronodular disease in the context of McCune-Albright syndrome, ACTH-independent macronodular hyperplasia, and, rarely, cortisol-producing adenomas. OBJECTIVE AND DESIGN: The whole-genome expression profile (WGEP) of normal (pooled) adrenals, PRKAR1A- (3) and GNAS-mutant (3) was studied. Quantitative RT-PCR and Western blot were used to validate WGEP findings. RESULTS: MAPK and p53 signaling pathways were highly overexpressed in all lesions against normal tissue. GNAS-mutant tissues were significantly enriched for extracellular matrix receptor interaction and focal adhesion pathways when compared with PRKAR1A-mutant (fold enrichment 3.5, P < 0.0001 and 2.1, P < 0.002, respectively). NFKB, NFKBIA, and TNFRSF1A were higher in GNAS-mutant tumors (P < 0.05). Genes related to the Wnt signaling pathway (CCND1, CTNNB1, LEF1, LRP5, WISP1, and WNT3) were overexpressed in PRKAR1A-mutant lesions. CONCLUSION: WGEP analysis revealed that not all cAMP activation is the same: adrenal lesions harboring PRKAR1A or GNAS mutations share the downstream activation of certain oncogenic signals (such as MAPK and some cell cycle genes) but differ substantially in their effects on others.

Integrated genomic analysis of nodular tissue in macronodular adrenocortical hyperplasia: progression of tumorigenesis in a disorder associated with multiple benign lesions.

CONTEXT: Massive macronodular adrenocortical disease or ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a clinically and genetically heterogeneous disorder. OBJECTIVE AND DESIGN: Whole-genome expression profiling and oligonucleotide array comparative genomic hybridization changes were analyzed in samples of different nodules from the same patients with AIMAH. Quantitative RT-PCR and staining were employed to validate the mRNA array data. RESULTS: Chromosomal gains were more frequent in larger nodules when compared with smaller nodules from the same patients. Among the 50 most overexpressed genes, 50% had a chromosomal locus that was amplified in the comparative genomic hybridization data. Although the list of most over- and underexpressed genes was similar between the nodules of different size, the gene set enrichment analysis identified different pathways associated with AIMAH that corresponded to the size; the smaller nodules were mainly enriched for metabolic pathways, whereas p53 signaling and cancer genes were enriched in larger nodules. Confirmatory studies demonstrated that BCL2, E2F1, EGF, c-KIT, MYB, PRKCA, and CTNNB1 were overexpressed in the larger nodules at messenger and/or protein levels. Chromosomal enrichment analysis showed that chromosomes 20q13 and 14q23 might be involved in progression of AIMAH from smaller to larger tumors. CONCLUSION: Integrated transcriptomic and genomic data for AIMAH provides supporting evidence to the hypothesis that larger adrenal lesions, in the context of this chronic, polyclonal hyperplasia, accumulate an increased number of genomic and, subsequently, transcript abnormalities. The latter shows that the disease appears to start with mainly tissue metabolic derangements, as suggested by the study of the smaller nodules, but larger lesions showed aberrant expression of oncogenic pathways.

MicroRNA signature in massive macronodular adrenocortical disease and implications for adrenocortical tumourigenesis.

PURPOSE: Massive macronodular adrenocortical disease (MMAD) may be caused by aberrant microRNA expression. To determine the microRNA profile in MMAD and identify putative microRNA-gene target pairs involved in adrenal tumourigenesis. EXPERIMENTAL DESIGN: We performed microRNA microarray analysis in 10 patients with ACTH-independent Cushing syndrome caused by MMAD (ages 39-60 years) and four normal adrenal cortex samples were used as controls. Microarray data were validated by real-time polymerase chain reaction (qRT-PCR). Identification of potential microRNA-gene target pairs implicated in MMAD pathogenesis has been performed by integrating our microRNA data with previously obtained cDNA microarray data. Experimental validation of specific microRNA gene targets was performed by transfection experiments and luciferase assay. RESULTS: A total of 37 microRNAs were differentially expressed between MMAD and normal tissues; 16 microRNAs were down-regulated, including miR-200b and miR-203, whereas 21 microRNAs were up-regulated, miR-210 and miR-484 among them. Comparison of microRNA data with different clinicopathological parameters revealed miR-130a and miR-382 as putative diagnostic MMAD markers. Interestingly, we detected miR-200b targeting directly Matrin 3 (MATR3) expression in an adrenocortical cancer cell line (H295R). CONCLUSIONS: MicroRNAs appear to have distinct regulatory effects in MMAD, including an association with clinical presentation and severity of the disease, expressed by the degree of hypercortisolism. This is the first investigation of microRNAs in MMAD, a disease with complex pathogenesis; the data indicate that specific microRNAs such as miR-200b may play a significant role in MMAD formation and/or progression.

Abnormalities of cAMP signaling are present in adrenocortical lesions associated with ACTH-independent Cushing syndrome despite the absence of mutations in known genes.

CONTEXT: Bilateral adrenal hyperplasias (BAHs) may be caused by mutations of genes that code for molecules that participate in cAMP signaling. Little is known about cAMP signaling in adrenal lesions associated with ACTH-independent Cushing syndrome (AICS) that do not harbor mutations in known genes. OBJECTIVE: We assessed the cAMP-signaling pathway by enzymatic and molecular studies. DESIGN: Samples from 27 patients (ages 5-60 years) were studied and compared with normal adrenocortical tissue (n=4) and aldosterone-producing adenomas (APA, n=5). All samples were sequenced for GNAS, PRKAR1A, PDE11A, and PDE8B sequencing defects. cAMP levels and binding, protein kinase A, and phosphodiesterase (PDE) activities were assayed. Immunohistochemistry was used for certain studies and the phosphorylation status of CREB was studied. PATIENTS: A total of 36 samples from patients were used. RESULTS: Cortisol-producing adenomas (CPAs) and other lesions that were GNAS, PRKAR1A, PDE11A, and PDE8B gene mutation-negative were compared with PRKAR1A mutation-positive lesions, normal tissue, and APAs; abnormalities of the cAMP-signaling pathway were found in both BAHs and CPAs. Interestingly, mutation-negative CPAs had significantly decreased PDE activity. CONCLUSION: Lesions of the adrenal associated with AICS, independently of their GNAS, PRKAR1A, PDE11A, and PDE8B mutation status, have functional abnormalities of cAMP signaling. It is probable that epigenetic events or additional defects of genes involved in this pathway are responsible for this phenomenon.

MicroRNA signature of primary pigmented nodular adrenocortical disease: clinical correlations and regulation of Wnt signaling.

MicroRNAs comprise a novel group of gene regulators implicated in the development of different types of cancer; however, their role in primary pigmented nodular adrenocortical disease (PPNAD) has not been investigated. PPNAD is a bilateral adrenal hyperplasia often associated with Carney complex, a multiple neoplasia syndrome; both disorders are caused by protein kinase A (PKA) regulatory subunit type 1A (PRKARIA)-inactivating mutations. We identified a 44-microRNA gene signature of PPNAD after comparing PPNAD with normal adrenal samples. Specifically, 33 microRNAs were up-regulated and 11 down-regulated in PPNAD relative to normal tissues. These results were validated by stem loop real-time PCR analysis. Comparison of microRNA microarray data with clinicopathologic variables revealed a negative correlation (r = -0.9499) between let-7b expression and cortisol levels in patients with PPNAD. Integration of microRNA microarray with serial analysis of gene expression data together with bioinformatic algorithm predictions revealed nine microRNA-gene target pairs with a potential role in adrenal pathogenesis. Using a PPNAD cell line, we showed that miR-449 was up-regulated and identified its direct target, WNT1-inducible signaling pathway protein 2 (WISP2); in addition, pharmacologic inhibition of PKA resulted in the up-regulation of miR-449 leading to the suppression of WISP2. Overall, we investigated, for the first time, the microRNA profile and its clinical significance in PPNAD; these data also suggest that PKA, via microRNA regulation, affects the Wnt signaling pathway, which through expression and clinical studies is suspected to be a primary mediator of PRKAR1A-related tumorigenesis.