Normal Adrenocortical Tissue Research Articles

Detection of near-infrared autofluorescence from adrenal neoplasms: An initial experience.

Fluorescence from adrenal tumors can be detected with near-infrared imaging after injection with indocyanine green. However, it is unknown if adrenal tumors exhibit autofluorescence. The aim of this study was to determine whether adrenal tumors emit near-infrared autofluorescence (NIRAF). This was a prospective study of patients who underwent minimally invasive adrenalectomy at a tertiary center. Intraoperative images were analyzed to detect NIRAF with a 750 nm camera. Descriptive and comparative statistical analyses were performed. Twenty-five adrenalectomies were examined. Only 11tumors (44%), that originated from the cortex exhibited autofluorescence. A contrast distinction between the tumor and retroperitoneum was observed in 23 patients, whereas a contrast distinction between the tumor and normal adrenocortical tissue was seen in 12 patients. The overall fluorescence intensity of adrenal tumors was found to be variable and ranging between 0.3 and 5.6 times that of the background tissue. Pheochromocytoma, malignancy and adrenal cyst did not demonstrate NIRAF. This is the first study to show that adrenocortical tissue can demonstrate NIRAF. The pattern of fluorescence was similar to that observed after indocyanine green injection in our historical experience. NIRAF has a potential to be used as an intraoperative optical adjunct during adrenalectomy.

Expression of clock-related genes in benign and malignant adrenal tumors.

Although the effect of the central clock system on adrenal function has been extensively studied, the role of the peripheral clock system in adrenal tumorigenesis remains largely unexplored. In this study we investigated the expression of clock-related genes in normal adrenocortical tissue and adrenocortical tumors. Twenty-seven fresh frozen human adrenal tissues including 13 cortisol secreting adenomas (CSA), seven aldosterone producing adenomas (APA), and seven adrenocortical carcinomas (ACC) were collected. CLOCK, BMAL1, PER1, CRY1, Rev-ERB, and RORα mRNA and protein expression were determined by qPCR and immunoblotting in pathological tissues and compared with the adjacent normal adrenal tissues. A significant downregulation of PER1, CRY1, and Rev-ERB compared with their normal tissue was demonstrated in CSA. All clock-related genes were overexpressed in APA compared with their normal tissue, albeit not significantly. A significant upregulation of CRY1 and PER1 and downregulation of BMAL1, RORα, and Rev-ERB compared with normal adrenal tissue was observed in ACC. BMAL1 and PER1 were significantly downregulated in APA compared with CSA. CLOCK, CRY1, and PER1 were upregulated, whereas BMAL1, RORα, and Rev-ERB were downregulated in ACC compared with CSA. Our study demonstrated the expression of CLOCK, BMAL1, PER1, CRY1, Rev-ERB, and RORα in normal and pathological human adrenal tissues. Adrenal tumors exhibited altered expression of these genes compared with normal tissue, with specific differences between benign and malignant lesions and between benign tumors arising from glomerulosa vs fasciculata zone. Further studies should clarify whether these alterations could be implicated in adrenocortical tumorigenesis.

SAT-343 Immunohistochemical Expression of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor (LH/hCG Receptor) in Adrenal Adenomas of Postmenopausal Women

Background: Increasingly, patients are found to have incidentally found adrenal adenomas on imaging performed for other purposes. Further biochemical work-up results in a diagnosis of mild hypercortisolism in as many as 30% of these patients. The incidence of adrenal adenomas and Cushing’s is higher in women than men with peak incidence ages 50-70. Elevated expression of gonadal markers such as LH/hCG receptor and the gonadal transcription factor GATA-4 has been well described in post-gonadectomy induced adrenal cortical neoplasia in neutered ferrets and certain mouse strains. The majority of these neoplasms of the adrenal cortex, which share a common embryologic progenitor with the gonads in the urogenital ridge, produce sex steroids and appear to differentiate towards a “gonadal” molecular profile. Isolated case reports have demonstrated LH/hCG receptor expression in adrenal secretory tumors in postmenopausal women and pregnant females. Taken together, there is strong evidence that a subset of adrenal tumors is driven by LH in postmenopausal women. Hypothesis: We hypothesized that adrenal adenomas producing secretory syndromes in postmenopausal women express LH/hCG receptor. Methods: 24 archival adrenal tissues from women >50 years of age with hypersecretory syndromes (primary aldosteronism and Cushing’s) were obtained with IRB approval. The samples were immunostained for expression of the LH/hCG receptor (monoclonal unconjugated LHR Antibody (LHCGR/1417), Novus Biologicals, CO) utilizing the Streptavidin–Biotin staining method. The slides were reviewed in a blinded fashion, and cytoplasmic, membrane, and nuclear expression of the markers were graded as no immunoreactivity, or 1+, 2+, or 3+ staining. Results: All 24 adenomas had demonstrated expression of LH/hCG receptor in both normal and adenomatous tissue. The staining in adenomas was heterogeneous, with clusters of densely stained LH/hCG receptor positive cells found selectively in areas of the adenomas. In addition, these clusters were found in normal adjacent adrenal tissue primarily in the subcapsular, zona glomerulosa region, an area where the putative adrenal cortical stem cells are found. Conclusion: LH/hCG receptor was found to be expressed in normal adrenocortical and adenomatous tissue in postmenopausal women, a population that has elevated serum LH levels. These findings may have clinical implications and provide a rationale for the treatment of secretory adrenal adenomas with GnRH agonists or antagonists in postmenopausal women.

A comparative proteomic study identified calreticulin and prohibitin up-regulated in adrenocortical carcinomas

BackgroundIdentifying novel tumor biomarkers to develop more effective diagnostic and therapeutic strategies for patients with ACC is urgently needed. The aim of the study was to compare the proteomic profiles between adrenocortical carcinomas (ACC) and normal adrenocortical tissues in order to identify novel potential biomarkers for ACC.MethodsThe protein samples from 12 ACC tissues and their paired adjacent normal adrenocortical tissues were profiled with two-dimensional electrophoresis; and differentially expressed proteins were identified by mass spectrometry. Expression patterns of three differently expressed proteins calreticulin, prohibitin and HSP60 in ACC, adrenocortical adenomas (ACA) and normal adrenocortical tissues were further validated by immunohistochemistry.ResultsIn our proteomic study, we identified 20 up-regulated and 9 down-regulated proteins in ACC tissues compared with paired normal controls. Most of the up-regulated proteins were focused in protein binding and oxidoreductase activity in Gene Ontology (GO) molecular function classification. By immunohistochemistry, two biomarkers calreticulin and prohibitin were validated to be overexpressed in ACC compared with adrenocortical adenomas (ACA) and normal tissues, but also calreticulin overexpression was significantly associated with tumor stages of ACC.ConclusionFor the first time, calreticulin and prohibitin were identified to be novel candidate biomarkers for ACC, and their roles during ACC carcinogenesis and clinical significance deserves further investigation.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1897372598927465

Abstract 3543: TOP2A is a therapeutic target for adrenocortical carcinoma .

Abstract Adrenocortical carcinoma (ACC) represents one of the most resistant human cancers to systemic chemotherapy and a major clinical challenge in the field of endocrine oncology. Recent molecular characterizations of ACC suggest new opportunities for targeted therapy. The current study aimed to evaluate expression and function of TOP2A in human adrenocortical neoplasm, and anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples. In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cell lines. TOP2A mRNA expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (p < 0.001); likewise, TOP2A protein expression was significantly higher in ACC (p < 0.05). Knockdown of TOP2A gene expression decreased cell proliferation, anchorage-independent growth and invasion (p < 0.05). Aclarubicin had the most potent antiproliferative activity out of 11 active TOP2A inhibitors, which significantly decreased proliferation and tumor spheroid size in ACC cell lines (p< 0.05). Our data indicate that most TOP2A inhibitors are effective compounds for ACC, with aclarubicin having the best anticancer activity. Thus, TOP2A inhibitors should be tested in future clinical trials for patients with locally advanced and metastatic ACC. Citation Format: Meenu Jain, Lisa Zhang, Mei He, Ya-Qin Zhang, Naris Nilubol, Min Shen, Electron Kebebew. TOP2A is a therapeutic target for adrenocortical carcinoma . [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 3543. doi:10.1158/1538-7445.AM2013-3543

TOP2A is overexpressed and is a therapeutic target for adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with unresectable disease. The aim of the current study was i) to evaluate TOP2A expression and function in human adrenocortical neoplasm and ACC cells and ii) to determine the anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 ACCs). In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth, and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cells. TOP2A mRNA and protein expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (P<0.05). Knockdown of TOP2A gene expression in ACC cell lines significantly decreased cell proliferation, anchorage-independent growth, and invasion (P<0.05). A screening assay in NCI-H295R cells showed that 11 of 14 TOP2A inhibitors had antiproliferative activity, 5 of the 14 TOP2A inhibitors had a higher antiproliferative activity than mitotane, and aclarubicin was the agent with the highest activity. Aclarubicin was validated to significantly decrease proliferation and tumor spheroid size in both NCI-H295R and SW13 ACC cell lines (P<0.05). Our results suggest that TOP2A is overexpressed in ACC, regulates cellular proliferation and invasion in ACC cells, and is an attractive target for ACC therapy. Of the TOP2A inhibitors screened, aclarubicin is a good candidate agent to test in future clinical trials for patients with locally advanced and metastatic ACC.

Partial adrenalectomy minimizes the need for long-term hormone replacement in pediatric patients with pheochromocytoma and von Hippel-Lindau syndrome

PurposeChildren with von Hippel-Lindau syndrome (VHL) are at an increased risk for developing bilateral pheochromocytomas. In an effort to illustrate the advantage of partial adrenalectomy (PA) over total adrenalectomy in children with VHL, we report the largest single series on PA for pediatric patients with VHL, demonstrating a balance between tumor removal and preservation of adrenocortical function. MethodsFrom 1994 to 2011, a prospectively maintained database was reviewed to evaluate 10 pediatric patients with hereditary pheochromocytoma for PA. Surgery was performed if there was clinical evidence of pheochromocytoma and if normal adrenocortical tissue was evident on preoperative imaging and/or intraoperative ultrasonography. Perioperative data were collected, and patients were observed for postoperative steroid use and tumor recurrence. ResultsTen pediatric patients with a diagnosis of VHL underwent 18 successful partial adrenalectomies (4 open, 14 laparoscopic). The median tumor size removed was 2.6 cm (range, 1.2-6.5 cm). Over a median follow-up of 7.2 years (range, 2.6-15.8 years), additional tumors in the ipsilateral adrenal gland were found in 2 patients. One patient underwent completion adrenalectomy, and 1 underwent a salvage PA with resection of the ipsilateral lesion. One patient required short-term steroid replacement therapy. At last follow-up, 7 patients had no radiographic or laboratory evidence of pheochromocytoma. ConclusionAt our institution, PA is the preferred form of management for pheochromocytoma in the (VHL) pediatric population. This surgical approach allows for removal of tumor while preserving adrenocortical function and minimizing the adverse effects of long-term steroid replacement on puberty and quality of life.

Glucocorticoid Receptor and Molecular Chaperones in the Pathogenesis of Adrenal Incidentalomas: Potential Role of Reduced Sensitivity to Glucocorticoids

Glucocorticoid (GC) sensitivity depends on glucocorticoid receptor (GR) and heat shock proteins (Hsps). We investigated whether common GR genes (ER22/23EK, N363S, Bcl I, and 9β) and adrenocorticotropin receptor promoter polymorphisms influence susceptibility for unilateral adrenal incidentaloma (AI), plus GR and Hsp expression in tumorous (n = 19), peritumorous (n = 13) and normal adrenocortical (n = 11) tissues. Patients (n = 112), population-matched controls (n = 100) and tumor tissues (n = 32) were genotyped for these polymorphisms. Postdexamethasone serum cortisol was higher in patients (p < 0.001). GR gene variants, larger allele of Bcl I (odds ratio [OR] 2.9; 95% confidence interval [CI] 1.7-5.1; p < 0.001] and minor allele of 9β (OR 3.0; 95% CI 1.6-5.7; p < 0.001) were independent predictors of AI. In patients, the first allele is linked with larger tumors (p = 0.002) and the latter with higher postdexamethasone cortisol levels (p = 0.025). Both allele carriers had lesser waist circumference (p = 0.02), similar adrenocorticotropin and higher basal (p = 0.024) and postdexamethasone cortisol concentrations (p < 0.001). Tumorous and constitutional genotypes were similar. GR-D is the major receptor isoform in normal adrenal cortex by Western blotting. Loss of other receptor isoforms, decrease in immunostaining for GR (p < 0.0001), underexpression of chaperones (p ≤ 0.01) and the presence of inducible Hsp70 were found in adenomas. In conclusion, GR gene variants, C allele of Bcl I and minor allele of 9β, are associated with AIs. Their concurrent presence in patients reduces GC sensitivity. Normal adrenal cortex preferentially expresses GR-D. In adenomas, the lack of other GR isoforms and underexpression of heat shock proteins perhaps permanently impair GC signaling, which could promote dysregulated cortisol production and tumor growth. The innate GC sensitivity probably modifies these effects.

Comprehensive DNA methylation analysis of benign and malignant adrenocortical tumors

The molecular pathogenesis of benign and malignant adrenocortical tumors (ACT) is incompletely clarified. The role of DNA methylation in adrenocortical tumorigenesis has not been analyzed in an unbiased, systematic fashion. Using the Infinium HumanMethylation27 BeadChip, the DNA methylation levels of 27,578 CpG sites were investigated in bisulfite-modified DNA from 6 normal adrenocortical tissue samples, 27 adrenocortical adenomas (ACA), and 15 adrenocortical carcinomas (ACC). Genes involved in cell cycle regulation, apoptosis, and transcriptional regulation of known or putative importance in the development of adrenal tumors showed significant and frequent hypermethylation. Such genes included CDKN2A, GATA4, BCL2, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC. Gene expression studies of selected hypermethylated genes (CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, SCGB3A1/HIN1) in 6 normal and 16 neoplastic adrenocortical tissues (10 ACA and 6 ACC), displayed reduced gene expression in benign and malignant ACT versus normal adrenocortical tissue. Treatment with 5-aza-2'-deoxycytidine of adrenocortical cancer H-295R cells increased expression of the hypermethylated genes CDKN2A, GATA4, DLEC1, HDAC10, PYCARD, and SCGB3A1/HIN1. In conclusion, the current study represents the first unbiased, quantitative, genome-wide study of adrenocortical tumor DNA methylation. Genes with altered DNA methylation patterns were identified of putative importance to benign and malignant adrenocortical tumor development.

Interleukin-13 receptor alpha2 is a novel therapeutic target for human adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a relatively rare but aggressive malignancy with limited therapeutic options. Previous genome-wide expression studies have demonstrated the overexpression of interleukin-13 receptor alpha2 (IL13Rα2) in some human malignancies. The authors evaluated IL13Rα2 mRNA and protein expression in 21 normal samples, 78 benign samples, 10 primary malignant samples, and 25 metastatic/recurrent samples and performed functional analyses with IL13 ligand and IL13 Rα2 knockdown in vitro. The sensitivity of 2 ACC cell lines (NCI-H295R [high IL13Rα2 expression] and SW13 [low IL13Rα2 expression]) to a highly specific IL-13 conjugated with Pseudomonas exotoxin (IL-13-PE) also was evaluated in both in vitro and in vivo models. IL13Rα2 was overexpressed in malignant tumors compared with benign and normal samples (15-fold higher; P < .05). Immunohistochemistry also confirmed higher protein expression in malignant and benign tumors than in normal adrenocortical tissues (P < .05). The half-maximal inhibitory concentration for IL-13-PE was 1.3 ng/mL in the NCI-H295R cell line and 1000 ng/mL in the SW13 cell line. Mice that received intratumoral or intraperitoneal IL-13-PE injection had a significant reduction in tumor size and increased tumor necrosis compared with control groups (P < .05) and also had prolonged survival (P < .05). IL13Rα2 protein expression increased in cells that were treated with IL-13 ligand along with cell invasion (P < .05). Direct IL13Rα2 knockdown decreased cellular proliferation and invasion (P < .05). The current results indicated that IL13Rα2 is overexpressed in ACC and regulates cell invasion and proliferation. IL13Rα2 is a novel therapeutic target for the treatment of human ACC.

Abstract 3932: IL13Rα2 regulates cell invasion, and is a therapeutic target for adrenocortical carcinoma

Abstract Adrencortical neoplasms are one of the most common human neopolasms. Furthermore, it is difficult to distinguish localized benign from malignant adrenal tumors. Adrenocortical carcinoma (ACC) is a relatively rare but aggressive malignancy with limited therapeutic options for patients with advanced and metastatic disease. Several genome wide expression studies have shown overexpression of Interleukin-13 Receptor alpha2 (IL13Rα2) in some human malignancies but the function of this gene in cancer cells is poorly understood. We evaluated IL13Rα2 mRNA expression in normal (n=21), and in benign (n=78) and malignant (n=10) adrenocortical tumors. We found 11-fold higher expression in malignant tumors compared to benign (p&amp;lt;0.05) and absent expression in normal adrenocortical tissue. Immunohistochemistry analysis confirmed high protein expression in malignant and benign tumors and absent expression in normal adrenocortical tissues (p&amp;lt;0.05). IL13Rα2 mRNA expression level also had 90% accuracy for distinguishing benign from malignant adrenocortical tumors. We used IL13Rα2 siRNA knockdown to assess its effect on cell proliferation and cell invasion in the NCI H295R cell line, which expressed high levels of IL13Rα2. IL13Rα2 knockdown did not significantly affect cell proliferation but did decrease invasion by 60% as compared to negative control (p&amp;lt;0.05). To determine the significance of IL13Rα2 as a therapeutic target for ACC, we evaluated the sensitivity in two ACC cell lines (NCI-H295R with high IL13Rα2 expression and SW13 with low IL13Rα2 expression) to a highly specific IL-13 targeting immunotoxin (IL-13-PE). We found the IC50 for IL-13-PE was 1.3ng/ml (NCI-H295R) and 1000ng/ml (SW13) in the cell lines. Based on this observation, we then tested the efficacy of IL-13-PE therapy in an ACC xenograft model in athymic nude mice. Mice receiving either intratumoral or intraperitoneal IL-13-PE therapy as compared to control showed a significant reduction in tumor size as well as tumor necrosis (p&amp;lt;0.05), and had prolonged survival (p&amp;lt;0.05). Both routes of IL-13-PE administration were well tolerated with no significant toxicity. Our study indicates that IL13Rα2 is overexpressed in 100% of ACC, is a good diagnostic marker for ACC, regulates ACC cell invasion, and is a novel therapeutic target for the treatment of ACC. 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 3932. doi:1538-7445.AM2012-3932

The DNA Methylome Of Benign And Malignant Adrenocortical Tumors

Introduction: Epigenetic mechanisms participate along with genetic abnormalities to cause altered patterns of gene expression and function in tumors. The molecular pathogenesis of benign and malignant adrenocortical tumors (ACT) is incompletely clarified. The role of DNA methylation in adrenocortical tumorigenesis has not been analyzed in an unbiased, systematic fashion. Methods: High molecular weight genomic DNA was isolated from normal adrenocortical tissue (n=6), aldosterone-producing adrenocortical adenomas (ACA; n=9), cortisol-producing ACA (n=9)., non-functional ACA (n=9) and adrenocortical carcinomas (ACC; n=15). DNA was bisulphite modified using the EZ DNA Methylation kit (Zymo Research, Orange, CA) and analyzed using the Infinium HumanMethylation27 BeadChip (Illumina, San Diego, CA). This genome-wide approach allows interrogation of 27,578 highly informative CpG sites from more than 14,000 genes per sample at single-nucleotide resolution. The 12-sample Bead Chip features content derived from the well-annotated NCBI CCDS database and is supplemented with more than 1,000 cancer-related genes. The methylation module in the GenomeStudio (Illumina) software was used to analyze the results. Wilcoxon and Fisher's exact test was used for statistical calculations and p<0.005 was considered significant. Results: Distinct hierarchical clustering of genes with altered DNA methylation levels in normal adrenocortical tissue, aldosterone-producing ACA, cortisol-producing ACA, non-functional ACA and ACC was evident. Interestingly, the DNA methylome of aldosterone-producing ACA and cortisol-producing ACA were similar, whereas the DNA methylome of ACC was significantly aberrant. Comparing benign versus malignant ACT, a total of 212 CpG islands were identified as significantly hypermethylated in ACC (p<0.005). Genes involved in cell cycle regulation and apoptosis of known or putative importance in the development of adrenal tumors showed significant and frequent hypermethylation. Such genes included BCL2, DLEC1, GATA4, HDAC10, HOXD3, PYCARD and RASSF2. Conclusions: This represents the first unbiased, quantitative, genome-wide study of adrenocortical tumor DNA methylation profiles. Distinct clustering of genes with altered DNA methylation levels in normal adrenocortical tissue, aldosterone-producing ACA, cortisol-producing ACA, non-functional ACA and ACC was evident. Genes with altered DNA methylation patterns were identified of putative importance to benign and malignant adrenocortical tumor development. These studies may not only help characterize the pathogenesis of adrenocortical tumors, but also may play an important role in tumor classification and diagnosis.

ALDOSTERONE-PRODUCING ADENOMA CAUSING HUMAN PRIMARY ALDOSTERONISM ARE CHARACTERISED BY UNDER- EXPRESSION OF THE TWIK-RELATED ACID-SENSITIVE K+ CHANNEL 2 (TASK-2) GENE: PP.21.321

Background: Primary aldosteronism is a common cause of arterial hypertension, but its underlying molecular mechanisms are unknown. However, it is well known that K+ is a key regulator of aldosterone secretion, which interacts with physiological secretagogues of aldosterone as angiotensin II (Ang II) and endothelin-1. Genetic manipulation of a widely distributed channels class, which generate background or “leak” potassium (K+) currents, the Twik-related Acid-Sensitive K channel 1 (TASK-1) and 3 (TASK-3), generated a phenotype that mimics human primary aldosteronism. Objective: To measure the expression of the TASK family in a series of aldosterone producing adenoma (APA). Methods: Whole transcriptome of 24 APA was compared to that of a normal adrenocortical tissue pool (n = 10) by oligomicroarray technique. Data were validated by quantitative real time RT PCR. Immunohistochemistry was used to investigate TASKs protein expression by adrenocortical tissues. Results and Conclusions: The most expressed TASK channel gene in both the normal ZG and in APAs was TASK-1, followed by TASK-2 while the expression of TASK-3 was scant. With a whole transcriptome analysis followed by confirmation with quantitative real time RT-PCR we found a consistent and marked under expression of TASK-2 channel in all studied APA (n = 24), while TASK-1 and TASK-3 were heterogeneously expressed, as compared to the normal adrenal cortex. Immunohistochemistry was used to confirm the expression of the 3 TASK K+ channels in the adrenal cortex at the protein level and quantitative immnunoblotting will be used to confirm the differences detected at the transcript level. The underexpression of TASK-2 channels in APA suggests blunted leak” K+ currents, with ensuing lowering of the membrane potential and thereby increased likelihood of opening of the voltage-gated T-type calcium channels. The latter is known to render the adrenocortical zona glomerulosa cells more sensitive to aldosterone secretagogues. Hence, our ex vivo findings, while pointing to TASK-2 channels under-expression as an important mechanism of primary aldosteronism, mandates further studies to ascertain the molecular mechanisms by which this underexpression develops.

Expression of glucocorticoid receptor isoforms in human adrenocortical adenomas

Introduction Glucocorticoid receptor (GR) is expressed in the normal human adrenal gland, however, no study has been performed to evaluate the separate expression of α- and β-isoforms (GRα and GRβ) in normal human adrenals and in adrenocortical adenomas. Experimental GRα and GRβ mRNA expression was examined by quantitative real-time PCR in 31 adrenal tissues including 19 non-functioning adenomas (NFA), 6 cortisol-producing adenomas (CPA) and 6 normal adrenocortical tissues. In addition, the presence and cellular localization of GRα and GRβ proteins in adrenal tissues were studied by immunohistochemistry. Results Compared to normal adrenocortical tissues, both GRα and GRβ mRNAs were significantly increased in CPA but not in NFA. Using anti-GRα antibody a strong nuclear staining was observed in NFA and CPA, and a less remarkable immunoreactivity was detected in some nuclei of normal adrenocortical cells. GRβ immunostaining was absent in normal adrenal tissues and NFA, while a strong cytoplasmic and nuclear immunoreaction was found in CPA. Conclusions Altered expression of GRα and GRβ in CPA raises their possible role in the pathophysiology of these adrenal tumors, although further studies are needed to elucidate the potential significance of these findings.

Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR-mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

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

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. We assessed the cAMP-signaling pathway by enzymatic and molecular studies. 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. A total of 36 samples from patients were used. 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. 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.

Expression of the zinc-finger transcription factor Snail in adrenocortical carcinoma is associated with decreased survival

In this study, we evaluate whether Snail is expressed in adrenocortical cancer (ACC) and if its expression is related to patient outcome. One of the best known functions of the zinc-finger transcription factor Snail is to induce epithelial-to-mesenchymal transition (EMT). Increasing evidence suggests that EMT plays a pivotal role in tumour progression and metastatic spread. Snail and E-cadherin expression were assessed by immunohistochemistry in 26 resected ACCs and real-time quantitative RT–PCR expression analysis was performed. Data were correlated with clinical outcome and in particular with overall patient survival. Seventeen of 26 (65%) ACC tumour samples expressed Snail when assessed by immunohistochemistry. Snail expression was neither detected in normal adrenocortical tissue, nor in benign adrenocortical adenomas. Expression levels were confirmed on the mRNA level by Real-Time–PCR. Survival rates were significantly decreased in Snail-positive tumours compared to Snail-negative tumours: 10 out of 16 vs one out of eight patients succumbed to disease after a median follow up of 14.5 and 28.5 months, respectively (P=0.03). Patients with Snail-expressing ACCs presented in advanced disease (11 out of 12 vs 6 out of 14, P=0.01) and tend to develop distant metastases more frequently than patients with negative staining (7 out of 11 vs two out of eight, P=0.19). In conclusion, we describe for the first time that Snail is expressed in a large subset of ACCs. Furthermore, Snail expression is associated with decreased survival, advanced disease and higher risk of developing distant metastases.

Expression and Functional Role of Urotensin-II and Its Receptor in the Adrenal Cortex and Medulla: Novel Insights for the Pathophysiology of Primary Aldosteronism

The involvement of urotensin II, a vasoactive peptide acting via the G protein-coupled urotensin II receptor, in arterial hypertension remains contentious. We investigated the expression of urotensin II and urotensin II receptor in adrenocortical and adrenomedullary tumors and the functional effects of urotensin II receptor activation. The expression of urotensin II and urotensin II receptor was measured by real time RT-PCR in aldosterone-producing adenoma (n = 22) and pheochromocytoma (n = 10), using histologically normal adrenocortical (n = 6) and normal adrenomedullary (n = 5) tissue as control. Urotensin II peptide and urotensin II receptor protein were investigated with immunohistochemistry and immunoblotting. To identify urotensin II-related and urotensin II receptor-related pathways, a whole transcriptome analysis was used. The adrenocortical effects of urotensin II receptor activation were also assessed by urotensin II infusion with/without the urotensin II receptor antagonist palosuran in rats. Urotensin II was more expressed in pheochromocytoma than in aldosterone-producing adenoma tissue; the opposite was seen for the urotensin II receptor expression. Urotensin II receptor activation in vivo in rats enhanced (by 182 +/- 9%; P < 0.007) the adrenocortical expression of immunoreactive aldosterone synthase. Urotensin II is a putative mediator of the effects of the adrenal medulla and pheochromocytoma on the adrenocortical zona glomerulosa. This pathophysiological link might account for the reported causal relationship between pheochromocytoma and primary aldosteronism.

Phosphodiesterase 11A Expression in the Adrenal Cortex, Primary Pigmented Nodular Adrenocortical Disease, and other Corticotropin-independent Lesions

A variety of adrenal tumors and bilateral adrenocortical hyperplasias (BAH) leading to Cushing syndrome (CS) may be caused by aberrant cAMP signaling. We recently identified patients with a micronodular form of BAH that we have called "isolated micronodular adrenocortical disease" (iMAD) in whom CS was associated with inactivating mutations in phosphodiesterase (PDE) 11A ( PDE11A). In the present study, we examined PDE11A expression in normal adrenocortical tissue, sporadic tumors, and hyperplasias without PDE11A mutations, and primary pigmented nodular adrenocortical disease (PPNAD) and adenomas from patients with PRKAR1A and a single tumor with a GNAS mutation. The total number of the tumor samples that we studied was 22. Normal human tissues showed consistent PDE11A expression. There was variable expression of PDE11A in sporadic adrenocortical hyperplasia or adenomas; PPNAD tissues from patients with PRKAR1A mutations expressed consistently high levels of PDE11A in contrast to adenomas caused by GNAS mutations. Phosphorylated CREB was the highest in tissues from patients with iMAD compared to all other forms of BAH and normal adrenal tissue. We conclude that PDE11A is expressed widely in adrenal cortex. Its expression appears to be increased in PPNAD but varies widely among other adrenocortical tumors. PRKAR1A expression appears to be higher in tissues with PDE11A defects. Finally, sequencing defects in PDE11A are associated with a high state of CREB phosphorylation, just like PRKAR1A mutations. These preliminary data suggest that these two molecules are perhaps regulated in a reverse manner in their control of cAMP signaling in adrenocortical tissues.

Cellular and molecular abnormalities of a macronodular adrenal hyperplasia causing beta-blocker-sensitive Cushing's syndrome

Cushing's syndrome due to ACTH-independent macronodular adrenal hyperplasia (AIMAH) can be associated with abnormal responses of aberrantly expressed adrenocortical receptors. This study aimed to characterize in vitro the pathophysiology of hypercortisolism in a beta-blocker-sensitive Cushing's syndrome due to AIMAH. Cortisol secretion profile under aberrant receptors stimulation revealed hyperresponsiveness to salbutamol (beta2-adrenoceptor agonist), cisapride (5-HT4 receptor agonist), and vasopressin in AIMAH cultured cells, but not in normal adrenocortical cells. By RT-PCR, AIMAH tissues revealed beta2-adrenoceptor overexpression rather than ectopical expression. MC2R expression was similar in both AIMAH and normal adrenocortical tissues. Curiously, cortisol levels of AIMAH cells under basal condition were 15-fold higher than those of control cells and were not responsive to ACTH. Analysis of culture medium from AIMAH cells could detect the presence of ACTH, which was immunohistochemically confirmed. Finally, the present study of AIMAH cells has identified: a) cortisol hyperresponsiveness to catecholamines, 5-HT4 and vasopressin in vitro, in agreement with clinical screening tests; b) abnormal expression of beta2-adrenoceptors in some areas of the hyperplastic adrenal tissue; c) autocrine loop of ACTH production. Altogether, the demonstration of aberrant responses to hormonal receptors and autocrine hormone production in the same tissue supports the assumption of multiple molecular alterations in adrenal macronodular hyperplasia.