Human Adrenocortical Adenomas Research Articles

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Wnt-Signaling Regulated by Glucocorticoid-Induced miRNAs.

Glucocorticoids (GCs) are pleiotropic hormones which regulate innumerable physiological processes. Their comprehensive effects are due to the diversity of signaling mechanism networks. MiRNAs, small, non-coding RNAs contribute to the fine tuning of signaling pathways and reciprocal regulation between GCs and miRNAs has been suggested. Our aim was to investigate the expressional change and potential function of GC mediated miRNAs. The miRNA expression profile was measured in three models: human adrenocortical adenoma vs. normal tissue, steroid-producing H295R cells and in hormonally inactive HeLa cells before and after dexamethasone treatment. The gene expression profile in 82 control and 57 GC-affected samples was evaluated in GC producing and six different GC target tissue types. Tissue-specific target prediction (TSTP) was applied to identify the most relevant miRNA−mRNA interactions. Glucocorticoid treatment resulted in cell type-dependent miRNA expression changes. However, 19.5% of the influenced signaling pathways were common in all three experiments, of which the Wnt-signaling pathway seemed to be the most affected. Transcriptome data and TSTP showed similar results, as the Wnt pathway was significantly altered in both the GC-producing adrenal gland and all investigated GC target tissue types. In different cell types, different miRNAs led to the regulation of similar pathways. Wnt signaling may be one of the most important signaling pathways affected by hypercortisolism. It is, at least in part, regulated by miRNAs that mediate the glucocorticoid effect. Our findings on GC producing and GC target tissues suggest that the alteration of Wnt signaling (together with other pathways) may be responsible for the leading symptoms observed in Cushing’s syndrome.

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging.

Introduction: [11C]Metomidate ([11C]MTO), the methyl ester analogue of etomidate, was developed as a positron emission tomography (PET) radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). A disadvantage of [11C]MTO is the rather high non-specific binding in the liver, which impacts both visualization and quantification of the uptake in the right adrenal gland. Furthermore, the short 20-minute half-life of carbon-11 is a logistic challenge in the clinical setting.Objectives: The aim of this study was to further evaluate the previously published fluorine-18 (T1/2=109.5 min) etomidate analogue, para-chloro-2-[18F]fluoroethyl etomidate; [18F]CETO, as an adrenal PET tracer.Methods: In vitro experiments included autoradiography on human and cynomolgus monkey (non-human primate, NHP) tissues and binding studies on adrenal tissue from NHPs. In vivo studies with [18F]CETO in mice, rats and NHP, using PET and CT/MRI, assessed biodistribution and binding specificity in comparison to [11C]MTO.Results: The binding of [18F]CETO in the normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas, was shown to be specific, both in vitro (in humans) and in vivo (in rats and NHP) with an in vitro Kd of 0.66 nM. Non-specific uptake of [18F]CETO in NHP liver was found to be low compared to that of [11C]MTO.Conclusions: High specificity of [18F]CETO to the adrenal cortex was demonstrated, with in vivo binding properties qualitatively surpassing those of [11C]MTO. Non-specific binding to the liver was significantly lower than that of [11C]MTO. [18F]CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.

ITRAQ-based proteomic analysis reveals possible target-related proteins in human adrenocortical adenomas

BackgroundAdrenocortical adenomas (ACAs) can lead to the autonomous secretion of aldosterone responsible for primary aldosteronism (PA), which is the most common form of secondary arterial hypertension. However, the authentic fundamental mechanisms underlying ACAs remain unclear.ObjectiveIsobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics and bioinformatics analyses from etiological studies of ACAs were performed to screen the differentially expressed proteins (DEPs) and investigate the relevant mechanisms of their occurrence and development. Results could help determine therapeutic targets of clinical significance.MethodsIn the present study, iTRAQ-based proteomics was applied to analyze ACA tissue samples from normal adrenal cortex tissues adjacent to the tumor. Using proteins extracted from a panel of four pairs of ACA samples, we identified some upregulated proteins and other downregulated proteins in all four pairs of ACA samples compared with adjacent normal tissue. Subsequently, we predicted protein–protein interaction networks of three DEPs to determine the authentic functional factors in ACA.ResultsA total of 753 DEPs were identified, including 347 upregulated and 406 downregulated proteins. The expression of three upregulated proteins (E2F3, KRT6A, and ALDH1A2) was validated by Western blot in 24 ACA samples. Our data suggested that some DEPs might be important hallmarks during the development of ACA.ConclusionsThis study is the first proteomic research to investigate alterations in protein levels and affected pathways in ACA using the iTRAQ technique. Thus, this study not only provides a comprehensive dataset on overall protein changes but also sheds light on its potential molecular mechanism in human ACAs.

Transcription factors GATA-6, SF-1, and cell proliferation in human adrenocortical tumors

Transcription factor GATA-6 is expressed in fetal and adult human adrenal cortex and has been suggested to have a role in adrenal androgen synthesis. In other tissues GATA-6 has been linked to the cell cycle regulation and the dedifferentiation of carcinoma cells. GATA-6 has been shown to be downregulated in mouse adrenocortical tumors, but has not been studied in human adrenocortical tumors in detail. We have now analyzed GATA-6 expression in 20 human adrenocortical adenomas and 16 carcinomas using Northern blot analysis and immunohistochemistry. GATA-6 mRNA and protein expression was remarkably diminished in adrenocortical carcinomas as compared to normal adrenal cortex and adenomas ( p < 0.05). In opposite to other tumor types GATA-6 expression was, however, high in virilizing carcinomas. Steroidogenic factor 1 (SF-1) has been functionally linked to GATA-6, and the expression of these two factors correlated in the adrenal tumors. Furthermore, GATA-6 immunoreactivity was linked to P450c17 expression. In contrast to GATA-6, we found upregulated cyclin-dependent kinase inhibitor p21 and proliferation marker Ki67 in adrenocortical carcinomas indicating that GATA-6 is not linked to cell proliferation in human adrenal tumors. Taken together, the present and earlier results link GATA-6 to adrenocortical steroidogenesis and to the benign adrenocortical phenotype.

Plasma and tissue chromogranin in patients with adrenocortical adenomas

Adrenal adenomas frequently arise from cortical islets in the medulla, and these islets seem to present a greater risk for pathological growth than cortical cells within the adrenal cortex. Chromogranin A (CgA), a glycoprotein co-stored in secreting granules and co-released with resident hormones of chromaffin cells, behaves as a prohormone, generating several biologically active peptides capable of influencing growth, morphogenesis and progression of endocrine tumors. The aim of our study was to investigate whether chromaffin cells may be involved in the development and growth of adrenocortical adenomas. We enrolled 19 patients (12 females and 7 males, mean+/-SD age 54.9+/-11.2 yr, age range 34-75 yr) with incidental, non-functioning, benign adrenocortical adenomas, and measured circulating levels of CgA, catecholamines and creatinine before and 2 months after surgery. Plasma CgA was evaluated by immunoradiometric assay. Testing for CgA immunoreactivity in the removed tissues was performed by immunohistochemical analysis. Mean plasma CgA did not significantly change following surgery (before 73.7+/-15.2 ng/ml; after 68.9+/-14.8 ng/ml). Individual CgA values indicated that 4 patients had plasma CgA levels above our cut-off of normality. After mass removal, CgA further increased in 2 cases, decreased in 1 and normalized in 1. No variation in CgA levels was found in the other patients. No correlation was observed between CgA and the variables measured, except between CgA and plasma creatinine (r=0.472, p<0.05). Histopathological evaluation revealed adrenocortical adenomas in all cases and immunohistochemical analysis detected no CgA immunoreactivity in any specimen. Our results show that in human adrenocortical adenomas CgA is not expressed and that removal of the mass does not modify plasma CgA levels. For these reasons the endocrine involvement of local CgA in adrenocortical tumorigenesis is unlikely.

Leptin inhibits cortisol and corticosterone secretion in pathologic human adrenocortical cells.

Regulation of adrenal corticosteroid secretion by leptin may involve interactions at multiple levels of the hypothalamic-pituitary-adrenal axis. To investigate the possible direct effects of leptin on corticosteroid secretion of human adrenocortical adenomas, cells from adrenocortical adenomas causing primary aldosteronism (n = 1) and Cushing's syndrome (n = 1), as well as cells from nonhyperfunctioning adrenocortical adenomas (n = 5) were isolated and incubated for 2 h with human recombinant leptin (1-1000 ng/ml) in the presence and absence of adrenocorticotrop hormone (ACTH), then cortisol, corticosterone and aldosterone concentrations in incubating media were determined using radioimmunoassays. It was found that leptin effectively and dose-dependently inhibited basal and ACTH-stimulated cortisol and corticosterone secretion in the three types of human adrenocortical adenoma cells. The inhibiting effect of basal corticosterone secretion was detectable in the presence of leptin concentration as low as 1 ng/ml, with decreases of corticosterone secretion to 34+/-4%, 57+/-11% and 79+/-9% in Cushing's syndrome, primary aldosteronism, and nonhyperfunctioning adrenocortical adenoma cells, respectively. The inhibition of basal cortisol secretion in the presence of low concentration of leptin was less prominent, but 10 ng/ml leptin significantly diminished basal cortisol secretion to 81+/-9% in adrenocortical adenoma cells from Cushing's syndrome, to 68+/-6% in adenoma cells from primary aldosteronism, and to 83+/-8% in cells from nonhyperfunctioning adenomas. The inhibition of ACTH-stimulated cortisol and corticosterone secretion by leptin was similar to those found in cells without ACTH stimulation. By contrast, leptin even at 1000 ng/ml concentration exerted no clear effect on basal and ACTH-stimulated aldosterone secretion in cells from primary aldosteronism and in those nonhyperfunctioning adenoma cells in which aldosterone secretion was detectable. These results indicate that leptin is a potent inhibitor of cortisol and corticosterone secretion in human adenomatous adrenocortical cells. The inhibition of these corticosteroids by leptin may represent a potentially important interaction that exists between leptin and the hypothalamic-pituitary-adrenal axis.

Expression and Regulation of BCL-2 Family Genes in Human Adrenocortical Adenomas in Comparison with Adrenal Hyperplasia of Cushing's Disease

The significance of increases in the expression of apoptosis-suppressing genes such as bcl-2 and mcl-1/EAT in human adrenal tumors has not yet been fully elucidated. Furthermore the roles of these genes in cell proliferation may involve interaction with steroidogenesis in the tumors via intracellular second messengers. Cyclic AMP (cAMP) caused human adrenocortical H295R cells to overexpress hCYP17 resulting in hypersecretion of cortisol. At the same time, however, expression of bcl-2, which has a cAMP response element (CRE), was not affected. Furthermore, in vivo Bcl-2 protein analysis showed its down-regulation in adrenal hyperplasia of Cushing's disease despite ACTH stimulation. Exogenous addition of glucocorticoid did not affect the expression of bcl-2 family genes. Expressions of McL-1/EAT and Bax did not differ markedly among human adrenal glands affected by various pathologies. In conclusion the down-regulation of Bcl-2 in Cushing's disease did not agree with no induction of this gene by cAMP in H295R cells, suggesting that expression of Bcl-2 protein was not regulated mainly by cAMP-protein kinase (PKA) pathways in human adrenal hyperplasia.

COUP-TFI Expression in Human Adrenocortical Adenomas: Possible Role in Steroidogenesis

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan nuclear receptor essential for neurogenesis, organogenesis, and cell fate determination. CYP17 gene transcription has recently been shown to be activated by SF-1 (steroidogenic factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and inhibited by COUP-TF binding to the sequence. Thus, COUP-TF and SF-1 act as a transcriptional repressor and activator, respectively, of CYP17 gene expression. Transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor-corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone-receptor), whereas transcriptional activation by SF-1 is mediated by coactivator SRC-1 (steriod receptor coactivator-1). We therefore examined the expression of COUP-TFI, SF-1, SRC-1, N-CoR, and SMRT in a variety of adrenocortical adenomas and compared the results with CYP17 mRNA levels. We found significantly high COUP-TFI mRNA expression in nonfunctional adenomas (n=8: 220±16%; normal 96±4%), a deoxycorticosterone-producing adenoma (n=1: 200%), and a pre-clinical Cushing’s adenoma (n=1: 280%), intermediate COUP-TFI expression in cortisol-producing adenomas (n=8: 63±5%), and low COUP-TFI expression in aldosterone-producing adenomas (n=8: 49±4%). In contrast to COUP-TFI, SF-1 mRNA expression did not vary significantly among adrenals. We did not detect the expected negative correlation between COUP-TFI and CYP17 mRNA levels in adrenocortical adenomas. High COUP-TFI expression was associated with a nonfunctioning phenotype. Interestingly, the pattern of COUP-TFI expression was similar to the profile of N-CoR expression, but not of SMRT expression. These results indicate that COUP-TFI and N-CoR may play a role in steroidogenesis by human adrenocortical adenomas.

COUP-TFI expression in human adrenocortical adenomas: possible role in steroidogenesis.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan nuclear receptor essential for neurogenesis, organogenesis, and cell fate determination. CYP17 gene transcription has recently been shown to be activated by SF-1 (steroidogenic factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and inhibited by COUP-TF binding to the sequence. Thus, COUP-TF and SF-1 act as a transcriptional repressor and activator, respectively, of CYP17 gene expression. Transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor-corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone-receptor), whereas transcriptional activation by SF-1 is mediated by coactivator SRC-1 (steroid receptor coactivator-1). We therefore examined the expression of COUP-TFI, SF-1, SRC-1, N-CoR, and SMRT in a variety of adrenocortical adenomas and compared the results with CYP17 mRNA levels. We found significantly high COUP-TFI mRNA expression in nonfunctional adenomas (n=8: 220+/-16%; normal 96+/-4%), a deoxycorticosterone-producing adenoma (n=1: 200%), and a pre-clinical Cushing's adenoma (n=1: 280%), intermediate COUP-TFI expression in cortisol-producing adenomas (n=8: 63+/-5%), and low COUP-TFI expression in aldosterone-producing adenomas (n=8: 49+/-4%). In contrast to COUP-TFI, SF-1 mRNA expression did not vary significantly among adrenals. We did not detect the expected negative correlation between COUP-TFI and CYP17 mRNA levels in adrenocortical adenomas. High COUP-TFI expression was associatedwith a nonfunctioning phenotype. Interestingly, the pattern of COUP-TFI expression was similar to the profile of N-CoR expression, but not of SMRT expression. These results indicate that COUP-TFI and N-CoR may play a role in steroidogenesis by human adrenocortical adenomas.

In vivo and in vitro evidence for the production of inhibin-like immunoreactivity in human adrenocortical adenomas and normal adrenal glands: relatively high secretion from adenomas manifesting Cushing's syndrome.

To clarify whether adrenal gland secretes inhibin in vivo in physiological or pathological conditions, we measured the levels of inhibin-like immunoreactivity (inhibin-LI) in adrenal veins (A-vein) and compared them with those in inferior vena cava (IVC) using blood samples obtained at catheterization of adrenal vein in the patients with adrenal adenoma manifesting Cushing's syndrome (Cs), aldosterone-producing adenoma, clinically non-functioning adenoma and normal adrenal gland. The tumor sides of A-veins in the patients with adenomas and also both sides of A-veins in subjects with normal adrenal glands showed significantly higher contents of inhibin-LI than their IVC. When the inhibin-LI secretion rate from adrenal gland was estimated by the difference between the levels of A-vein (tumor side) and IVC, Cs adenomas showed the highest secretion rate. Similarly, the tissue inhibin-LI content and the basal secretion rate of inhibit-LI from primary cultured cells were the highest in Cs adenomas. These findings indicated that normal adrenal glands and adrenocortical adenomas produced and secreted inhibin-LI into the general circulation in vivo and Cs adenomas have relatively high capacity for secreting inhibin-LI, and the present study provided the first in vivo evidence for adrenal inhibin-LI production in pathological conditions.

P53 Mutations in sporadic adrenocortical tumors

Non-familial human adrenocortical adenomas and carcinomas were screened for mutations in exons 5-8 of the p53 tumor suppressor gene by single-strand-conformation-polymorphism (SSCP) analysis, followed by direct sequencing of PCR-amplified DNA. Point mutations in codons 12, 13 and 61 in H-ras, K-ras and N-ras proto-oncogenes were similarly assessed by direct DNA sequencing. Three out of 15 primary adrenocortical carcinomas (20%) contained a mis-sense point mutation in the conserved regions (exons 5 and 8) of the p53 gene. Mutations were located in codon 157 (GTC-->TTC; Val-->Phe), codon 163 (TAC-->AAC; Tyr-->Asn), and codon 273 (CGT-->TGT; Arg-->Cys). The mutation in codon 157 was detected in the primary tumor as well as in brain and lymph-node metastases. Among 18 adrenocortical adenomas, there was only a single non-miscoding mutation in codon 295 (CCT-->CCC; Pro-->Pro). These data suggest that mutational inactivation of the p53 gene occurs in a minority (20%) of sporadic adrenocortical carcinomas and that these mutations constitute a late event in the multi-step process of malignant transformation. No ras mutations were detected in any of these tumors, suggesting that these genes are not involved in the development of tumors originating from the adrenal cortex.

Expression of progesterone-binding protein in normal and neoplastic human adrenals

Specific progesterone-binding protein (P4-BP) is demonstrated in adrenocortical nuclei of the guinea pig, but, not in nuclei of other animals. We tried to demonstrate the progesterone-binding activity in nuclei of human normal adrenals and adrenal tumors. Normal adrenals were obtained from six patients with renal cell carcinomas undergoing radical nephrectomy. Seven adrenocortical adenomas were obtained: five tumors from patients with Cushing's syndrome, one tumor from non-functioning adenoma, and one from aldosteronoma. Nuclei were purified from the tissues, and progesterone binding assay was performed. We could not demonstrated progesterone-binding activity in nuclei of six normal human adrenals. However, we demonstrated progesterone-binding activity in nuclei purified from human adrenocortical adenomas associated with Cushing's syndrome. Saturation analysis revealed a Kd of 13.85 +/- 1.99 nM (mean +/- SD, n = 5) and a binding capacity of 1.95 +/- 0.37 pmol/mg DNA (mean +/- SD, n = 5). A Kd of progesterone-binding activity in human adrenocortical adenoma was similar to that of guinea pig P4-BP, and a binding capacity was about one-fifteenth of guinea pig P4-BP. However, nuclei purified from a non-functioning adrenocortical adenoma and an aldosteronoma failed to demonstrate progesterone-binding activity. The binding activity was specific for progesterone. 5 alpha-pregnane-3,20-dione was a modest competitor, while 17 beta-estradiol, testosterone, cortisol, and other related steroids were poor competitors. Thus the progesterone-binding activity in human adrenals was similar to guinea pig P4-BP in the affinity and specificity of binding.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effects of Metoclopramide and Dopamine on Aldosterone Secretion in Cultured Adrenocortical Adenoma Cells and Adjacent Non-adenoma Cells from Patients with Primary Aldosteronism

Several authors have reported that metoclopramide (MCP), a dopaminergic antagonist, stimulates aldosterone secretion and that dopamine (DA) inhibits the MCP-mediated aldosterone secretion in man. However, many controversial results have been reported relating to the direct effect of MCP and DA on the secretion of aldosterone by adrenocortical cells in in vitro experiments. The present studies were designed to determine whether or not MCP and DA exerts its effect directly on the cultured human adrenocortical adenoma cells and adjacent non-adenoma cells obtained from patients with primary aldosteronism. A bolus intravenous injection of 10 mg MCP significantly increased the plasma aldosterone concentration (PAC) from 255 +/- 57 pg/ml (mean +/- SD) to 386 +/- 98 pg/ml after 15 min in patients with primary aldosteronism. But MCP (10(10)-10(-6) M) failed to increase aldosterone secretion from both adenoma cells and non-adenoma cells in culture. DA (10(-9) and 10(-6) M) did not suppress the basal secretion and the enhanced secretion of aldosterone by A II or ACTH in cells of either culture. The analysis by HPLC showed that 57% of dopamine hydrochloride added in the medium was preserved after 60 min incubation. These results suggest that MCP and DA do not act directly on the human adrenal glomerulosa cells in the regulation of aldosterone secretion.