Zsófia Tömböl

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.

Meta-analysis of adrenocortical tumour genomics data: Novel pathogenic pathways revealed

Sporadic adrenocortical tumours are common, but their pathogenesis is poorly elucidated. In this study, we present a meta-analysis and review of gene expression microarray and comparative genome hybridization (CGH) studies performed to date on these tumours, including our own data. Data of whole genome microarray studies from altogether 164 tumours (97 benign, 67 malignant) and 18 normal tissues were reclassified and reanalysed. Significant gene sets and cytogenetic changes from publications without available genomic data were also examined including 269 benign, 215 malignant tumour and 30 normal tissues. In our experimental study, 11 tumour and four normal samples were analysed by parallel mRNA and CGH profiling. Data were examined by an integrative bioinformatics approach (GeneSpring, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis softwares) searching for common gene expression changes and paralleling chromosome aberrations. Both meta-analysis of available mRNA and CGH profiling data and our experimental study revealed three major pathogenetic pathways: (1) cell cycle, (2) retinoic acid signalling (including lipopolysaccharide/Toll like receptor 4 pathway), (3) complement system and antigen presentation. These pathways include novel, previously undescribed pathomechanisms of adrenocortical tumours, and associated gene products may serve as diagnostic markers of malignancy and therapeutic targets.

MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas

MicroRNAs are involved in the pathogenesis of several tumors, however, there have been no data on microRNA expression in pheochromocytomas to date. The objective of our study was to perform microRNA expression profiling in sporadic and hereditary benign, and recurring adrenomedullary tumors. Furthermore, the applicability of formalin-fixed paraffin-embedded tissue samples for the analysis of microRNA expression in pheochromocytomas was examined. MicroRNA expression data of three matched frozen and formalin-fixed paraffin-embedded samples were correlated. A total of 21 formalin-fixed paraffin-embedded samples (sporadic benign, multiple endocrine neoplasia 2, von Hippel-Lindau disease, sporadic recurring) were subjected to microRNA expression profiling using microarrays. MicroRNAs with significant differences in expression were validated and sample sizes were extended including tumors from neurofibromatosis type 1 patients by real-time quantitative reverse-transcription PCR (n=33). MicroRNA target prediction was carried out by TargetScan and MicroCosm Targets. Pathway analysis of targets was performed by Ingenuity Pathway Analysis and DIANA mirPath. Furthermore, microRNA expression profiles of a malignant pheochromocytoma and a pair of primary and recurrent tumors were studied by TaqMan Human MicroRNA Cards. MicroRNA expression correlated well between frozen and formalin-fixed paraffin-embedded samples (70–92%). Microarray analysis revealed 16 significantly differentially expressed microRNAs. Five of these were validated by real-time RT-PCR. miR-139-3p, miR-541 and miR-765 were significantly differentially expressed between sporadic benign and von Hippel-Lindau-related pheochromocytomas. Significantly higher expression of miR-885-5p and miR-1225-3p was found in multiple endocrine neoplasia type 2 and sporadic recurring pheochromocytomas, respectively. Pathway analysis revealed the possible involvement of Notch- and G-protein-coupled receptor signaling in tumor recurrence. MicroRNA expression profiles in the primary recurrent and recurring malignant comparisons have been similar. In conclusion, we have proved that formalin-fixed paraffin-embedded samples can be used for the analysis of microRNA expression in pheochromocytomas. MicroRNA expression patterns differ between various sporadic, hereditary and recurring tumors and miR-1225-3p may be useful for identifying recurring pheochromocytomas.

MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3′UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA.

High prevalence of PROP1 gene mutations in hungarian patients with childhood-onset combined anterior pituitary hormone deficiency

Combined pituitary hormone deficiency is characterized by the impaired production of pituitary hormones, commonly including growth hormone. The pathomechanism of the childhood-onset form of this disorder may involve germline mutations of genes encoding pituitary transcription factors, of which PROP1 gene mutations have been studied most extensively. However, controversy exists about the significance of PROP1 gene mutations, as both low and high frequencies have been reported in these patients. Because the different results may be related to differences in patient populations and/or the variability of clinical phenotypes, we performed the present study to examine the prevalence and spectrum of PROP1 gene mutations in 35 patients with non-acquired childhood-onset growth hormone deficiency combined with at least one other anterior pituitary hormone deficiency. Genetic testing indicated the presence of disease-causing mutations in exons 2 and 3 of the PROP1 gene in 15 patients (43% of all patients; homozygous mutations in 10 patients and compound heterozygous mutations in 5 patients). Comparison of clinical data of patients with and without PROP1 gene mutations failed to show significant differences, except an earlier growth retardation detected in patients with PROP1 gene mutations. In one patient with PROP1 gene mutation, radiologic imaging showed an enlargement of the anterior lobe of the pituitary, whereas the other patients had hypoplastic or normal pituitary gland. All patients with PROP1 gene mutations had normal posterior pituitary lobe by radiologic imaging. These results indicate that using our inclusion criteria for genetic testing, PROP1 gene mutations can be detected in a high proportion of Hungarian patients with non-acquired childhood-onset growth hormone deficiency combined with at least one other anterior pituitary hormone defect.

Marked chromogranin A elevation in a patient with bilateral adrenal incidentalomas, and its rapid normalization after discontinuation of proton pump inhibitor therapy

Chromogranin A (CgA) is a well-recognized tumour marker for various neuroendocrine tumours. 1 In their recent article Grossrubatscher et al. demonstrate the utility of CgA in the diagnosis and follow-up of phaeochromocytoma. 2 As the secretory activity of these tumours can be episodic, CgA measurement is of great value in cases where urinary catecholamines are negative. False-positive elevated CgA, however, can be related to various conditions, 1 including proton pump inhibitor (PPI) and steroid therapy, or be associated with impaired renal function and type A gastritis, and can lead to serious differential diagnostic problems in patients with adrenal tumours. Here, we report a case of highly elevated serum CgA level in a patient with bilateral adrenal adenomas, that was clearly associated with proton pump inhibitor therapy. As the patient also suffered from hypertension, the possibility of phaeochromocytoma had to be excluded. Discontinuation of PPI intake for 5 days normalized serum CgA, indicating that even a short interruption of PPI therapy can solve the differential diagnostic dilemma. A 73-year-old woman with a history of hypertension and gastrooesophageal reflux disease was examined for bilateral adrenal incidentalomas revealed by ultrasonography and subsequent computed tomography (CT). Although the clinical picture did not clearly suggest endocrine disease, routine endocrinological examination was undertaken. Hypercortisolism and primary aldosteronism were excluded. Serum CgA determined by radioimmunoassay (CISBio International, Gil-Sur-Yvette, France), however, was highly elevated (728 ng/ml, normal range 18–98·1 ng/ml). By contrast, urinary catecholamine metabolites (vanillylmandelic acid, normetanephrine, metanephrine, homovanillic acid) were normal, and 131 I-metaiodobenzylguanidine (MIBG) scintigraphy was also negative. Abdominal MRI showed that the signal intensities of bilateral adrenal tumours were different from those seen in phaeochromocytomas. 3 Serum creatinine and blood urea nitrogen were normal. As the patient had been treated with PPI for at least 8 years (omeprazole, and presently lansoprazole in a daily dose of 2 × 30 mg), iatrogenic elevation of CgA was suspected. To solve the serious differential diagnostic problem, we decided to suspend PPI therapy and replace it with sucralphate (4 × 1 g/day). Serum CgA returned to normal within 5 days (Fig. 1), and after a single dose (30 mg) of lansoprazole it increased again above the upper normal limit (132·4 ng/ml). Immunohistochemical analysis for CgA in mucosal biopsy samples from the gastric corpus indicated mild, simple (diffuse), enterochromaffin cell-like (ECL) hyperplasia. Although our patient did not present symptoms characteristic of phaeochromocytoma, suspicion was raised because of the highly elevated CgA together with the history of hypertension. As all other diagnostic examinations characteristic of phaeochromocytoma were negative, the possibility of PPI-related CgA elevation was raised. Short-, mediumand long-term PPI therapy has been shown to raise serum CgA. 4,5 Giusti et al . described the elevation of CgA in response to short-term (5–90 days) PPI therapy. 4 Our observation showing that a single dose of PPI may raise CgA above normal also supports a high sensitivity of serum CgA to PPI therapy. Elevation of CgA, however, was unusually high in the case presented. Available literature describes CgA levels up to 400 ng/ml (approx. 8 n m ), even after long-term PPI therapy. 5

Effects of mitotane on gene expression in the adrenocortical cell line NCI-H295R: A microarray study

AIM The adrenolytic agent mitotane is widely used in the treatment of adrenocortical cancer; however, its mechanism of action is poorly elucidated. We have studied mitotane-induced mRNA expression changes in the NCI-H295R adrenocortical cancer cell line. MATERIALS & METHODS Cell viability and hormone assays were used to select the optimal mitotane concentration effectively inhibiting hormone secretion without affecting cell viability. RNA isolated from cultures treated for 48 and 72 h was subjected to Agilent 4×44K microarray platforms. Microarray results were validated by quantitative reverse-transcription PCR. RESULTS Altogether, 117 significantly differentially expressed genes were detected at 48 h and 72 h (p < 0.05) in mitotane-treated samples relative to controls. Three significantly underexpressed genes involved in steroid hormone biosynthesis (HSD3B1, HSD3B2 and CYP21A2) and four significantly overexpressed genes (GDF15, ALDH1L2, TRIB3 and SERPINE2) have been validated. CONCLUSION Gene-expression changes might be involved in the adrenal action of mitotane and in the inhibition of hormone secretion.

Relevance of MicroRNA-s in Neoplastic Diseases

MicroRNA molecules consisting of 19-23 nucleotides influence numerous basic physiological and pathophysiological processes as endogenous mediators of RNA interference. These molecules are capable of specifically inhibiting the translation of messenger RNA molecules, but in some cases also promote the degradation of mRNA-s. Altered microRNA expression profiles were noted in several human diseases, most data, however, are known for neoplasms. Characteristic microRNA profiles are known both in solid and haematologic malignancies. MicroRNA profiles enable the distinction of benign follicular adenomas from follicular neoplasms of the thyroid. The micro-RNA expression patterns could be associated with the clinical behaviour of certain neoplasms (e.g. lung tumours and chronic lymphocytic leukemia) as well. It is possible that small molecular weight RNA-s may be used for therapeutical purposes in the future.

Differences in the expression of histamine-related genes and proteins in normal human adrenal cortex and adrenocortical tumors

Histamine is involved in the pathogenesis of several tumors; however, there are no data on its possible involvement in human adrenocortical tumorigenesis. The expression of genes and proteins involved in the biosynthesis (histidine decarboxylase, HDC), action (histamine receptors: HRH1–HRH4), and metabolism of histamine is largely unknown both in the normal human adrenal cortex and in adrenocortical tumors. In this study, we examined the expression of histamine-related genes and proteins and histamine content in normal adrenal cortex, benign adrenocortical adenomas, and malignant adrenocortical cancer (ACC). Fifteen normal adrenals and 43 tumors were studied. mRNA expression was examined by real time RT-PCR. Western-blotting and immunohistochemistry were used for the study of proteins. Tissue histamine content was determined by enzyme-linked immunosorbent assay. We found that all proteins involved in histamine biosynthesis and action are present both in the normal adrenal cortex and in the tumors studied. HDC expression and histamine content was highest in the normal tissues and lower in benign tumors, whereas it was significantly less in ACCs. HRH3 expression was significantly higher in ACC samples than in the other groups. Adrenocortical tumorigenesis might, thus, be characterized by reduced histamine biosynthesis; furthermore, different adrenocortical tumor subtypes may show unique histamine receptor expression profiles.

Differences in MicroRNA expression profiles of adrenocortical tumors--letter.

In their elegant study published in the December 15, 2009 issue of Clinical Cancer Research , Soon and colleagues described differences in the microRNA expression profiles of adrenocortical tumors (ACT) and established predictors for poor prognosis. Twenty-three microRNAs were found to be significantly differentially expressed between adrenocortical adenomas (ACA) and carcinomas (ACC) but only two between normal adrenal cortices (NA) and ACTs by microarray analysis. Four microRNAs were validated by quantitative real-time PCR (1). In our previous study published in the September 2009 issue of Endocrine-Related Cancer (epub 22 June; ref. 2), we have performed an integrative bioinformatic analysis on ACTs including parallel microRNA (Taqman TLDA Panel) and mRNA profiling and pathway analysis. We have found 22 microRNAs with significant differences in expression among NA, hormonally inactive ACA, cortisol-producing ACA, and ACC. Six microRNAs were validated by quantitative real-time PCR. dCT miR-511 − dCT miR-503 (dCT, delta cycle threshhold) was identified as the best predictor of malignancy. Whereas the number of significantly differentially expressed microRNAs was highly similar in the two studies, only two were common in both: miR-503 and miR-181b (both overexpressed in ACCs). There are several differences between the structures of the two studies that might have accounted for these discrepancies. Although the number of tumors included in the study by Soon et al. was higher (27 ACA and 22 ACC) than in ours (19 ACA and 7 ACC), the groups have not been subdivided based on hormone secretion in the former. We have studied only cortisol-secreting ACCs, and ACAs have been subdivided into inactive ACA ( n = 10) and cortisol-producing ACA ( n = 9) groups. Both ACAs and ACCs express glucocorticoid receptors that are even overexpressed in ACCs (3). Adrenocortical microRNA expression patterns might be influenced by glucocorticoids. 1 1 A. Patocs, Z. Tombol, P.M. Szabo, P. Igaz, K. Racz, unpublished results. This might argue against the establishment of tumor groups irrespective of their hormonal activity. Furthermore, we have studied intact NA ( n = 10) removed from patients operated for malignant kidney tumors, whereas Soon et al. used NA samples ( n = 6) from adrenalectomy specimens away from the site of the adenoma. He et al. reported that the overexpression of miR-221 characteristic for papillary thyroid cancer can be detected in the unaffected, histologically normal tissue surrounding the tumor (4). It is possible that a similar phenomenon might also exist in ACTs, and this could be related to the finding that only two microRNAs were significantly differentially expressed between NA and tumors. Further studies will be necessary to clarify the relevance and potential diagnostic applicability of microRNAs for adrenocortical tumors. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.