Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas.

Microarray, RT-qPCR based arrays and next-generation-sequencing (NGS) are available high-throughput methods for miRNA profiling (miRNome). Analytical and biological performance of these methods were tested in identification of biologically relevant miRNAs in non-functioning pituitary adenomas (NFPA). miRNome of 4 normal pituitary (NP) and 8 NFPA samples was determined by these platforms and expression of 21 individual miRNAs was measured on 30 (20 NFPA and 10 NP) independent samples. Complex bioinformatics was used. 132 and 137 miRNAs were detected by all three platforms in NP and NFPA, respectively, of which 25 were differentially expressed (fold change > 2). The strongest correlation was observed between microarray and TaqMan-array, while the data obtained by NGS were the most discordant despite of various bioinformatics settings. As a technical validation we measured the expression of 21 selected miRNAs by individual RT-qPCR and we were able to validate 35.1%, 76.2% and 71.4% of the miRNAs revealed by SOLiD, TLDA and microarray result, respectively. We performed biological validation using an extended number of samples (20 NFPAs and 8 NPs). Technical and biological validation showed high correlation (p < 0.001; R = 0.96). Pathway and network analysis revealed several common pathways but no pathway showed the same activation score. Using the 25 platform-independent miRNAs developmental pathways were the top functional categories relevant for NFPA genesis. The difference among high-throughput platforms is of great importance and selection of screening method can influence experimental results. Validation by another platform is essential in order to avoid or to minimalize the platform specific errors.

MEN1 and microRNAs: The link between sporadic pituitary, parathyroid and adrenocortical tumors?

Sporadic tumors of the pituitary, parathyroids and adrenal cortex are unique, as their benign forms are very common, but malignant forms are exceptionally rare. Hereditary forms of these tumors occur in multiple endocrine neoplasia syndrome type 1 (MEN1). We hypothesize that the pathogenic link among the sporadic tumors of these organs of different germ layers might be represented by common molecular pathways involving the MEN1 gene and microRNAs (miR). miR-24 might be a microRNA linking the three tumor entities, but other candidates such as miR-142-3p and microRNAs forming the DLK1-MEG3 miRNA cluster might also be of importance.

In silico analysis of pathways affected by differentially expressed microRNA in adrenocortical tumors.

BACKGROUND: MicroRNA are involved in the pathogenesis of several tumors, and several studies have been performed on the microRNA profile of adrenocortical tumors to date. The pathways affected by these microRNA, however, have not been analyzed yet by a systematic approach. AIM: To perform an in silico bioinformatics analysis of microRNA commonly altered in at least two studies and to decipher the pathways affected by microRNA in adrenocortical tumors. METHODS: Datasets on microRNA and mRNA expression have been retrieved from 5 and 3 studies, respectively. MicroRNA mRNA targets have been identified by our tissue specific target prediction pipeline, and mRNA have been subjected to Ingenuity Pathway Analysis. RESULTS: Thirty- nine microRNA were identified as commonly altered in two studies. Altogether 49,817 mRNA targets have been found for these microRNA. One-hundred and seventy-eight significant pathways associating with these have been identified and were found in all studies. We have selected 12 pathways involving retinoic acid signaling (lipopolysaccharide/ interleukin-1 mediated inhibition of retinoic X receptor (RXR) function, peroxisome proliferator-activated receptor (PPAR)α/RXRα activation, retinoic A receptor activation and PPAR signaling pathways) and cell cycle alterations (aryl hydrocarbon receptor signaling, growth arrest and DNA damage-inducible 45 signaling, integrin signaling, G2/M DNA damage checkpoint regulation, cyclins and cell cycle regulation and cell cycle control of chromosomal replication pathways) as these have been also established in our previous study on the functional genomics meta-analysis of adrenocortical tumors. Several microRNA have been identified that could affect these pathways. CONCLUSIONS: MicroRNA might affect several pathogenic pathways in adrenocortical tumors. Validation studies are required to confirm the biological relevance of these findings.

Underexpression of C-myc in adrenocortical cancer: a major pathogenic event?

The protooncogene c-myc is a major factor in tumourigenesis. Whereas c-myc overexpression is considered to be a general feature of many tumours, we have recently demonstrated c-myc underexpression in adrenocortical cancer by a meta-analysis. We hypothesise that c-myc underexpression might be a central event in adrenortical tumourigenesis based on network topology modelling and previous experimental observations. In this brief hypothesis, we present our arguments and their possible relevance.

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.

Steroid biosynthesis inhibitors in the therapy of hypercortisolism: theory and practice.

Cushing's syndrome is a rare disease with significant morbidity and mortality. Surgical intervention represents the most effective treatment option in both adrenocorticotropin-dependent and -independent forms of hypercortisolism. It is not uncommon, however, that surgery fails to cure or control the disease. Pharmacotherapy with drugs inhibiting steroid biosynthesis can be effectively used in these cases in order to alleviate symptoms or even to induce chemical adrenalectomy. A few drugs inhibiting single or multiple steps in adrenal steroid biosynthesis can be used in clinical practice. Drugs predominantly inhibiting single enzymatic steps include the 11beta-hydroxylase inhibitor metyrapone and the 3beta-hydroxysteroid dehydrogenase inhibitor trilostane, whereas mitotane, aminoglutethimide, ketoconazole and etomidate block multiple enzymatic reactions. Etomidate is the only agent available for parenteral administration that renders it as a treatment of choice in critically ill patients requiring a rapid control of hypercortisolemia. Ketoconazole, metyrapone and aminoglutethimide can be used alone or in combination for the treatment of hypercortisolism caused by benign adrenocorticotropin- or cortisol-secreting tumors. The clinical utility of trilostane is variable. Besides blocking multiple steps in adrenal steroid biosynthesis, the DDT (insecticide) analogue mitotane also has adrenolytic properties by inducing mitochondrial degeneration that renders it superior to other drugs in the treatment of adrenocortical cancer. Severe side effects may develop during therapy with each aforementioned drug that include hepatic, endocrine and neurological toxicity. After summarizing the chemical and biological properties of steroid biosynthetic inhibitors, the authors describe their possible clinical applications and limitations.