Henriett Butz

Crosstalk between TGF-β signaling and the microRNA machinery.

The activin/transforming growth factor-β (TGF-β) pathway plays an important role in tumorigenesis either by its tumor suppressor or tumor promoting effect. Loss of members of the TGF-β signaling by somatic mutations or epigenetic events, such as DNA methylation or regulation by microRNA (miRNA), may affect the signaling process. Most members of the TGF-β pathway are known to be targeted by one or more miRNAs. In addition, the biogenesis of miRNAs is also regulated by TGF-β both directly and through SMADs. Based on these interactions, it appears that autoregulatory feedback loops between TGF-β and miRNAs influence the fate of tumor cells. Our aim is to review the crosstalk between TGF-β signaling and the miRNA machinery to highlight potential novel therapeutic targets.

MicroRNA profile indicates downregulation of the TGFβ pathway in sporadic non-functioning pituitary adenomas

MicroRNAs (miRs) are small, 16–29 nucleotide long, non-coding RNA molecules which regulate the stability or translational efficiency of targeted mRNAs via RNA interference. MiRs participate in the control of cell proliferation, cell differentiation, signal transduction, cell death, and they play a role in carcinogenesis. The aims of our study were to analyse the expression profile of miRs in sporadic clinically non-functioning pituitary adenomas (NFPA) and in normal pituitary tissues, and to identify biological pathways altered in these pituitary tumors. MiR expression profiles of 12 pituitary tissue specimens (8 NFPA and 4 normal pituitary tissues) were determined using miR array based on quantitative real-time PCR with 678 different primers. Five overexpressed miRs and mRNA expression of Smads (Smad1-9), MEG and DLK1 genes were evaluated with individual Taqman assays in 10 NFPA and 10 normal pituitary tissues. Pathway analysis was performed by the DIANA-mirPath tool. Complex bioinformatical analysis by multiple algorithms and association studies between miRs, Smad3 and tumor size was performed. Of the 457 miRs expressed in both NFPA and normal tissues, 162 were significantly under- or overexpressed in NFPA compared to normal pituitary tissues Expression of Smad3, Smad6, Smad9, MEG and DLK1 was significantly lower in NFPA than in normal tissues. Pathway analysis together with in silico target prediction analysis indicated possible downregulation of the TGFβ signaling pathway in NFPA by a specific subset of miRs. Five miRs predicted to target Smad3 (miR-135a, miR-140-5p, miR-582-3p, miR-582-5p and miR-938) were overexpressed. Correlation was observed between the expression of seven overexpressed miRs and tumor size. Downregulation of the TGFβ signaling through Smad3 via miRs may have a possible role in the complex regulation of signaling pathways involved in the tumorigenesis process of NFPA.

The rs4844880 polymorphism in the promoter region of the HSD11B1 gene associates with bone mineral density in healthy and postmenopausal osteoporotic women

INTRODUCTION The 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1) plays an important role in the regulation of local glucocorticoid concentration in a tissue specific manner. Previous studies indicated associations between polymorphisms (SNPs) of the HSD11B1 gene and laboratory as well as osteodensitometric parameters of bone metabolism. In our present work we examined whether the tagging HSD11B1 gene polymorphisms could influence bone metabolism in healthy and postmenopausal osteoporotic women. EXPERIMENTAL HapMap database was used for identification and selection of SNPs located in the 38kb range of the HSD11B1 gene. Twelve SNPs were selected and genotyped in 209 healthy control women using Taqman SNP assays on Real-Time PCR and direct DNA sequencing. Of these SNPs, the rs4844880 was genotyped in 154 women with postmenopausal osteoporosis. Functional characterization of the rs4844880 was performed by in vitro luciferase assay. RESULTS One of the 12 HSD11B1 SNPs, the rs4844880 showed a significant association with higher bone mineral density and/or T- and Z-scores at lumbar spine in healthy women. When data from 154 postmenopausal osteoporotic women were compared to those obtained from 101 age-matched postmenopausal healthy women selected from our healthy control group this association was strongly significant at the femoral neck region. In vitro luciferase assay demonstrated that the polymorphic rs4844880 allele inhibited the luciferase activity more significantly than the major allele. CONCLUSIONS The rs4844880 polymorphism in the promoter region of the HSD11B1 gene resulting in a reduced expression of the enzyme may exert a beneficial effect on bone in healthy and postmenopausal osteoporotic women.

Minireview: MIRomics in Endocrinology: A Novel Approach for Modeling Endocrine Diseases

MicroRNAs (miRNAs) are small (16-24 nucleotides) noncoding RNAs that negatively regulate gene expression. Growing evidence demonstrates that miRNAs participate in the regulation of numerous physiological and pathological processes. The clinical utility of the cell-type-specific miRNA expression profile (miRomics) has been directly demonstrated in molecular classification of tumor samples and in prediction of prognosis or therapeutic responsiveness. Identification of the relevant miRNAs and their targets requires both in silico and molecular biological methods. In this review, we summarize the methodological arsenal used in miRNA-related research, and through our own data on adrenal tumors, we present how miRNA could be integrated into omics-based networks. The expanding knowledge obtained from miRNA research may lead to the development of novel diagnostic and treatment modalities in future.

Evaluation and diagnostic potential of circulating extracellular vesicle-associated microRNAs in adrenocortical tumors.

There is no available blood marker for the preoperative diagnosis of adrenocortical malignancy. The objective of this study was to investigate the expression of extracellular vesicle-associated microRNAs and their diagnostic potential in plasma samples of patients suffering from adrenocortical tumors. Extracellular vesicles were isolated either by using Total Exosome Isolation Kit or by differential centrifugation/ultracentrifugation. Preoperative plasma extracellular vesicle samples of 6 adrenocortical adenomas (ACA) and 6 histologically verified adrenocortical cancer (ACC) were first screened by Taqman Human Microarray A-cards. Based on the results of screening, two miRNAs were selected and validated by targeted quantitative real-time PCR. The validation cohort included 18 ACAs and 16 ACCs. Beside RNA analysis, extracellular vesicle preparations were also assessed by transmission electron microscopy, flow cytometry and dynamic light scattering. Significant overexpression of hsa-miR-101 and hsa-miR-483-5p in ACC relative to ACA samples has been validated. Receiver operator characteristics of data revealed dCThsa-miR-483-5p normalized to cel-miR-39 to have the highest diagnostic accuracy (area under curve 0.965), the sensitivity and the specifity were 87.5 and 94.44, respectively. Extracellular vesicle-associated hsa-miR-483-5p thus appears to be a promising minimally invasive biomarker in the preoperative diagnosis of ACC but needs further validation in larger cohorts of patients.

Circulating miRNAs as biomarkers for endocrine disorders

Specific, sensitive and non-invasive biomarkers are always needed in endocrine disorders. miRNAs are short, non-coding RNA molecules with well-known role in gene expression regulation. They are frequently dysregulated in metabolic and endocrine diseases. Recently it has been shown that they are secreted into biofluids by nearly all kind of cell types. As they can be taken up by other cells they may have a role in a new kind of paracrine, cell-to-cell communication. Circulating miRNAs are protected by RNA-binding proteins or microvesicles hence they can be attractive candidates as diagnostic or prognostic biomarkers. In this review, we summarize the characteristics of extracellular miRNA’s and our knowledge about their origin and potential roles in endocrine and metabolic diseases. Discussions about the technical challenges occurring during identification and measurement of extracellular miRNAs and future perspectives about their roles are also highlighted.

Novel Genetic Mutation in the Background of Carney Complex

Carney complex is a rare disease inherited in an autosomal dominant manner. It is mostly caused by inactivating mutations of the subunit of protein kinase A. Carney complex is associated with atrial myxoma, nevi or myxomas of the skin, breast tumor and endocrine overactivity. Primary pigmented nodular adrenocortical disease is the specific endocrine manifestation. The authors present the history of a 53-year-old female patient who had undergone surgery for atrial myxomas, thyroid tumor and breast cancer. She was also operated for an adrenal adenoma causing Cushing’s syndrome. Genetic study revealed a novel mutation in the regulatory subunit of protein kinase A (ivs2-1G>A splice mutation in intron 2). Her heterozygous twins were also genetically screened and one of them carried the same mutation. The authors emphasize that despite the absence of specific treatment for patients with Carney complex, confirmation of the diagnosis by genetic studies is important for the close follow-up of the patient and early identification of novel manifestations.

Analysis of Circulating MicroRNAs in Vivo following Administration of Dexamethasone and Adrenocorticotropin

Purpose. The interaction of hormones of the pituitary-adrenal axis and adrenal cortex-associated circulating microRNAs is mostly unknown. We have studied the effects of dexamethasone and adrenocorticotropin on the expression of five circulating microRNAs (hsa-miR-27a, hsa-miR-200b, hsa-miR-214, hsa-miR-483-5p, and hsa-miR-503) reported to be related to the adrenal cortex in plasma samples. Methods. Expression of microRNAs was studied in plasma samples of 10 individuals examined by 1 mg dexamethasone suppression test and another 10 individuals stimulated by 250 μg tetracosactide (adrenocorticotropin). Total RNA was isolated and microRNA expression was analyzed by real-time reverse transcription quantitative polymerase chain reaction normalized to cel-miR-39 as reference. Results. Only circulating hsa-miR-27a proved to be significantly modulated in vivo by hormonal treatments: its expression was upregulated by dexamethasone whereas it was suppressed by adrenocorticotropin. Secreted hsa-miR-27a was significantly induced by dexamethasone in vitro in NCI-H295R cells, as well. The expression of hsa-miR-483-5p proposed as diagnostic marker for adrenocortical malignancy was not affected by dexamethasone or tetracosactide administration. Conclusions. hsa-miR-27a expression is modulated by hormones of the hypothalamic-pituitary-adrenal axis both in vitro and in vivo. The biological relevance of hsa-miR-27a modulation by hormones is unclear, but the responsiveness of circulating microRNAs to hormones of the pituitary-adrenal axis is noteworthy.

Technical Aspects Related to the Analysis of Circulating microRNAs

Specific and sensitive noninvasive biofluid-based biomarkers are always needed in the laboratory diagnosis of diseases. Biomarkers are applied not only for diagnostic purposes but for stratifying a disease and for assessing the therapy response or disease progression. MicroRNAs (miRNAs) are short noncoding RNA molecules regulating gene expression posttranscriptionally. They are frequently dysregulated in many physiological and pathophysiological conditions. miRNAs are present in the circulation and in other biofluids that are common matrices for clinical laboratory testing that has raised the possibility that miRNAs may serve as novel biomarkers. Their excellent stability also supports the possibility that miRNAs once will be routinely used biomarkers in clinical practice. From an analytical point of view, there are many factors (starting material, sample storage and processing, different RNA extraction and detection methods, intra- and interassay variability, and assay interferences) to consider if a miRNA as biomarker is aimed to be introduced as a clinical laboratory test. Despite several pre-analytical and analytical factors that still need standardization, a significant number of studies have been published about the potential role of circulating miRNAs as biomarkers. Due to the lack of standardization of methods, there are a lot of discrepancies among results. In this chapter, we aimed to summarize the current findings about circulating miRNAs focusing on the analytical points related to miRNAs measurements from biofluids.

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes’ expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially regulated by miRNAs.