Difference in miRNA Expression in Functioning and Silent Corticotroph Pituitary Adenomas Indicates the Role of miRNA in the Regulation of Corticosteroid Receptors.

Corticotroph pituitary adenomas commonly cause Cushing's disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3' UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids.

Telomere length and TERT abnormalities in pituitary adenomas.

OBJECTIVES: Pituitary adenomas (PAs) are among the most frequent intracranial tumors in humans. Abnormal telomerase activity and telomere lengthening are features of tumor cells. They may result from mutations in TERT promoter region, gene amplification or aberrant DNA methylation pattern. Such changes were found in variety of tumors including those of brain. Aim of the study was to evaluate the incidence of TERT abnormalities and to assess their role in telomere lengthening in PAs. METHODS: Study involved 101 patients with PA including both nonfunctioning and functioning subtypes. Telomerase length as well as TERT mRNA level and gene amplification were estimated using quantitative PCR (qPCR). Promoter mutations were assessed using Sanger sequencing. The results from genome-wide DNA methylation profiling with HumanMethylation 450K (Illumina) were used for the analysis of TERT locus. RESULTS: Variable telomere length was observed in patients, however no relationship with clinicopathological features was found. We observed a missense variant in TERT promoter in one patient only whereas increased TERT copy number were identified in 6 patients (5.6%). However no relationship between these results and telomere length or TERT expression was found. DNA methylation at TERT locus was not found to be changed when adenoma samples and normal tissue sections were compared. CONCLUSION: The results indicate that telomerase abnormalities do not play a role in pathogenesis of pituitary tumors.

Invasive and Noninvasive Nonfunctioning Gonadotroph Pituitary Tumors Differ in DNA Methylation Level of LINE-1 Repetitive Elements.

PURPOSE: Epigenetic dysregulation plays a role in pituitary tumor pathogenesis. Some differences in DNA methylation were observed between invasive and noninvasive nonfunctioning gonadotroph tumors. This study sought to determine the role of DNA methylation changes in repetitive LINE-1 elements in nonfunctioning gonadotroph pituitary tumors. METHODS: We investigated LINE-1 methylation levels in 80 tumors and normal pituitary glands with bisulfite-pyrosequencing. Expression of two LINE-1 open reading frames (L1-ORF1 and L1-ORF2) was analyzed with qRT-PCR in tumor samples and mouse gonadotroph pituitary cells treated with DNA methyltransferase inhibitor. Immunohistochemical staining against L1-ORF1p was also performed in normal pituitary glands and tumors. RESULTS: Hypomethylation of LINE-1 was observed in pituitary tumors. Tumors characterized by invasive growth revealed lower LINE-1 methylation level than noninvasive ones. LINE-1 methylation correlated with overall DNA methylation assessed with HM450K arrays and negatively correlated with L1-ORF1 and L1-ORF2 expression. Treatment of αT3-1 gonadotroph cells with 5-Azacytidine clearly increased the level of L1-ORF1 and L1-ORF2 mRNA; however, its effect on LßT2 cells was less pronounced. Immunoreactivity against L1-ORF1p was higher in tumors than normal tissue. No difference in L1-ORF1p expression was observed in invasive and noninvasive tumors. CONCLUSION: Hypomethylation of LINE-1 is related to invasive growth and influences transcriptional activity of transposable elements.

Circulating Hsa-miR-431-5p as Potential Biomarker for Squamous Cell Vulvar Carcinoma and Its Premalignant Lesions.

Vulvar squamous cell carcinoma (VSCC) develops from high-grade squamous intraepithelial lesions (HSIL) and differentiated vulvar intraepithelial neoplasia (dVIN). This study aimed to assess the diagnostic value of circulating hsa-miR-431-5p in vulvar precancers and VSCC. Expression levels of hsa-miR-431-5p were analyzed by quantitative RT-PCR in plasma samples of 29 patients with vulvar precancers (HSIL or dVIN), 107 with VSCC as well as 15 healthy blood donors. We used hsa-miR-93-5p and hsa-miR-425-5p as normalizers. The levels of miR-431-5p were increased in the blood of patients with VSCC compared to those with vulvar precancers. Statistically significant differences in the survival rates (time to progression) were revealed for VSCC patients categorized by miR-431-5p levels. Low levels of circulating miR-431-5p were found to be indicative of unfavorable survival rates. In summary, our data reveal the diagnostic potential of circulating miR-431-5p in patients with vulvar precancers and VSCC.

Differential microRNA Expression in USP8-Mutated and Wild-Type Corticotroph Pituitary Tumors Reflect the Difference in Protein Ubiquitination Processes.

BACKGROUND: USP8 mutations are the most common driver changes in corticotroph pituitary tumors. They have direct effect on cells' proteome through disturbance of ubiquitination process and also influence gene expression. The aim of this study was to compare microRNA profiles in USP8-mutated and wild-type tumors and determine the probable role of differential microRNA expression by integrative microRNA and mRNA analysis. METHODS: Patients with Cushing's disease (n = 28) and silent corticotroph tumors (n = 20) were included. USP8 mutations were identified with Sanger sequencing. MicroRNA and gene expression was determined with next-generation sequencing. RESULTS: USP8-mutated patients with Cushing's disease showed higher rate of clinical remission and trend towards lower tumor volume than wild-type patients. Comparison of microRNA profiles of USP8-mutated and wild-type tumors revealed 68 differentially expressed microRNAs. Their target genes were determined by in silico prediction and microRNA/mRNA correlation analysis. GeneSet Enrichment analysis of putative targets showed that the most significantly overrepresented genes are involved in protein ubiquitination-related processes. Only few microRNAs influence the expression of genes differentially expressed between USP8-mutated and wild-type tumors. CONCLUSIONS: Differences in microRNA expression in corticotropinomas stratified according to USP8 status reflect disturbed ubiquitination processes, but do not correspond to differences in gene expression between these tumors.

The Search of miRNA Related to Invasive Growth of Nonfunctioning Gonadotropic Pituitary Tumors.

PURPOSE: Nonfunctioning gonadotropic pituitary neuroendocrine tumors (PitNETs) are among the most frequent neoplasms of pituitary gland. Although PitNETs are commonly considered benign, a notable part of patients suffer from tumor recurrence after treatment. Invasive growth of pituitary tumor is among the most important prognostic factors. Since molecular features of invasiveness are of potential clinical usefulness, this study was aimed to verify whether invasive and noninvasive nonfunctioning gonadotropic PitNETs differ in the miRNA expression profile and whether the differences could provide a possible molecular classifier. METHODS: miRNA profiles were determined in 20 patients (11 invasive and 9 noninvasive tumors) using next-generation sequencing. The expression of selected miRNAs was assessed in the independent cohort of 80 patients with qRT-PCR. RESULTS: When miRNA profiles of invasive and noninvasive tumors were compared, 29 miRNAs were found differentially expressed. Hsa-miR-184, hsa-miR-181a-2-3p, hsa-miR-93-3p, hsa-miR-574-5p, hsa-miR-185-5p, and hsa-miR-3200-5p showed a potential clinical value according to ROC curve analysis. Unfortunately, differential expression of only hsa-miR-185-5p was confirmed in the validation cohort, with AUG at 0.654. CONCLUSION: Differences in miRNAs expression profiles in invasive and noninvasive gonadotropic PitNETs are slight and the level of miRNA expression seems not to be applicable as useful classifier of tumor invasiveness.

DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors.

microRNAs are involved in pathogenesis of cancer. DNA methylation plays a role in transcription of miRNA-encoding genes and may contribute to changed miRNA expression in tumors. This issue was not investigated in pituitary neuroendocrine tumors (PitNETs) previously. DNA methylation patterns, assessed with HumanMethylation450K arrays in 34 PitNETs and five normal pituitaries, were used to determine differentially methylated CpGs located at miRNA genes. It showed aberrant methylation in regions encoding for 131 miRNAs. DNA methylation data and matched miRNA expression profiles, determined with next-generation sequencing (NGS) of small RNAs, were correlated in 15 PitNETs. This showed relationship between methylation and expression levels for 12 miRNAs. DNA methylation and expression levels of three of them (MIR145, MIR21, and MIR184) were determined in the independent group of 80 tumors with pyrosequencing and qRT-PCR and results confirmed both aberrant methylation in PitNETs and correlation between methylation and expression. Additionally, in silico target prediction was combined with analysis of established miRNA profiles and matched mRNA expression pattern, assessed with amplicon-based NGS to indicate putative target genes of epigenetically deregulated miRNAs. This study reveals aberrant DNA methylation in miRNA-encoding genes in gonadotroph PitNETs. Methylation changes affect expression level of miRNAs that regulate putative target genes with tumorigenesis-relevant functions.

USP8 mutations in corticotroph adenomas determine a distinct gene expression profile irrespective of functional tumour status.

OBJECTIVE: Pituitary corticotroph adenomas commonly cause Cushing's disease (CD) but part of these tumours are hormonally inactive (silent corticotroph adenomas, SCA). USP8 mutations are well-known driver mutations in corticotrophinomas. Differences in transcriptomic profiles between functioning and silent tumours or tumours with different USP8 status have not been investigated. DESIGN AND METHODS: Forty-eight patients (28 CD, 20 SCA) were screened for USP8 mutations with Sanger sequencing. Twenty-four patients were included in transcriptomic profiling with Ampliseq Transcriptome Human Gene Expression Core Panel. The entire patients group was included in qRT-PCR analysis of selected genes expression. Immunohistochemistry was used for visualization of selected protein. RESULTS: We found USP8 mutation in 15 patients with CD and 4 SCAs. USP8 mutations determine molecular profile of the tumours as showed by hierarchical clustering and identification of 1648 genes differentially expressed in USP8-mutated and USP8-wild-type tumours. Mutations affect many molecular pathways as observed in Gene Set Enrichment analysis. USP8-mutated adenomas showed higher level of POMC, CDC25A, MAPK4 but lower level of CCND2, CDK6, CDKN1B than USP8-wt tumours. Eighty-seven genes differentially expressed between CD-related adenomas and SCAs were found, including those involved in cell signalling (GLI2, DLC1, TBX2, RASSF6), cell adhesion (GJA1, CDH6), ion transport (KCNN4, KCNJ5) and GABA signalling (GABBR2, GABRD). CONCLUSION: USP8 mutations occur in functioning and silent corticotrophinomas. They have pleiotropic effect, not limited to EGFR signalling, and affect expression levels of many genes involved in different pathways. Expression of GABA-related genes GABBR2, GNAL, GABARD and KCNJ5 correspond to functional status of the tumours.

The Role of Aberrant DNA Methylation in Misregulation of Gene Expression in Gonadotroph Nonfunctioning Pituitary Tumors.

Gonadotroph nonfunctioning pituitary adenomas (NFPAs) are common intracranial tumors, but the role of aberrant epigenetic regulation in their development remains poorly understood. In this study, we investigated the effect of impaired CpG methylation in NFPAs. We determined DNA methylation and transcriptomic profiles in 32 NFPAs and normal pituitary sections using methylation arrays and sequencing, respectively. Ten percent of differentially methylated CpGs were correlated with gene expression, and the affected genes are involved in a variety of tumorigenesis-related pathways. Different proportions of gene body and promoter region localization were observed in CpGs with negative and positive correlations between methylation and gene expression, and different proportions of CpGs were located in 'open sea' and 'shelf/shore' regions. The expression of ~8% of genes differentially expressed in NFPAs was related to aberrant methylation. Methylation levels of seven CpGs located in the regulatory regions of FAM163A, HIF3A and PRSS8 were determined by pyrosequencing, and gene expression was measured by qRT-PCR and immunohistochemistry in 83 independent NFPAs. The results clearly confirmed the negative correlation between methylation and gene expression for these genes. By identifying which aberrantly methylated CpGs affect gene expression in gonadotrophinomas, our data confirm the role of aberrant methylation in pathogenesis of gonadotroph NFPAs.

DNA methylation profiling in nonfunctioning pituitary adenomas.

Nonfunctioning pituitary adenomas (NFPAs) are among the most frequent intracranial tumors but their molecular background, including changes in epigenetic regulation, remains poorly understood. We performed genome-wide DNA methylation profiling of 34 NFPAs and normal pituitary samples. Methylation status of the selected genomic regions and expression level of corresponding genes were assessed in a group of 75 patients. NFPAs exhibited distinct global methylation profile as compared to normal pituitary. Aberrant DNA methylation appears to contribute to deregulation of the cancer-related pathways as shown by preliminary functional analysis. Promoter hypermethylation and decreased expression level of SFN, STAT5A, DUSP1, PTPRE and FGFR2 was confirmed in the enlarged group of NFPAs. Difference in the methylation profiles between invasive and non-invasive NFPAs is very slight. Nevertheless, invasiveness-related aberrant epigenetic deregulation of the particular genes was found including upregulation of ITPKB and downregulation CNKSR1 in invasive tumors.