Meningiomas are common intracranial tumors in adults. Abnormal microRNA (miRNA) expression plays a role in their pathogenesis. Change in miRNA expression level can be caused by impaired epigenetic ...regulation of miRNA-encoding genes. We found the genomic region covering the MIR193B gene to be DNA hypermethylated in meningiomas based on analysis of genome-wide methylation (HumanMethylation450K Illumina arrays). Hypermethylation of MIR193B was also confirmed via bisulfite pyrosequencing. Both hsa-miR-193b-3p and hsa-miR-193b-5p are downregulated in meningiomas. Lower expression of hsa-miR-193b-3p and higher MIR193B methylation was observed in World Health Organization (WHO) grade (G) II/III tumors as compared to GI meningiomas. CCND1 mRNA was identified as a target of hsa-miR-193b-3p as further validated using luciferase reporter assay in IOMM-Lee meningioma cells. IOMM-Lee cells transfected with hsa-miR-193b-3p mimic showed a decreased cyclin D1 level and lower cell viability and proliferation, confirming the suppressive nature of this miRNA. Cyclin D1 protein expression (immunoreactivity) was higher in atypical than in benign meningiomas, accordingly to observations of lower hsa-miR-193b-3p levels in GII tumors. The commonly observed hypermethylation of MIR193B in meningiomas apparently contributes to the downregulation of hsa-miR-193b-3p. Since hsa-miR-193b-3p regulates proliferation of meningioma cells through negative regulation of cyclin D1 expression, it seems to be an important tumor suppressor in meningiomas.
Chordomas are rare tumors of notochord remnants, occurring mainly in the sacrum and skull base. Despite of their unusually slow growth, chordomas are highly invasive and the involvement of adjacent ...critical structures causes treatment challenges. Due to the low incidence, the molecular pathogenesis of this entity remains largely unknown. This study aimed to investigate DNA methylation abnormalities and their impact on gene expression profiles in skull base chordomas. 32 tumor and 4 normal nucleus pulposus samples were subjected to DNA methylation and gene expression profiling with methylation microarrays and RNA sequencing. Genome-wide DNA methylation analysis revealed two distinct clusters for chordoma (termed subtypes C and I) with different patterns of aberrant DNA methylation. C Chordomas were characterized by general hypomethylation with hypermethylation of CpG islands, while I chordomas were generally hypermethylated. These differences were reflected by distinct distribution of differentially methylated probes (DMPs). Differentially methylated regions (DMRs) were identified, indicating aberrant methylation in known tumor-related genes in booth chordoma subtypes and regions encoding small RNAs in subtype C chordomas. Correlation between methylation and expression was observed in a minority of genes. Upregulation of TBXT in chordomas appeared to be related to lower methylation of tumor-specific DMR in gene promoter. Gene expression-based clusters of tumor samples did not overlap with DNA methylation-based subtypes. Nevertheless, they differ in transcriptomic profile that shows immune infiltration in I chordomas and up-regulation of cell cycle in C chordomas. Immune enrichment in chordomas I was confirmed with 3 independent deconvolution methods and immunohistochemistry. Copy number analysis showed higher chromosomal instability in C chordomas. Nine out of eight had deletion of CDKN2A/B loci and downregulation of genes encoded in related chromosomal band. No significant difference in patients' survival was observed between tumor subtypes, however, shorter survival was observed in patients with higher number of copy number alterations.
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 (
= 28) and silent ones (
= 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
and
. The role of hsa-miR-124-3p in the regulation of
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
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.
Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent
,
,
and
...mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs.
Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in
,
and
with Sanger sequencing, while 128 of these patients were screened for
mutations with next generation sequencing and immunohistochemistry.
mutations were found in 41% CD and 8,5% silent tumors, while
mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No
mutations were found. Four
mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense
mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift
variant had low protein expression.
mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency.
We confirmed high prevalence of
mutations and low incidence of
and
mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD.
mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.
Protein deubiquitinases
and
are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs).
mutations have pleiotropic effects that include notable changes in genes' expression. ...Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that
-mutated tumors have lower
,
,
and higher
expression.
-mutated tumors have lower
and
expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of
mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated
in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.
Pituitary neuroendocrine corticotroph tumors commonly cause Cushing's disease (CD) that results from increased adrenocorticotropic hormone (ACTH) secretion by the pituitary tumor and consequent ...increase of cortisol levels in blood. However, in some patients, corticotroph tumors remain clinically non-functioning. Cortisol secretion is regulated by the hypothalamic-pituitary-adrenal axis and includes a negative feedback between cortisol and ACTH secretion. Glucocorticoids reduce ACTH level both by hypothalamic regulation and acting on corticotrophs
glucocorticoid (GR) and mineralocorticoid (MR) receptors. The aim of the study was to determine the role of GR and MR expression at mRNA and protein levels in both functioning and silent corticotroph tumors.
Ninety-five patients were enrolled, including 70 with CD and 25 with silent corticotroph tumors. Gene expression levels of
and
coding for GR and MR, respectively, were determined with qRT-PCR in the two tumor types. GR and MR protein abundance was assessed with immunohistochemistry.
Both GR and MR were expressed in corticotroph tumors. Correlation between
and
expression levels was observed.
expression was higher in silent than in functioning tumors. In CD patients
and
levels were negatively correlated with morning plasma ACTH levels and tumor size. Higher
was confirmed in patients with remission after surgery and in densely granulated tumors. Expression of both genes and GR protein was higher in
-mutated tumors. Similar relationship between
mutations and expression levels were observed in analysis of silent tumors that also revealed a negative correlation between GR and tumor size and higher
expression in densely granulated tumors.
Although the associations between gene/protein expression and patients clinical features are not strong, they consistently show an evident trend in which higher receptor expression corresponds to more favorable clinical characteristics.
Acromegaly results from growth hormone hypersecretion, predominantly caused by a somatotroph pituitary neuroendocrine tumor (PitNET). Acromegaly-causing tumors are histologically diverse. Our aim was ...to determine transcriptomic profiles of various somatotroph PitNETs and to evaluate clinical implication of differential gene expression. A total of 48 tumors were subjected to RNA sequencing, while expression of selected genes was assessed in 134 tumors with qRT-PCR. Whole-transcriptome analysis revealed three transcriptomic groups of somatotroph PitNETs. They differ in expression of numerous genes including those involved in growth hormone secretion and known prognostic genes. Transcriptomic subgroups can be distinguished by determining the expression of marker genes. Analysis of the entire cohort of patients confirmed differences between molecular subtypes of tumors. Transcriptomic group 1 includes ~20% of acromegaly patients with
mutations-negative, mainly densely granulated tumors that co-express
and
(SF-1). SF-1 expression was verified with immunohistochemistry. Transcriptomic group 2 tumors are the most common (46%) and include mainly
-mutated, densely granulated somatotroph and mixed PitNETs. They have a smaller size and express favorable prognosis-related genes. Transcriptomic group 3 includes predominantly sparsely granulated somatotroph PitNETs with low
mutations frequency causing ~35% of acromegaly. Ghrelin signaling is implicated in their pathogenesis. They have an unfavorable gene expression profile and higher invasive growth rate.
Histone post-translational modifications (PTMs) play an important role in the regulation of the expression of genes, including those involved in cancer development and progression. However, our ...knowledge of PTM patterns in human tumours is limited.
MS-based analyses were used to quantify global alterations of histone PTMs in colorectal cancer (CRC) samples. Histones isolated from 12 CRCs and their corresponding normal mucosa by acidic extraction were separated by SDS-PAGE and analysed by liquid chromatography-mass spectrometry.
Among 96 modified peptides, 41 distinct PTM sites were identified, of which 7, 13, 11, and 10 were located within the H2A, H2B, H3, and H4 sequences, respectively, and distributed among the amino-terminal tails and the globular domain of the four histones. Modification intensities were quantified for 33 sites, of which 4 showed significant (p-value ≤ 0.05) differences between CRC tissues and healthy mucosa samples. We identified histone H3 lysine 27 acetylation (H3K27Ac) as a modification upregulated in CRC, which had not been shown previously.
The present results indicate the usefulness of a bottom-up proteomic approach for the detection of histone modifications at a global scale. The differential abundance of H3K27Ac mark in CRC, a PTM associated with active enhancers, suggests its role in regulating genes whose expression changes in CRC.
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
,
and
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.
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
-mutated and wild-type tumors and determine the probable role of differential microRNA expression by integrative microRNA and mRNA analysis.
Patients with Cushing's disease (
= 28) and silent corticotroph tumors (
= 20) were included.
mutations were identified with Sanger sequencing. MicroRNA and gene expression was determined with next-generation sequencing.
-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
-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
-mutated and wild-type tumors.
Differences in microRNA expression in corticotropinomas stratified according to
status reflect disturbed ubiquitination processes, but do not correspond to differences in gene expression between these tumors.
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