Epigenetic control of adamantinomatous craniopharyngiomas Marrero-Gutiérrez, Junier; Bueno, Ana Carolina; Martins, Clarissa Silva ...
The journal of clinical endocrinology and metabolism,
2024-Jan-05, 2024-01-05, 20240105
Journal Article
Recenzirano
Odprti dostop
Studies addressing the methylation pattern in adamantinomatous craniopharyngioma (ACP) are lacking.
To identify methylation signatures in ACPs regarding clinical presentation and outcome.
Clinical ...and pathology data were collected from 35 ACP patients (54% male; 18.1 years 2-68). CTNNB1 mutations and methylation profile (MethylationEPIC/Array-Illumina) were analyzed in tumoral DNA. Unsupervised machine learning analysis of this comprehensive methylome sample was achieved using hierarchical clustering and multi-dimensional scaling. Statistical associations between clusters and clinical features were achieved using Fisher's test and global biological process interpretations were aided by Gene Ontology enrichment analyses.
Two clusters were revealed consistently by all unsupervised methods (ACP-1: n = 18; ACP-2: n = 17) with strong bootstrap statistical support. ACP-2 was enriched by CTNNB1 mutations (100% vs 56%, P = 0.0006), hypomethylated in CpG Island (CGI), non-CGI sites, and globally (P < 0.001), and associated with greater tumor size (24.1 vs 9.5cm3, P = 0.04). Enrichment analysis highlighted pathways on signaling transduction, transmembrane receptor, development of anatomical structures, cell-adhesion, cytoskeleton organization, and cytokine binding, and also cell-type specific biological processes as regulation of oligodendrocytes, keratinocyte, and epithelial cells differentiation.
Two clusters of ACP patients were consistently revealed by unsupervised machine learning methods, being one of them significantly hypomethylated, enriched by CTNNB1 mutated ACPs, and associated with increased tumor size. Enrichment analysis reinforced pathways involved in tumor proliferation and in cell-specific tumoral microenvironment.
To evaluate the therapeutic potential of vitamin D receptor (VDR) signaling in adrenocortical carcinoma (ACC) cells.
We evaluated VDR's methylation pattern in H295R ACC cells, and investigated the ...effects of calcitriol and seocalcitol treatments on adrenocortical tumorigenesis.
VDR was hypermethylated and underexpressed in basal H295R cells. Treatments with calcitriol and seocalcitol restored VDR signaling, resulted in antiproliferative effects, and impaired Wnt/B-catenin signaling. RNAseq of treated cells demonstrated VDR activation on steroid hormones biosynthesis and Rap1 signaling, among others. In vivo, seocalcitol constrained the growth of H295R xenografts and reduced autonomous tumor steroid secretion without hypercalcemia-associated side effects.
H295R cells present VDR hypermethylation, which can be responsible for its underexpression and signaling inactivation under basal conditions. VDR signaling promoted antiproliferative effects in vitro and in vivo, suggesting that it may be a potential therapeutic target for ACC and a valuable tool for patient's clinical management.
•Resembling adrenocortical carcinomas, VDR DNA is hypermethylated in H295R cells.•VDR is underexpressed and inactive in H295R cells under basal conditions.•Vitamin D treatment restores VDR signaling in H295R cells and impairs tumorigenesis.•Cell-active VDR transcriptome landscape reinforces its influence in steroid synthesis and reveals novel pathways associated with ACC.•Seocalcitol inhibits H295R xenograft autonomous steroid secretion.
Objective Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter ...hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance. Design Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls. Results Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival. Conclusions VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.
Children diagnosed with pediatric adrenocortical tumors (pACT) have variable outcomes, and, to date, the disease lacks robust prognostic biomarkers. The prognostic potential of tumor methylation has ...been demonstrated in several cancers. We aimed to evaluate the pACT methylation profile and its association with disease presentation and survival. In this cross-sectional study, we accessed the DNA methylation (MethylationEPIC Array, Illumina) of 57 primary pACT from Southeastern Brazil and the respective patients’ clinicopathological features. We also applied our analysis in an independent 48 pACT methylation dataset. Unsupervised learning whole-methylome analysis showed two groups with distinct methylation signatures: pACT-1 and pACT-2. Compared to pACT-2, pACT-1 tumors were enriched with higher methylation in CpG islands, mainly in gene promoter regions. The topmost hypermethylated gene in these samples was shown to be underexpressed. Patients in the pACT-1 group were older at diagnosis and were more likely to have carcinomas and nonlocalized/advanced and recurrent/metastatic disease. Univariate and bivariate regressions showed that pACT-1 methylation signature confers superior hazard ratio of disease progression and death than known prognostic features. The methylation groups had similar frequencies of germline mutations in the TP53 gene, including the regionally frequent p.R337H. Our analysis replication validated our findings and reproduced those recently described in pACT. We demonstrated the existence of different tumor methylation signatures associated with pACT presentation and clinical evolution, even in the context of germline TP53 mutations. Our data support tumor methylation profiling as a robust and independent prognostic biomarker for pACT and suggest a list of candidate genes for further validation.
A collection of 237,954 sugarcane ESTs was examined in search of signal transduction genes. Over 3500 components involved in several aspects of signal transduction, transcription, development, cell ...cycle, stress responses and pathogen interaction were compiled into the Sugarcane Signal Transduction (SUCAST) Catalogue. Sequence comparisons and protein domain analysis revealed 477 receptors, 510 protein kinases, 107 protein phosphatases, 75 small GTPases, 17 G-proteins, 114 calcium and inositol metabolism proteins, and over 600 transcription factors. The elements were distributed into 29 main categories subdivided into 409 sub-categories. Genes with no matches in the public databases and of unknown function were also catalogued. A cDNA microarray was constructed to profile individual variation of plants cultivated in the field and transcript abundance in six plant organs (flowers, roots, leaves, lateral buds, and 1st and 4th internodes). From 1280 distinct elements analyzed, 217 (17%) presented differential expression in two biological samples of at least one of the tissues tested. A total of 153 genes (12%) presented highly similar expression levels in all tissues. A virtual profile matrix was constructed and the expression profiles were validated by real-time PCR. The expression data presented can aid in assigning function for the sugarcane genes and be useful for promoter characterization of this and other economically important grasses.
Purpose: This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast
cancer patients.
Experimental Design: Biopsy samples were obtained ...before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified
and gene expression was analyzed using cDNA microarrays.
Results: Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines,
42 patients, who presented at least a partial response (≥30% reduction in tumor dimension), were classified as responsive.
Gene profile of samples, divided into training set ( n = 38) and independent validation set ( n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not
separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B , and PBEF , which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile
in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples
of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group
all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1 , and CLPTM1 , could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and
one resistant tumor. The robustness of this classifier is 2.5 greater than the first one.
Conclusion: A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by
a larger number of samples is still needed.
Abstract
Pediatric adrenocortical tumors (pACTs) display complex genomic background and lack robust prognostic biomarkers. However, pACT methylation profiling was recently associated with patient ...prognosis. In order to evaluate whether tumor DNA methylation is associated with patient prognosis, we studied a Brazilian cohort of pACT in which most of the patients were carriers of the germline P53 p.R337H mutation (86%). We analyzed the DNA methylation profile of 57 tumor samples (MethylationEPIC BeadChip Array, Illumina) and the respective patients’ clinicopathological features associated with outcome and overall survival. Median age at diagnosis of the 40 girls and 17 boys was 2.1 years (range: 0.2–16.6). Reduced overall survival was observed in patients diagnosed after 4 years of age (n=43, HR=26.5, p<0.0001, 5-year survival (5ys)=43%), in those presenting with Cushing’s syndrome (n=7, HR=12.5, p<0.0001, 5ys=28%), advanced/non localized disease (IPACTR stages III and IV; n=11, HR=12.9, p<0.0001, 5ys=33%) or carcinomas (n=10, HR=4.9, p=0.04 5ys=56%). Unsupervised analysis of methylation data (Euclidean distance and Ward’s method; Pvclust package, R, version 4.0.3) included all 766,031 probes filtered in by the quality control step, and revealed two groups with distinct methylation signatures with a bootstrap support of 99%, namely pACT1 and pACT2. The overall variance of differentially methylated probes (DMPs; FDR p-values <10–6 and, simultaneously, median M-value difference >2) between the groups was 0.084% (n=642 probes). Tumors from pACT1 were enriched for hypomethylation in genes’ body region and for hypermethylation in promoter regions (p<0.0001). Gene set analysis showed that DMPs were mostly enriched in gene sets involved in nervous system differentiation and development, mRNA binding, and peptide secretion and related to pathways in cancer, cell adhesion, and calcium signaling. Methylation was not associated with germline P53 p.R337H mutation (p=0.57). Compared to pACT2, pACT1 group comprised patients older at diagnosis (p=0.0004) and higher frequencies of patients diagnosed after 4 years of age (80 vs 13%; p<0.0001), presenting with Cushing syndrome (40 vs. 4%; p=0.01), non-localized/advanced disease (70 vs 8%; p=0.0001), carcinomas (71 vs 17%. p=0.009), who needed adjuvant chemotherapy (90 vs. 9%; p<0.0001), and who experienced post-surgery recurrence or metastasis (90 vs 6%; p<0.0001). pACT1 methylation signature was associated with reduced overall survival from the disease (HR=51, p=0.0001, 5ys: 20 vs 98%), and remained associated after adjusting for prognostic features. Interestingly, different tumor methylation signatures were associated with pACT clinical presentation and evolution, regardless the presence of the P53 p.R337H mutation. Our data supports the analysis of methylation profiling as a robust and independent prognostic biomarker for pACT.
O objetivo deste trabalho é apresentar os métodos originais em Bioinformática desenvolvidos para a análise estatística na interpretação dos dados de duas técnicas baseadas em imagens: a técnica de ...microarranjos de DNA e a técnica de ressonância magnética funcional. O interesse principal é abordar essas técnicas experimentais quando enfrenta-se uma situação clara de amostras escassas, isto é, quando existem relativamente poucas observações experimentais do fenômeno estudado, sendo a análise individual/personalizada o representante extremo desta situação, que tem que ser resolvida. Para tanto, opta-se pelo uso da Inferência Bayesiana no contexto da Teoria da Decisão sob Incerteza, implementada computacionalmente sob o arcabouço dos Sistemas de Suporte à Decisão. Ambas as tecnologias estudadas produzem dados complexos, baseados na interpretação das diferenças entre imagens obtidas da resposta do sistema a um estímulo e da resposta numa situação controle. O resultado deste trabalho é o desenvolvimento de dois sistemas de suporte à decisão, chamados HTself e Dotslashen, para a análise de dados de microarranjos e ressonância magnética funcional, respectivamente; e de seus métodos matemáticos/computacionais subjacentes. Os sistemas desenvolvidos extraem conhecimento racional de bancos-de-dados normativos, através de modelos matemáticos específicos, contornando então o problema de amostras escassas. Finalmente, neste trabalho são descritas aplicações a problemas reais, para destacar a utilidade dos sistemas de suporte à decisão desenvolvidos nas áreas de Biologia Molecular e Neuroimagem Funcional.
The goal of this work is to present the novel Bioinformatics methods that were developed aiming the statistical analysis of two image-based techniques: DNA microarrays and functional magnetic resonance imaging. The main interest is to approach these experimental techniques in small sample size situations, i.e., when there are relatively few experimental observations of the phenomena of interest, for which the case of single subject/datum analysis is its most extreme. In order to approach these problems we chose to use Bayesian Inference in the context of the Decision Theory under Uncertainty, computationally implemented under the Decision Support Systems framework. Both technologies produce complex data, based on the interpretation of differences between images from the response to a given stimulus and the control situation. The result of this work is the development of two decision support systems, called HTself and Dotslashen, to analyze microarray and functional magnetic resonance imaging data, respectively; and the underling mathematical and computational methods. These systems use the rational knowledge from normative databases implemented in specific mathematical models, overcoming the problem of small sample size. Finally, in this work it is described applications to real problems in order to stress the utility for Molecular Biology and Functional Neuroimaging of the developed decision support systems.
Sucrose is the major product of photosynthesis in many higher plants. It is transported from the source tissue through the phloem to various sink tissues to support plant growth, development and ...reproduction. Knowledge on the signal transduction pathways involved in sucrose synthesis in mature leaves is limited. Using a microarray approach, we analyzed the expression profiles of 1920 sugarcane genes encoding signal transduction elements, transcription factors and stress-related proteins. We used individuals from a population segregating for sugar content and gene expression profiles were obtained from seven individuals with highest and seven with lowest sugar content. Surprisingly, from the 24 differentially expressed genes, 19 were more expressed in plants containing low-sugar content. Three of these genes encoded 14-3-3 like proteins, which have been found to reduce sucrose phosphate synthase (SPS) activity. Another encoded an SNF1-related protein similar to a protein kinase that phosphorylates SPS
in vitro
making it a target for the interaction with 14-3-3 proteins. The up-regulation of eight stress related genes in the lower sugar content plants supports a view that sugar levels modulate a complex signal transduction network that seems to involve responses that are related to stress. Evidence that hormone signaling is related to the sucrose content was also found. These data reinforced the usefulness of genomic approaches to uncover how sucrose metabolism can be regulated in sugarcane.
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