Expression and regulatory roles of lncRNAs in G-CIMP-low vs G-CIMP-high Glioma: an in-silico analysis

BACKGROUND: Clinically relevant glioma subtypes, such as the glioma-CpG island methylator phenotype (G-CIMP), have been defined by epigenetics. In this study, the role of long non-coding RNAs in association with the poor-prognosis G-CMIP-low phenotype and the good-prognosis G-CMIP-high phenotype was investigated. Functional associations of lncRNAs with mRNAs and miRNAs were examined to hypothesize influencing factors of the aggressive phenotype. METHODS: RNA-seq data on 250 samples from TCGA\u27s Pan-Glioma study, quantified for lncRNA and mRNAs (GENCODE v28), were analyzed for differential expression between G-CIMP-low and G-CIMP-high phenotypes. Functional interpretation of the differential lncRNAs was performed by Ingenuity Pathway Analysis. Spearman rank order correlation estimates between lncRNA, miRNA, and mRNA nominated differential lncRNA with a likely miRNA sponge function. RESULTS: We identified 4371 differentially expressed features (mRNA = 3705; lncRNA = 666; FDR ≤ 5%). From these, the protein-coding gene TP53 was identified as an upstream regulator of differential lncRNAs PANDAR and PVT1 (p = 0.0237) and enrichment was detected in the development of carcinoma (p = 0.0176). Two lncRNAs (HCG11, PART1) were positively correlated with 342 mRNAs, and their correlation estimates diminish after adjusting for either of the target miRNAs: hsa-miR-490-3p, hsa-miR-129-5p. This suggests a likely sponge function for HCG11 and PART1. CONCLUSIONS: These findings identify differential lncRNAs with oncogenic features that are associated with G-CIMP phenotypes. Further investigation with controlled experiments is needed to confirm the molecular relationships

Noninvasive approaches to detect methylation-based markers to monitor gliomas

In this review, we summarize the current approaches used to detect glioma tissue-derived DNA methylation markers in liquid biopsy specimens with the aim to diagnose, prognosticate and potentially track treatment response and evolution of patients with gliomas

SIRT1 regulates Mxd1 during malignant melanoma progression

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, gamma H2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.FAPESP [2011/0166-38, 2011/12306-1, 2014/13663-0, 2015/07925-5, 2016/06488-3]DAAD [PKZ A/12/79134]FAPESP/BAYLAT [2012/51300-7]Univ Fed Sao Paulo UNIFESP, Dept Pharmacol, Ontogeny & Epigenet Lab, Sao Paulo, SP, BrazilUniv Sao Paulo, Ribeirao Preto Med Sch, Dept Genet, Ribeirao Preto, SP, BrazilFriedrich Alexander Univ Erlangen Nurnberg FAU, Inst Pathol, Expt Tumorpathol, Erlangen, GermanyFriedrich Alexander Univ Erlangen Nurnberg FAU, Dept Pediat & Adolescent Med, Erlangen, GermanyUniv Fed Sao Paulo UNIFESP, Dept Pharmacol, Ontogeny & Epigenet Lab, Sao Paulo, SP, BrazilFAPESP [2011/0166-38, 2011/12306-1, 2014/13663-0, 2015/07925-5, 2016/06488-3]DAAD [PKZ A/12/79134]FAPESP/BAYLAT [2012/51300-7]Web of Scienc

Comprehensive functional annotation of 77 prostate cancer risk loci.

Genome-wide association studies (GWAS) have revolutionized the field of cancer genetics, but the causal links between increased genetic risk and onset/progression of disease processes remain to be identified. Here we report the first step in such an endeavor for prostate cancer. We provide a comprehensive annotation of the 77 known risk loci, based upon highly correlated variants in biologically relevant chromatin annotations--we identified 727 such potentially functional SNPs. We also provide a detailed account of possible protein disruption, microRNA target sequence disruption and regulatory response element disruption of all correlated SNPs at r(2) ≥ 0.88%. 88% of the 727 SNPs fall within putative enhancers, and many alter critical residues in the response elements of transcription factors known to be involved in prostate biology. We define as risk enhancers those regions with enhancer chromatin biofeatures in prostate-derived cell lines with prostate-cancer correlated SNPs. To aid the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancers, as well as the transcription factor TCF7L2. We analyzed in depth three variants in risk enhancers, two of which show significantly altered androgen sensitivity in LNCaP cells. This includes rs4907792, that is in linkage disequilibrium (r(2) = 0.91) with an eQTL for NUDT11 (on the X chromosome) in prostate tissue, and rs10486567, the index SNP in intron 3 of the JAZF1 gene on chromosome 7. Rs4907792 is within a critical residue of a strong consensus androgen response element that is interrupted in the protective allele, resulting in a 56% decrease in its androgen sensitivity, whereas rs10486567 affects both NKX3-1 and FOXA-AR motifs where the risk allele results in a 39% increase in basal activity and a 28% fold-increase in androgen stimulated enhancer activity. Identification of such enhancer variants and their potential target genes represents a preliminary step in connecting risk to disease process

Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion

Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial–mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS–PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2's transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion

Molecular landscape of IDH-mutant primary astrocytoma Grade IV/glioblastomas

WHO 2016 classified glioblastomas into IDH-mutant and IDH-wildtype with the former having a better prognosis but there was no study on IDH-mutant primary glioblastomas only, as previous series included secondary glioblastomas. We recruited a series of 67 IDH-mutant primary glioblastomas/astrocytoma IV without a prior low-grade astrocytoma and examined them using DNA-methylation profiling, targeted sequencing, RNA sequencing and TERT promoter sequencing, and correlated the molecular findings with clinical parameters. The median OS of 39.4 months of 64 cases and PFS of 25.9 months of 57 cases were better than the survival data of IDH-wildtype glioblastomas and IDH-mutant secondary glioblastomas retrieved from datasets. The molecular features often seen in glioblastomas, such as EGFR amplification, combined +7/-10, and TERT promoter mutations were only observed in 6/53 (11.3%), 4/53 (7.5%), and 2/67 (3.0%) cases, respectively, and gene fusions were found only in two cases. The main mechanism for telomere maintenance appeared to be alternative lengthening of telomeres as ATRX mutation was found in 34/53 (64.2%) cases. In t-SNE analyses of DNA-methylation profiles, with an exceptional of one case, a majority of our cases clustered to IDH-mutant high-grade astrocytoma subclass (40/53; 75.5%) and the rest to IDH-mutant astrocytoma subclass (12/53; 22.6%). The latter was also enriched with G-CIMP high cases (12/12; 100%). G-CIMP-high status and MGMT promoter methylation were independent good prognosticators for OS (p = 0.022 and p = 0.002, respectively) and TP53 mutation was an independent poor prognosticator (p = 0.013) when correlated with other clinical parameters. Homozygous deletion of CDKN2A/B was not correlated with OS (p = 0.197) and PFS (p = 0.278). PDGFRA amplification or mutation was found in 16/59 (27.1%) of cases and was correlated with G-CIMP-low status (p = 0.010). Aside from the three well-known pathways of pathogenesis in glioblastomas, chromatin modifying and mismatch repair pathways were common aberrations (88.7% and 20.8%, respectively), the former due to high frequency of ATRX involvement. We conclude that IDH-mutant primary glioblastomas have better prognosis than secondary glioblastomas and have major molecular differences from other commoner glioblastomas. G-CIMP subgroups, MGMT promoter methylation, and TP53 mutation are useful prognostic adjuncts

A Distinct DNA Methylation Shift in a Subset of Glioma CpG Island Methylator Phenotypes during Tumor Recurrence

Glioma diagnosis is based on histomorphology and grading; however, such classification does not have predictive clinical outcome after glioblastomas have developed. To date, no bona fide biomarkers that significantly translate into a survival benefit to glioblastoma patients have been identified. We previously reported that the IDH mutant G-CIMP-high subtype would be a predecessor to the G-CIMP-low subtype. Here, we performed a comprehensive DNA methylation longitudinal analysis of diffuse gliomas from 77 patients (200 tumors) to enlighten the epigenome-based malignant transformation of initially lower-grade gliomas. Intra-subtype heterogeneity among G-CIMP-high primary tumors allowed us to identify predictive biomarkers for assessing the risk of malignant recurrence at early stages of disease. G-CIMP-low recurrence appeared in 9.5% of all gliomas, and these resembled IDH-wild-type primary glioblastoma. G-CIMP-low recurrence can be characterized by distinct epigenetic changes at candidate functional tissue enhancers with AP-1/SOX binding elements, mesenchymal stem cell-like epigenomic phenotype, and genomic instability. Molecular abnormalities of longitudinal G-CIMP offer possibilities to defy glioblastoma progression