Abstract
Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery ...of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.
Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for ...biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also ...important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and ...contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary carcinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low- and high-grade lung neuroendocrine neoplasms.
Abstract Aim of the study A vast majority of human malignancies are associated with ageing, and age is a strong predictor of cancer risk. Recently, DNA methylation-based marker of ageing, known as ...‘epigenetic clock’, has been linked with cancer risk factors. This study aimed to evaluate whether the epigenetic clock is associated with breast cancer risk susceptibility and to identify potential epigenetics-based biomarkers for risk stratification. Methods Here, we profiled DNA methylation changes in a nested case–control study embedded in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (n = 960) using the Illumina HumanMethylation 450K BeadChip arrays and used the Horvath age estimation method to calculate epigenetic age for these samples. Intrinsic epigenetic age acceleration (IEAA) was estimated as the residuals by regressing epigenetic age on chronological age. Results We observed an association between IEAA and breast cancer risk (OR, 1.04; 95% CI, 1.007–1.076, P = 0.016). One unit increase in IEAA was associated with a 4% increased odds of developing breast cancer (OR, 1.04; 95% CI, 1.007–1.076). Stratified analysis based on menopausal status revealed that IEAA was associated with development of postmenopausal breast cancers (OR, 1.07; 95% CI, 1.020–1.11, P = 0.003). In addition, methylome-wide analyses revealed that a higher mean DNA methylation at cytosine-phosphate-guanine (CpG) islands was associated with increased risk of breast cancer development (OR per 1 SD = 1.20; 95 %CI: 1.03–1.40, P = 0.02) whereas mean methylation levels at non-island CpGs were indistinguishable between cancer cases and controls. Conclusion Epigenetic age acceleration and CpG island methylation have a weak, but statistically significant, association with breast cancer susceptibility.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Abstract
One of the ground-breaking discoveries of the international cancer genome sequencing endeavours is the high frequency of mutational and non-mutational changes in epigenetic regulator genes ...(ERGs), which constitute a “genetic smoking gun” that epigenetic mechanisms lie in the very heart of cancer biology. However, functional importance of disruption of ERGs in tumorigenesis and cancer phenotype is poorly understood. We hypothesise that these genes are candidates to be drivers («epidrivers») of cancer onset and progression, thus regulating mechanisms underpinning cancer development. Moreover, deregulation of epidrivers may play a role in epithelial-to-mesenchymal transition (EMT) and emergence of cancer resilience. The overarching aim of this study was to identify and functionally characterize “Epidrivers” in tumorigenesis and cancer cell plasticity. To this end, we have set up novel epigenome-wide functional screens in human cultured cells (and organoids) combined with state-of-the-art genome-editing approach (based on CRISPR/Cas9 screen) and multiparametric phenotyping. We designed a custom-made lentiviral CRISPR library that consists of 1,649 gRNAs targeting all known ERGs (426 genes). Lentiviral CRISPR library was used to deliver the ERG gRNAs to target cells expressing the RNA-guided DNA endonuclease Cas9. Independent clones (derived from transduced breast and lung cancer cell lines constitutively expressing Cas9) are screened for acquisition of distinct features of transformed cells. The results of the identification of Epidriver genes based on the analysis of deregulation of core cellular processes, epigenome (ChIP-seq) as well as EMT and multiparametric phenotyping, will be presented.
Citation Format: Andrea Halaburkova, Vincent Cahais, Cyrille Cuenin, Rita Khoueiry, Maria Ouzounova, Akram Ghantous, Zdenko Herceg. Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4313.
Puberty is a critical developmental period of life characterized by marked physiological changes, including changes in the immune system and gut microbiota development. Exposure to inflammation ...induced by immune stressors during puberty has been found to stimulate central inflammation and lead to immune disturbance at distant sites from the gut; however, its enduring effects on gut immunity are not well explored. Therefore, in this study, we used a pubertal lipopolysaccharides (LPS)-induced inflammation mouse model to mimic pubertal exposure to inflammation and dysbiosis. We hypothesized that pubertal LPS-induced inflammation may cause long-term dysfunction in gut immunity by enduring dysregulation of inflammatory signaling and epigenetic changes, while prebiotic/probiotic intake may mitigate the gut immune system deregulation later in life. To this end, four-week-old female Balb/c mice were fed prebiotics/probiotics and exposed to LPS in the pubertal window. To better decipher the acute and enduring immunoprotective effects of biotic intake, we addressed the effect of treatment on interleukin (IL)-17 signaling related-cytokines and pathways. In addition, the effect of treatment on gut microbiota and epigenetic alterations, including changes in microRNA (miRNA) expression and DNA methylation, were studied. Our results revealed a significant dysregulation in selected cytokines, proteins, and miRNAs involved in key signaling pathways related to IL-17 production and function, including IL-17A and F, IL-6, IL-1β, transforming growth factor-β (TGF-β), signal transducer and activator of transcription-3 (STAT3), p-STAT3, forkhead box O1 (FOXO1), and miR-145 in the small intestine of adult mice challenged with LPS during puberty. In contrast, dietary interventions mitigated the lasting adverse effects of LPS on gut immune function, partly through epigenetic mechanisms. A DNA methylation analysis demonstrated that enduring changes in gut immunity in adult mice might be linked to differentially methylated genes, including Lpb, Rorc, Runx1, Il17ra, Rac1, Ccl5, and Il10, involved in Th17 cell differentiation and IL-17 production and signaling. In addition, prebiotic administration prevented LPS-induced changes in the gut microbiota in pubertal mice. Together, these results indicate that following a healthy diet rich in prebiotics and probiotics is an optimal strategy for programming immune system function in the critical developmental windows of life and controlling inflammation later in life.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract
Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular ...circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance.
Background
DNA isolation from formalin-fixed paraffin-embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated ...DNA is often compromised, and commercial kits are used to overcome this to some extent.
Methods
We developed a new protocol (IARCp) to improve the quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp’s performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin® DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit.
Results
Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6–394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments by spectrophotometer, fluorometer, and real-time PCR assay.
Conclusion
Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. This protocol does not require the use of any commercial kits or phenol for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle-income countries.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
To study the interaction between HIV and other carcinogenic infections in conjunctival squamous cell carcinoma (SCC), we evaluated the presence of a broad spectrum of human viruses in conjunctiva ...specimens. Beta Human papillomavirus (HPV; n = 46), gamma HPV (n = 52), polyomaviruses (n = 12) and herpes viruses (n = 3) was determined in DNA extracted from 67 neoplastic and 55 non‐neoplastic conjunctival tissues of HIV‐positive and HIV negative subjects by Luminex‐based assays. Next‐generation sequencing (NGS) was also used to further characterize the presence of cutaneous HPVs. Detection of beta‐2 HPV infections was associated with the risk of neoplasia (adjusted odds ratio aOR 3.0; 95% confidence interval CI 1.3‐6.8), regardless of HIV status (HIV positive, aOR 2.6, 95% CI 0.9‐7.7; HIV negative, aOR 3.5, 95% CI 0.9‐14.4). EBV was strongly associated with the risk of neoplasia (aOR 12.0, 95% CI 4.3‐33.5; P < .01) mainly in HIV individuals (HIV positive, aOR 57.5; 95% CI: 10.1‐327.1; HIV negative aOR 2.6; 95% CI: 0.2‐34.7). NGS allowed to identify 13 putative novel HPVs in cases and controls. Our findings suggest a role of beta HPV types and EBV, in conjunctival SCC. However, additional studies of viral expression in tumor tissue are required to confirm the causal association.
What's new?
Risk of conjunctival squamous cell carcinoma (SCC) is strongly associated with ultraviolet radiation exposure and immune suppression. In Africa, SCC incidence has also been affected by the spread of HIV and may be impacted by other viral infections. Here, analyses of a large number of viruses in conjunctival non‐malignant and malignant tissues from patients in Southern Uganda reveal associations between conjunctival SCC and cutaneous beta human papillomavirus (HPV) and Epstein‐Barr virus (EBV) infection. Beta‐2 HPV infection was linked to neoplasia risk regardless of HIV status, whereas EBV was associated with neoplasia primarily in HIV‐infected individuals.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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