Genome-wide Methylation Changes Research Articles

P06-17 Assessment of genome-wide methylation changes caused by TiO2 nanoparticles on human intestinal caco-2 cells

P06-17 Assessment of genome-wide methylation changes caused by TiO2 nanoparticles on human intestinal caco-2 cells

DNA methylation analysis reveals local changes in resistant and susceptible soybean lines in response to Phytophthora sansomeana.

Phytophthora sansomeana is an emerging oomycete pathogen causing root rot in many agricultural species including soybean. However, as of now, only one potential resistance gene has been identified in soybean, and our understanding of how genetic and epigenetic regulation in soybean contributes to responses against this pathogen remains largely unknown. In this study, we performed whole genome bisulfite sequencing (WGBS) on two soybean lines, Colfax (resistant) and Williams 82 (susceptible), in response to P. sansomeana at two time points: 4 and 16 hours post-inoculation to compare their methylation changes. Our findings revealed that there were no significant changes in genome-wide CG, CHG (H = A, T, or C), and CHH methylation. However, we observed local methylation changes, specially an increase in CHH methylation around genes and transposable elements (TEs) after inoculation, which occurred earlier in the susceptible line and later in the resistant line. After inoculation, we identified differentially methylated regions (DMRs) in both Colfax and Williams 82, with a predominant presence in TEs. Notably, our data also indicated that more TEs exhibited changes in their methylomes in the susceptible line compared to the resistant line. Furthermore, we discovered 837 DMRs within or flanking 772 differentially expressed genes (DEGs) in Colfax and 166 DMRs within or flanking 138 DEGs in Williams 82. These DEGs had diverse functions, with Colfax primarily showing involvement in metabolic process, defense response, plant and pathogen interaction, anion and nucleotide binding, and catalytic activity, while Williams 82 exhibited a significant association with photosynthesis. These findings suggest distinct molecular responses to P. sansomeana infection in the resistant and susceptible soybean lines.

DNA Methylation Changes in Blood Cells of Fibromyalgia and Chronic Fatigue Syndrome Patients.

Fibromyalgia (FM) and Chronic Fatigue Syndrome (CFS) affect 0.4% and 1% of society, respectively, and the prevalence of these pain syndromes is increasing. To date, no strong association between these syndromes and the genetic background of affected individuals has been shown. Therefore, it is plausible that epigenetic changes might play a role in the development of these syndromes. Three previous studies have attempted to elaborate the involvement of genome-wide methylation changes in blood cells in the development of fibromyalgia and chronic fatigue syndrome. These studies included 22 patients with fibromyalgia and 127 patients with CFS, and the results of the studies were largely discrepant. Contradicting results of those studies may be attributed to differences in the omics data analysis approaches used in each study. We reanalyzed the data collected in these studies using an updated and coherent data-analysis framework. Overall, the methylation changes that we observed overlapped with previous results only to some extent. However, the gene set enrichment analyses based on genes annotated to methylation changes identified in each of the analyzed datasets were surprisingly coherent and uniformly associated with the physiological processes that, when affected, may result in symptoms characteristic of fibromyalgia and chronic fatigue syndrome. Methylomes of the blood cells of patients with FM and CFS in three independent studies have shown methylation changes that appear to be implicated in the pathogenesis of these syndromes.

Herbivory induced methylation changes in the Lombardy poplar: A comparison of results obtained by epiGBS and WGBS.

DNA cytosine methylation is an epigenetic mechanism involved in regulation of plant responses to biotic and abiotic stress and its ability to change can vary with the sequence context in which a cytosine appears (CpG, CHG, CHH, where H = Adenine, Thymine, Cytosine). Quantification of DNA methylation in model plant species is frequently addressed by Whole Genome Bisulfite Sequencing (WGBS), which requires a good-quality reference genome. Reduced Representation Bisulfite Sequencing (RRBS) is a cost-effective potential alternative for ecological research with limited genomic resources and large experimental designs. In this study, we provide for the first time a comprehensive comparison between the outputs of RRBS and WGBS to characterize DNA methylation changes in response to a given environmental factor. In particular, we used epiGBS (recently optimized RRBS) and WGBS to assess global and sequence-specific differential methylation after insect and artificial herbivory in clones of Populus nigra cv. 'italica'. We found that, after any of the two herbivory treatments, global methylation percentage increased in CHH, and the shift was detected as statistically significant only by epiGBS. As regards to loci-specific differential methylation induced by herbivory (cytosines in epiGBS and regions in WGBS), both techniques indicated the specificity of the response elicited by insect and artificial herbivory, together with higher frequency of hypo-methylation in CpG and hyper-methylation in CHH. Methylation changes were mainly found in gene bodies and intergenic regions when present at CpG and CHG and in transposable elements and intergenic regions at CHH context. Thus, epiGBS succeeded to characterize global, genome-wide methylation changes in response to herbivory in the Lombardy poplar. Our results support that epiGBS could be particularly useful in large experimental designs aimed to explore epigenetic changes of non-model plant species in response to multiple environmental factors.

On the thermodynamics of DNA methylation process

DNA methylation is an epigenetic mechanism that plays important roles in various biological processes including transcriptional and post-transcriptional regulation, genomic imprinting, aging, and stress response to environmental changes and disease. Consistent with thermodynamic principles acting within living systems and the application of maximum entropy principle, we propose a theoretical framework to understand and decode the DNA methylation process. A central tenet of this argument is that the probability density function of DNA methylation information-divergence summarizes the statistical biophysics underlying spontaneous methylation background and implicitly bears on the channel capacity of molecular machines conforming to Shannon’s capacity theorem. On this theoretical basis, contributions from the molecular machine (enzyme) logical operations to Gibb entropy (S) and Helmholtz free energy (F) are intrinsic. Application to the estimations of S on datasets from Arabidopsis thaliana suggests that, as a thermodynamic state variable, individual methylome entropy is completely determined by the current state of the system, which in biological terms translates to a correspondence between estimated entropy values and observable phenotypic state. In patients with different types of cancer, results suggest that a significant information loss occurs in the transition from differentiated (healthy) tissues to cancer cells. This type of analysis may have important implications for early-stage diagnostics. The analysis of entropy fluctuations on experimental datasets revealed existence of restrictions on the magnitude of genome-wide methylation changes originating by organismal response to environmental changes. Only dysfunctional stages observed in the Arabidopsis mutant met1 and in cancer cells do not conform to these rules.

Multimodal analysis of genome-wide methylation, copy number aberrations, and end motif signatures enhances detection of early-stage breast cancer.

Breast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes. Here, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects. We identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity. Our findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer.

Abstract 1747: Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

Abstract Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 412; RC, 12941 ± 1023; n=2; p=2.10e-8].Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p<.0001]. Both primary and recurrent GBM cells were subject to select MAT2A and AHCY inhibitor compounds and EC50s were determined. Recurrent cells demonstrated a vulnerability to TMZ-generated cell death both by genetically knocking down and pharmacologically inhibiting MAT2A and AHCY. MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to augment the practically negligible rate of remission for GBM and improve treatment outcomes for cancer patients. Citation Format: Emma Rowland, Jordan Walter, Anna Jermakowicz, Robert Suter, Rebecca Riggins, Nagi Ayad. Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1747.

337 Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

OBJECTIVES/GOALS: Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. METHODS/STUDY POPULATION: Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. RESULTS/ANTICIPATED RESULTS: Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 0.012; RC, 12925 ± 0.023; n=2; p=2.10e-8]. Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p DISCUSSION/SIGNIFICANCE: MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to increase remission and reduce recurrence for GBM patients.

Genome-Wide Methylation Changes Associated with Replicative Senescence and Differentiation in Endothelial and Bone Marrow Mesenchymal Stromal Cells.

Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/β-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent cells.

Genome-wide DNA methylation predicts environmentally driven life history variation in a marine fish.

Epigenetic modifications are thought to be one of the molecular mechanisms involved in plastic adaptive responses to environmental variation. However, studies reporting associations between genome-wide epigenetic changes and habitat-specific variations in life history traits (e.g., lifespan, reproduction) are still scarce, likely due to the recent application of methylome resequencing methods to non-model species. In this study, we examined associations between whole genome DNA methylation and environmentally driven life history variation in 2 lineages of a marine fish, the capelin (Mallotus villosus), from North America and Europe. In both lineages, capelin harbor 2 contrasting life history tactics (demersal vs. beach-spawning). Performing whole genome and methylome sequencing, we showed that life history tactics are associated with epigenetic changes in both lineages, though the effect was stronger in European capelin. Genetic differentiation between the capelin harboring different life history tactics was negligible, but we found genome-wide methylation changes in both lineages. We identified 9,125 European and 199 North American differentially methylated regions (DMRs) due to life history. Gene ontology (GO) enrichment analysis for both lineages revealed an excess of terms related to neural function. Our results suggest that environmental variation causes important epigenetic changes that are associated with contrasting life history tactics in lineages with divergent genetic backgrounds, with variable importance of genetic variation in driving epigenetic variation. Our study emphasizes the potential role of genome-wide epigenetic variation in adaptation to environmental variation.

Oil and hypoxia alter DNA methylation and transcription of genes related to neurological function in larval Cyprinodon variegatus

DNA methylation is an important epigenetic mark involved in modulating transcription. While multiple studies document the ability of environmental stressors to alter methylation patterns, there is little information regarding the effects of oil and hypoxia on the methylome. Oil and hypoxic stress are threats in coastal ecosystems, which act as nursery habitats for developing fish. To explore the methylation altering effects of oil and hypoxia on developing fish, we exposed larval Cyprinodon variegatus to oil, hypoxia, or both for 48 h followed by 48 h of depuration in clean, normoxic conditions. We then used immunoprecipitation coupled with high-throughput sequencing (MeDIP seq) to evaluate genome-wide methylation changes. We also performed RNA seq to associate methylation and altered transcription. Oil and hypoxia together elicited greater impacts to methylation than either stressor individually. Additionally, the oil+hypoxia treatment exhibited an overlap between differentially methylated regions and differential gene expression at 20 loci. Functional analyses of these loci revealed enrichment of processes related to neurological function and development. Two neurological genes (slc1a2, asxl2) showed altered methylation of promoter CpG islands and transcriptional changes, suggesting epigenetic modulation of gene expression. Our results suggest a possible mechanism explaining altered behavior patterns noted in fish following oil exposure.

Traffic-related air pollution and genome-wide DNA methylation: A randomized, crossover trial

BackgroundTraffic-related air pollution (TRAP) has been associated with changes in gene-specific DNA methylation. However, few studies have investigated impact of TRAP exposure on genome-wide DNA methylation in circulating blood of human. ObjectiveTo explore the association between TRAP exposure and genome-wide DNA methylation. MethodsWe conducted a randomized, crossover exposure trial among 35 healthy adults in Shanghai, China. All subjects were randomly allocated to a traffic-free park or a main road for consecutive 4 h, respectively. Blood genome-wide DNA methylation after each exposure session was measured by the Infinium Methylation EPIC BeadChip (850K). The differentially methylated CpGs loci associated with TRAP exposure were identified using linear mixed-effect model. ResultsThe average concentrations of traffic-related air pollutants including black carbon, ultrafine particles, carbon dioxide, and nitrogen dioxide were 2–3 times higher in the road compared to those in the park. Methylation levels of 68 CpG loci were significantly changed (false discovery rate < 0.05) following TRAP exposure, among which 49 were hypermethylated and 19 were hypomethylated. The annotated genes based on the differential CpGs loci were related to pathways in cardiovascular signaling, cytokine signaling, immune response, nervous system signaling, and metabolism. ConclusionsWe found that TRAP exposure was associated with DNA methylation in dozens of genes concerning cardiometabolic health. This trial for the first-time profiled genome-wide methylation changes induced by TRAP exposure using the 850K assay, providing epigenetic insights in understanding the cardiometabolic effects of TRAP exposure.

DNA methylation dynamics associated with long-term isolation of simulated space travel

Long-term isolation is one of the risk factors that astronauts will encounter in spaceflight. At present, few researches have explored DNA methylation dynamics during long-term isolation. In this study, using time series DNA methylation data from “Mars-500” mission, we conducted a multi-step analysis to investigate the characteristics and dynamic patterns of DNA methylation as well as their functional insights during long-term isolation. The results showed that genome-wide methylation changes were minimal. In the six identified DNA methylation dynamic patterns, most of significantly fluctuating CpG sites could be returned to the baseline in post-isolation, and the remaining sites persistently decreased during isolation. Next, functional enrichment analysis of genes with each pattern revealed strong functional specificity. Some patterns were also significantly associated with nervous system diseases, digestive system diseases and cancers. In conclusion, the DNA methylation dynamics during long-term isolation have great functional significance, and might be helpful for protection of astronaut health.

P083 An integrative analysis of DNA methylation and RNA-Seq data in human adipose-stem cells of Crohn’s Disease patients with different clinical activity

Abstract Background Crohn’s disease (CD) is characterized by the expansion of mesenteric fat attached to the inflamed segments of the intestine, also known as “creeping fat” which seems to be directly related to disease activity. Our group revealed that adipose-stem cells (hASCs) isolated from the creeping fat of CD subjects are dysfunctional (showing a high inflammatory profile, high invasive and phagocytic capacities, and worse immunosuppressive properties); and this dysfunction is maintained even in hASCs isolated from CD subjects in remission of the disease.1 Methods 1) Culture and characterization of hASCs isolated from adipose tissue biopsies of active CD, inactive CD, and healthy subjects (n=25). Visceral origin: creeping fat in active CD subjects and mesenteric fat in CD subjects in remission and healthy subjects. 2) Comparative DNA methylome analysis in hASCs isolated from the included population (Infinium Human-Methylation EPIC (850K) BeadChip; Illumina, Inc). 3) Transcriptomic study in hASCs isolated from the included population (Illumina HiSeq 2500 system). 4) Bioinformatic analysis &amp; data integration. Results Methylation and transcriptomic studies revealed a multi-omic profile of hASCs isolated from CD subjects compared to control individuals (Heatmaps Fig 1A). Indeed, the integration of genome-wide methylation changes and gene expression differences revealed the main candidate’s genes (Figure 1B). The most significant upregulated gene in CD was Mab-21 like 2 (MAB21L2) (Fig 1B-C). This gene encodes a downstream target of transforming growth factor-beta signaling. MAB21L2 was validated in an independent cohort of hASC isolated from control, active, and inactive CD subjects (n=7/each group) using qPCR. In agreement, MAB21L2 gene expression was significantly higher in hASCs isolated from the active and inactive group than in the control group (Fig 1D). Another interesting candidate gene found was tyrosine kinase 2 (TYK2) which is significantly upregulated in active CD subjects. This gene promulgates cytokine signals by phosphorylating receptor subunits. It is also a component of both the type I and III interferon signaling pathways.2 Finally, another gene upregulated in remission of the disease is the calcium voltage-gated channel subunit alpha 1H (CACNA1H), which has been involved in low-grade inflammation and has been proposed as an important player in IBD during the remission phase.3 Conclusion Integration of data revealed genes involved in the dysregulation of hASCs associated with CD and genes related to disease remission. Reference 1. Serena C Stem Cell Rep 2017 9(4):1109–23; 2De Vries LCS JCC 2021 617-30; 3Picard E Br J Pharmacol 2019;176:950–63

Longitudinal genome-wide DNA methylation changes in response to kidney failure replacement therapy

Chronic kidney disease (CKD) is an emerging public health priority associated with high mortality rates and demanding treatment regimens, including life-style changes, medications or even dialysis or renal transplantation. Unavoidably, the uremic milieu disturbs homeostatic processes such as DNA methylation and other vital gene regulatory mechanisms. Here, we aimed to investigate how dialysis or kidney transplantation modifies the epigenome-wide methylation signature over 12 months of treatment. We used the Infinium HumanMethylation450 BeadChip on whole blood samples from CKD-patients undergoing either dialysis (n = 11) or kidney transplantation (n = 12) and 24 age- and sex-matched population-based controls. At baseline, comparison between patients and controls identified several significant (PFDR < 0.01) CpG methylation differences in genes with functions relevant to inflammation, cellular ageing and vascular calcification. Following 12 months, the global DNA methylation pattern of patients approached that seen in the control group. Notably, 413 CpG sites remained differentially methylated at follow-up in both treatment groups compared to controls. Together, these data indicate that the uremic milieu drives genome-wide methylation changes that are partially reversed with kidney failure replacement therapy. Differentially methylated CpG sites unaffected by treatment may be of particular interest as they could highlight candidate genes for kidney disease per se.

Genome-wide host methylation profiling of anal and cervical carcinoma.

HPV infection results in changes in host gene methylation which, in turn, are thought to contribute to the neoplastic progression of HPV-associated cancers. The objective of this study was to identify joint and disease-specific genome-wide methylation changes in anal and cervical cancer as well as changes in high-grade pre-neoplastic lesions. Formalin-fixed paraffin-embedded (FFPE) anal tissues (n = 143; 99% HPV+) and fresh frozen cervical tissues (n = 28; 100% HPV+) underwent microdissection, DNA extraction, HPV genotyping, bisulfite modification, DNA restoration (FFPE) and analysis by the Illumina HumanMethylation450 Array. Differentially methylated regions (DMR; t test q<0.01, 3 consecutive significant CpG probes and mean Δβ methylation value>0.3) were compared between normal and cancer specimens in partial least squares (PLS) models and then used to classify anal or cervical intraepithelial neoplasia-3 (AIN3/CIN3). In AC, an 84-gene PLS signature (355 significant probes) differentiated normal anal mucosa (NM; n = 9) from AC (n = 121) while a 36-gene PLS signature (173 significant probes) differentiated normal cervical epithelium (n = 10) from CC (n = 9). The CC progression signature was validated using three independent publicly available datasets (n = 424 cases). The AC and CC progression PLS signatures were interchangeable in segregating normal, AIN3/CIN3 and AC and CC and were found to include 17 common overlapping hypermethylated genes. Moreover, these signatures segregated AIN3/CIN3 lesions similarly into cancer-like and normal-like categories. Distinct methylation changes occur across the genome during the progression of AC and CC with overall similar profiles and add to the evidence suggesting that HPV-driven oncogenesis may result in similar non-random methylomic events. Our findings may lead to identification of potential epigenetic drivers of HPV-associated cancers and also, of potential markers to identify higher risk pre-cancerous lesions.

Changes in Methylation across Structural and MicroRNA Genes Relevant for Progression and Metastasis in Colorectal Cancer.

Simple SummaryChanges in the expression of key molecules such as microRNAs (miRs) can drive or suppress carcinogenesis and metastasis. A number of established transcriptional and genetic mechanisms regulate miR gene expression, but methylation/epigenetics have been analyzed less. Here, we systematically evaluated genome-wide methylation changes, focusing on miR, downstream targets, and further genes relevant for metastasis in colorectal cancers (CRC), including CpG islands, open seas, and north and south shore regions. A number of miRs deregulated during CRC progression/metastasis were significantly affected by methylation changes, especially within CpG islands and open seas. Several of these miRs cooperate in cancer- and metastasis-related pathways, while methylation changes otherwise primarily affect protein-coding genes. Our results highlight alternative routes to the transcriptional and genetic control of miR and further gene expression relevant for CRC progression and metastasis by changes in gene methylation. They also bear important therapeutic implications since drugs that alter methylation states are now in clinical use.MiRs are important players in cancer and primarily genetic/transcriptional means of regulating their gene expression are known. However, epigenetic changes modify gene expression significantly. Here, we evaluated genome-wide methylation changes focusing on miR genes from primary CRC and corresponding normal tissues. Differentially methylated CpGs spanning CpG islands, open seas, and north and south shore regions were evaluated, with the largest number of changes observed within open seas and islands. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed several of these miRs to act in important cancer-related pathways, including phosphatidylinositol 3-kinase (PI3K)–protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. We found 18 miR genes to be significantly differentially methylated, with MIR124-2, MIR124-3, MIR129-2, MIR137, MIR34B, MIR34C, MIR548G, MIR762, and MIR9-3 hypermethylated and MIR1204, MIR17, MIR17HG, MIR18A, MIR19A, MIR19B1, MIR20A, MIR548F5, and MIR548I4 hypomethylated in CRC tumor compared with normal tissue, most of these miRs having been shown to regulate steps of metastasis. Generally, methylation changes were distributed evenly across all chromosomes with predominance for chromosomes 1/2 and protein-coding genes. Interestingly, chromosomes abundantly affected by methylation changes globally were rarely affected by methylation changes within miR genes. Our findings support additional mechanisms of methylation changes affecting (miR) genes that orchestrate CRC progression and metastasis.

METHYLATION PROFILING OF DAPK1, LRPPRC, RAB6C, AND ZNF471 IN SALIVA AND TISSUES AS NOVEL EPIGENETIC MARKERS OF ORAL SQUAMOUS CELL CARCINOMA

<h3>Background</h3> Despite rapid progress in the diagnosis and treatment of oral squamous cell carcinoma (OSCC), the survival rates remain poor. Early identification of premalignant lesions is critical in reducing mortality. Epigenetic changes, such as aberrant DNA methylation resulting in altered gene expression, contribute to oral carcinogenesis. <h3>Objective</h3> To identify aberrantly methylated genes in saliva and tissue of OSCC patients as novel biomarkers of patients' prognosis. <h3>Methods</h3> Genome-wide methylation changes were identified by differential methylation hybridization microarray. The results were compared against datasets from TCGA-HNSCC. Promoter sequences of <i>DAPK1, LRPPRC, RAB6C</i>, and <i>ZNF471</i> were validated by bisulfite genome sequencing. The methylation status of these genes was tested in saliva and tissue specimens by targeted next-generation sequence. <h3>Results</h3> Promoter sequences of <i>DAPK1, LRPPRC, RAB6C,</i> and <i>ZNF471</i> were significantly hypermethylated in tumors compared with matched normal tissue (<i>P</i> < .0001). Sensitivity and specificity of methylation markers for detection of OSCC were in the range of 70% to 100%, with AUC 0.83 and above. Salivary DNA methylation levels were higher in premalignant lesions and OSCC in comparison to healthy controls (<i>P</i> < .05). The r-value between premalignant tissue versus saliva and OSCC versus saliva were in the range of 0.6297 to 0.8023 and 0.7823 to 0.9419, respectively. <h3>Conclusions</h3> Our data confirms that the methylation pattern of these candidate genes are significantly higher in premalignant and OSCC tissues and saliva. Thus, the methylation profiling of these candidate genes has the potential to serve as novel non-invasive markers of OSCC.

An Epigenome Wide Association Study Reveals Timing of Physical Activity During Pregnancy Is Associated With Placental DNA Methylation

ObjectivesPhysical activity (PA) prior to and during pregnancy may influence offspring health through epigenetic modifications in the placenta. Prior studies had a single PA assessment in pregnancy limiting the ability to account for PA changes during pregnancy. We hypothesized that timing of PA may be associated with differential methylation and evaluated associations between multiple assessments of PA and genome-wide methylation changes in the placenta. MethodsPlacental tissues were obtained at delivery and DNA methylation was measured using the Illumina Human Methylation450 Beadchip for 301 mothers in the NICHD Fetal Growth Studies–Singleton cohort. Total PA (metabolic equivalent minutes/week) was assessed using the Pregnancy PA Questionnaire targeted for 8–13 (visit 0), 16–22 (visit 1), 24–29 (visit 2), 30–33 (visit 3), 34–37 (visit 4), 38–41 (visit 5) weeks’ gestation. For associations of PA at each visit with methylation, we conducted linear regression adjusting for potential confounders such as maternal age, race/ethnicity, pre-pregnancy body mass index. Genes annotating significant CpG sites (false discovery rate adjusted P < 0.05) were queried for enrichment of functional pathways using Ingenuity Pathway Analysis. ResultsPA in the 12 months prior to visit 0 was not significantly associated with methylation, whereas PA since last visit for visits 1–5 were associated with methylation of 1, 0, 2, 29, 30 CpG sites, respectively (P values ranging from 3.07 × 10–9 to 3.35 × 10–6). Thirteen CpG sites significantly related to PA overlapped at visits 4 & 5, with the most significant associations at cg21385047 located in sphingosine-1-phosphate receptor 1 (S1PR1, P = 3.07 × 10–9, P = 7.45 × 10–9, respectively). Five enriched pathways overlapped at visits 4 & 5 (P < 0.05): inositol pyrophosphates biosynthesis, gustation pathway, choline biosynthesis III, cAMP-mediated signaling, G-protein coupled receptor signaling. ConclusionsFindings suggest that PA during pregnancy is associated with placental DNA methylation changes at loci potentially related to cardiovascular and neurological system development/function. If replicated, our findings could shed light onto the mechanisms underlying changes in offspring epigenetic profile associated with maternal PA. Funding SourcesEunice Kennedy Shriver National Institute of Child Health and Human Development.

Bortezomib induces methylation changes in neuroblastoma cells that appear to play a significant role in resistance development to this compound

The anticancer activity of bortezomib (BTZ) has been increasingly studied in a number of indications and promising results for the use of this treatment have been shown in neuroblastoma. As BTZ treatment is usually administered in cycles, the development of resistance and side effects in patients undergoing therapy with BTZ remains a major challenge for the clinical usage of this compound. Common resistance development also means that certain cells are able to survive BTZ treatment and bypass molecular mechanisms that render BTZ anticancer activity. We studied the methylome of neuroblastoma cells that survived BTZ treatment. Our results indicate that BTZ induces pronounced genome wide methylation changes in cells which recovered from the treatment. Functional analyses of identified methylation changes demonstrated they were involved in key cancer pathology pathways. These changes may allow the cells to bypass the primary anticancer activity of BTZ and develop a treatment resistant and proliferative phenotype. To study whether cells surviving BTZ treatment acquire a proliferative phenotype, we repeatedly treated cells which recovered from the first round of BTZ treatment. The repetitive treatment led to induction of the extraordinary proliferative potential of the cells, that increased with subsequent treatments. As we did not observe similar effects in cells that survived treatment with lenalidomide, and non-treated cells cultured under the same experimental conditions, this phenomenon seems to be BTZ specific. Overall, our results indicate that methylation changes may play major role in the development of BTZ resistance.