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Background: Cancer is the leading cause of disease-related mortality in children. Causes of leukemia, the most common form, are largely unknown. Growing evidence points to an origin in-utero, when global redistribution of DNA methylation occurs driving tissue differentiation., Methods: Epigenome-wide DNA methylation was profiled in surrogate (blood) and target (bone marrow) tissues at birth, diagnosis, remission and relapse of pediatric pre-B acute lymphoblastic leukemia (pre-B ALL) patients. Double-blinded analyses was performed between prospective cohorts extending from birth to diagnosis and retrospective studies backtracking from clinical disease to birth. Validation was carried out using independent technologies and populations., Results: The imprinted and immuno-modulating VTRNA2-1 was hypermethylated (FDR<0.05) at birth in nested cases relative to controls in all tested populations (totaling 317 cases and 483 controls), including European and Hispanic ancestries. VTRNA2-1 methylation was stable over follow-up years after birth and across surrogate, target and other tissues (n=5,023 tissues; 30 types). When profiled in leukemic tissues from two clinical cohorts (totaling 644 cases), VTRNA2-1 methylation exhibited higher levels at diagnosis relative to controls, it reset back to normal levels at remission, and then re-increased to above control levels at relapse. Hypermethylation was significantly associated with worse pre-B ALL patient survival and with reduced VTRNA2-1 expression (n=2,294 tissues; 26 types), supporting a functional and translational role for VTRNA2-1 methylation., Conclusion: This study provides proof-of-concept to detect at birth epigenetic precursors of pediatric pre-B ALL. These alterations were reproducible with different technologies, in three continents and in two ethnicities, and can offer biomarkers for early detection and prognosis as well as actionable targets for therapy., (© 2024. The Author(s).)

Epigenetic alterations are ubiquitous across human malignancies. Thus, functional characterization of epigenetic events deregulated by environmental pollutants should enhance our understanding of the mechanisms of carcinogenesis and inform preventive strategies. Recent reports showing the presence of known cancer-driving mutations in normal tissues have sparked debate on the importance of non-mutational stressors potentially acting as cancer promoters. Here, we aimed to test the hypothesis that the presence of mutations in p53, a commonly mutated gene in human malignancies, may influence cellular response to an environmental non-mutagenic agent, potentially involving epigenetic mechanism. We used the CRISPR-Cas9 system to generate knockouts of p53 in MCF7 and T47D breast cancer cell lines and characterized DNA methylome changes by targeted pyrosequencing and methylome-wide Infinium MethylationEPIC BeadChip arrays after exposure to sodium arsenite, a well-established human carcinogen with documented effects on the epigenome. We found that the knockout of p53 alone was associated with extensive alterations in DNA methylation content, with predominant CpG hypermethylation concurrent with global demethylation, as determined by LINE-1 repetitive element pyrosequencing. While exposure to sodium arsenite induced little to no effects in parental cell lines, mutant cells, upon treatment with sodium arsenite, exhibited a markedly altered response in comparison to their wild-type counterparts. We further performed genome regional analyses and found that differentially methylated regions (DMRs) associated with exposure to sodium arsenite map to genes involved in chromatin remodeling and cancer development. Reconstitution of wild-type p53 only partially restored p53-mutant-specific differential methylation states in response to sodium arsenite exposure, which may be due to the insufficient reconstitution of p53 function, or suggestive of a potential exposure-specific epigenetic memory. Together, our results revealed wide-spread epigenetic alterations associated with p53 mutation that influence cellular response to sodium arsenite exposure, which may constate an important epigenetic mechanism by which tumour promoting agents synergize with driver mutations in cancer promotion., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Chronic arsenic exposure can lead to various health issues, including cancer. Concerns have been mounting about the enhancement of arsenic toxicity through co-exposure to various prevalent lifestyle habits. Smokeless tobacco products are commonly consumed in South Asian countries, where their use frequently co-occurs with exposure to arsenic from contaminated groundwater. To decipher the in vitro molecular and cellular responses to arsenic and/or smokeless tobacco, we performed temporal multi-omics analysis of the transcriptome and DNA methylome remodelling in exposed hTERT-immortalized human normal oral keratinocytes (NOK), as well as arsenic and/or smokeless tobacco genotoxicity and mutagenicity investigations in NOK cells and in human p53 knock-in murine embryonic fibroblasts (Hupki MEF). RNAseq results from acute exposures to arsenic alone and in combination with smokeless tobacco extract revealed upregulation of genes with roles in cell cycle changes, apoptosis and inflammation responses. This was in keeping with global DNA hypomethylation affecting genes involved in the same processes in response to chronic treatment in NOK cells. At the phenotypic level, we observed a dose-dependent decrease in NOK cell viability, induction of DNA damage, cell cycle changes and increased apoptosis, with the most pronounced effects observed under arsenic and SLT co-exposure conditions. Live-cell imaging experiments indicated that the DNA damage likely resulted from induction of apoptosis, an observation validated by a lack of exome-wide mutagenesis in response to chronic exposure to arsenic and/or smokeless tobacco. In sum, our integrative omics study provides novel insights into the acute and chronic responses to arsenic and smokeless tobacco (co-)exposure, with both types of responses converging on several key mechanisms associated with cancer hallmark processes. The generated rich catalogue of molecular programs in oral cells regulated by arsenic and smokeless tobacco (co-)exposure may provide bases for future development of biomarkers for use in molecular epidemiology studies of exposed populations at risk of developing oral cancer., Competing Interests: DECLARATION OF COMPETING INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

UVB significantly impacts the occurrence of cutaneous disorders, ranging from inflammatory to neoplastic diseases. Polyphenols derived from plants have been found to exhibit photoprotective effects against various factors that contribute to skin cancer. During the fermentation of the polyphenol-enriched blueberry preparation (PEBP), small oligomers of polyphenols were released, thus enhancing their photoprotective effects. This study aimed to investigate the protective effects of PEBP on UVB-induced skin inflammation. Topical preparations of polyphenols were applied to the skin of dorsally shaved mice. Mice were subsequently exposed to UVB and were sacrificed 90 min after UVB exposure. This study revealed that pretreatment with PEBP significantly inhibited UVB-induced recruitment of mast and neutrophil cells and prevented the loss of skin thickness. Furthermore, the findings show that PEBP treatment resulted in the downregulation of miR-210, 146a, and 155 and the upregulation of miR-200c and miR-205 compared to the UVB-irradiated mice. Additionally, PEBP was found to reduce the expression of IL-6, IL-1β, and TNFα, inhibiting COX-2 and increasing IL-10 after UVB exposure. Moreover, DNA methylation analysis indicated that PEBP might potentially reduce the activation of inflammation-related pathways such as MAPK, Wnt, Notch, and PI3K-AKT signaling. Our finding suggests that topical application of PEBP treatment may effectively prevent UVB-induced skin damage by inhibiting inflammation.

Waterpipe smoking is frequent in the Middle East and Africa with emerging prevalence worldwide. The epigenome acts as a molecular sensor to exposures and a crucial driver in several diseases. With the widespread use of waterpipe smoking, it is timely to investigate its epigenomic markers and their role in addiction, as a central player in disease prevention and therapeutic strategies. DNA methylome-wide profiling was performed on an exposure-rich population from the Middle East, constituting of 216 blood samples split equally between never, cigarette-only and waterpipe-only smokers. Waterpipe smokers showed predominantly distinct epigenetic markers from cigarette smokers, even though both smoking forms are tobacco-based. Moreover, each smoking form could be accurately (∼90 %) inferred from the DNA methylome using machine learning. Top markers showed dose-response relationship with extent of smoking and were validated using independent technologies and additional samples (total N = 284). Smoking markers were enriched in regulatory regions and several biological pathways, primarily addiction. The epigenetically altered genes were not associated with genetic etiology of tobacco use, and the methylation levels of addiction genes, in particular, were more likely to reverse after smoking cessation. In contrast, other epigenetic markers continued to feature smoking exposure after cessation, which may explain long-term health effects observed in former smokers. This study reports, for the first time, blood epigenome-wide markers of waterpipe smokers and reveals new markers of cigarette smoking, with implications in mechanisms of addiction and the capacity to discriminate between different smoking types. These markers may offer actionable targets to reverse the epigenetic memory of addiction and can guide future prevention strategies for tobacco smoking as the most preventable cause of illnesses worldwide., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive., Methods: To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS)., Results: Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205)., Conclusions: Our findings indicate that HPV types belonging to the mucosal alpha, and the 'cutaneous' beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings., (© 2023. The Author(s).)

Gut immune system homeostasis is crucial to overall host health. Immune disturbance at the gut level may lead to systemic and distant sites' immune dysfunction. Probiotics and prebiotics consumption have been shown to improve gut microbiota composition and function and enhance gut immunity. In the current study, the immunomodulatory and anti-inflammatory effects of viable and heat-inactivated forms of the novel probiotic bacterium Rouxiella badensis subsp. acadiensis (Canan SV-53), as well as the prebiotic protocatechuic acid (PCA) derived from the fermentation of blueberry juice by SV-53, were examined. To this end, female Balb/c mice received probiotic (viable or heat-inactivated), prebiotic, or a mixture of viable probiotic and prebiotic in drinking water for three weeks. To better decipher the immunomodulatory effects of biotics intake, gut microbiota, gut mucosal immunity, T helper-17 (Th17) cell-related cytokines, and epigenetic modulation of Th17 cells were studied. In mice receiving viable SV-53 and PCA, a significant increase was noted in serum IgA levels and the number of IgA-producing B cells in the ileum. A significant reduction was observed in the concentrations of proinflammatory cytokines, including interleukin (IL)-17A, IL-6, and IL-23, and expression of two proinflammatory miRNAs, miR-223 and miR425, in treated groups. In addition, heat-inactivated SV-53 exerted immunomodulatory properties by elevating the IgA concentration in the serum and reducing IL-6 and IL-23 levels in the ileum. DNA methylation analysis revealed the role of heat-inactivated SV-53 in the epigenetic regulation of genes related to Th17 and IL-17 production and function, including Il6 , Il17rc , Il9 , Il11 , Akt1 , Ikbkg , Sgk1 , Cblb , and Smad4 . Taken together, these findings may reflect the potential role of the novel probiotic bacterium SV-53 and prebiotic PCA in improving gut immunity and homeostasis. Further studies are required to ascertain the beneficial effects of this novel bacterium in the inflammatory state.

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.

Genetic and epigenetic alterations of the telomere maintenance machinery like telomere length and telomerase reverse transcriptase (encoded by TERT gene) are reported in several human malignancies. However, there is limited knowledge on the status of the telomere machinery in periampullary carcinomas (PAC) which are rare and heterogeneous groups of cancers arising from different anatomic sites around the ampulla of Vater. In the current study, we investigated the relative telomere length (RTL) and the most frequent genetic and epigenetic alterations in the TERT promoter in PAC and compared it with tumor-adjacent nonpathological duodenum (NDu). We found shorter RTLs (1.27 vs 1.33, P = 0.01) and lower TERT protein expression (p = 0.04) in PAC tissues as compared to the NDu. Although we did not find any mutation at two reactivating hotspot mutation sites of the TERT promoter, we detected polymorphism in 45% (9/20) of the cases at rs2853669 (T > C). Also, we found a hypermethylated region in the TERT promoter of PACs consisting of four CpGs (cg10896616 with Δβ 7%; cg02545192 with Δβ 9%; cg03323598 with Δβ 19%; and cg07285213 with Δβ 15%). In conclusion, we identified shorter telomeres with DNA hypermethylation in the TERT promoter region and lower TERT protein expression in PAC tissues. These results could be used further to investigate molecular pathology and develop theranostics for PAC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Epigenetic alterations are a near-universal feature of human malignancy and have been detected in malignant cells as well as in easily accessible specimens such as blood and urine. These findings offer promising applications in cancer detection, subtyping, and treatment monitoring. However, much of the current evidence is based on findings in retrospective studies and may reflect epigenetic patterns that have already been influenced by the onset of the disease., Methods: Studying breast cancer, we established genome-scale DNA methylation profiles of prospectively collected buffy coat samples (n = 702) from a case-control study nested within the EPIC-Heidelberg cohort using reduced representation bisulphite sequencing (RRBS)., Results: We observed cancer-specific DNA methylation events in buffy coat samples. Increased DNA methylation in genomic regions associated with SURF6 and REXO1/CTB31O20.3 was linked to the length of time to diagnosis in the prospectively collected buffy coat DNA from individuals who subsequently developed breast cancer. Using machine learning methods, we piloted a DNA methylation-based classifier that predicted case-control status in a held-out validation set with 76.5% accuracy, in some cases up to 15 years before clinical diagnosis of the disease., Conclusions: Taken together, our findings suggest a model of gradual accumulation of cancer-associated DNA methylation patterns in peripheral blood, which may be detected long before clinical manifestation of cancer. Such changes may provide useful markers for risk stratification and, ultimately, personalized cancer prevention., (© 2023. The Author(s).)

Background: Obesity is a multifactorial and chronic condition of growing universal concern. It has recently been reported that bariatric surgery is a more successful treatment for severe obesity than other noninvasive interventions, resulting in rapid significant weight loss and associated chronic disease remission. The identification of distinct epigenetic patterns in patients who are obese or have metabolic imbalances has suggested a potential role for epigenetic alterations in causal or mediating pathways in the development of obesity-related pathologies. Specific changes in the epigenome (DNA methylome), associated with metabolic disorders, can be detected in the blood. We investigated whether such epigenetic changes are reversible after weight loss using genome-wide DNA methylome analysis of blood samples from individuals with severe obesity (mean BMI ~ 45) undergoing bariatric surgery., Results: Our analysis revealed 41 significant (Bonferroni p < 0.05) and 1169 (false discovery rate p < 0.05) suggestive differentially methylated positions (DMPs) associated with weight loss due to bariatric surgery. Among the 41 significant DMPs, 5 CpGs were replicated in an independent cohort of BMI-discordant monozygotic twins (the heavier twin underwent diet-induced weight loss). The effect sizes of these 5 CpGs were consistent across discovery and replication sets (p < 0.05). We also identified 192 differentially methylated regions (DMRs) among which SMAD6 and PFKFB3 genes were the top hypermethylated and hypomethylated regions, respectively. Pathway enrichment analysis of the DMR-associated genes showed that functional pathways related to immune function and type 1 diabetes were significant. Weight loss due to bariatric surgery also significantly decelerated epigenetic age 12 months after the intervention (mean =  - 4.29; p = 0.02)., Conclusions: We identified weight loss-associated DNA-methylation alterations targeting immune and inflammatory gene pathways in blood samples from bariatric-surgery patients. The top hits were replicated in samples from an independent cohort of BMI-discordant monozygotic twins following a hypocaloric diet. Energy restriction and bariatric surgery thus share CpGs that may represent early indicators of response to the metabolic effects of weight loss. The analysis of bariatric surgery-associated DMRs suggests that epigenetic regulation of genes involved in endothelial and adipose tissue function is key in the pathophysiology of obesity., (© 2022. The Author(s).)

Background: DNA methylation in blood may reflect adverse exposures accumulated over the lifetime and could therefore provide potential improvements in the prediction of cancer risk. A substantial body of research has shown associations between epigenetic aging and risk of disease, including cancer. Here we aimed to study epigenetic measures of aging and lifestyle-related factors in association with risk of breast cancer., Methods: Using data from four prospective case-control studies nested in three cohorts of European ancestry participants, including a total of 1,655 breast cancer cases, we calculated three methylation-based measures of lifestyle factors (body mass index [BMI], tobacco smoking and alcohol consumption) and seven measures of epigenetic aging (Horvath-based, Hannum-based, PhenoAge and GrimAge). All measures were regression-adjusted for their respective risk factors and expressed per standard deviation (SD). Odds ratios (OR) and 95% confidence intervals (CI) were calculated using conditional or unconditional logistic regression and pooled using fixed-effects meta-analysis. Subgroup analyses were conducted by age at blood draw, time from blood sample to diagnosis, oestrogen receptor-positivity status and tumour stage., Results: None of the measures of epigenetic aging were associated with risk of breast cancer in the pooled analysis: Horvath 'age acceleration' (AA): OR per SD = 1.02, 95%CI: 0.95-1.10; AA-Hannum: OR = 1.03, 95%CI:0.95-1.12; PhenoAge: OR = 1.01, 95%CI: 0.94-1.09 and GrimAge: OR = 1.03, 95%CI: 0.94-1.12, in models adjusting for white blood cell proportions, body mass index, smoking and alcohol consumption. The BMI-adjusted predictor of BMI was associated with breast cancer risk, OR per SD = 1.09, 95%CI: 1.01-1.17. The results for the alcohol and smoking methylation-based predictors were consistent with a null association. Risk did not appear to substantially vary by age at blood draw, time to diagnosis or tumour characteristics., Conclusion: We found no evidence that methylation-based measures of aging, smoking or alcohol consumption were associated with risk of breast cancer. A methylation-based marker of BMI was associated with risk and may provide insights into the underlying associations between BMI and breast cancer., (© 2022. The Author(s).)

Ultraviolet radiation (UV) is causally linked to cutaneous melanoma, yet the underlying epigenetic mechanisms, known as molecular sensors of exposure, have not been characterized in clinical biospecimens. Here, we integrate clinical, epigenome (DNA methylome), genome and transcriptome profiling of 112 cutaneous melanoma from two multi-ethnic cohorts. We identify UV-related alterations in regulatory regions and immunological pathways, with multi-OMICs cancer driver potential affecting patient survival. TAPBP, the top gene, is critically involved in immune function and encompasses several UV-altered methylation sites that were validated by targeted sequencing, providing cost-effective opportunities for clinical application. The DNA methylome also reveals non UV-related aberrations underlying pathological differences between the cutaneous and 17 acral melanomas. Unsupervised epigenomic mapping demonstrated that non UV-mutant cutaneous melanoma more closely resembles acral rather than UV-exposed cutaneous melanoma, with the latter showing better patient prognosis than the other two forms. These gene-environment interactions reveal translationally impactful mechanisms in melanomagenesis., (© 2022. The Author(s).)

Burkitt lymphoma (BL) is a malignant B cell neoplasm that accounts for almost half of pediatric cancers in sub-Saharan African countries. Although the BL endemic prevalence is attributable to the combination of Epstein-Barr virus (EBV) infection with malaria and environmental carcinogens exposure, such as the food contaminant aflatoxin B1 (AFB1), the molecular determinants underlying the pathogenesis are not fully understood. Consistent with the role of epigenetic mechanisms at the interface between the genome and environment, AFB1 and EBV impact the methylome of respectively leukocytes and B cells specifically. Here, we conducted a thorough investigation of common epigenomic changes following EBV or AFB1 exposure in B cells. Genome-wide DNA methylation profiling identified an EBV-AFB1 common signature within the TGFBI locus, which encodes for a putative tumor suppressor often altered in cancer. Subsequent mechanistic analyses confirmed a DNA-methylation-dependent transcriptional silencing of TGFBI involving the recruitment of DNMT1 methyltransferase that is associated with an activation of the NF-κB pathway. Our results reveal a potential common mechanism of B cell transformation shared by the main risk factors of endemic BL (EBV and AFB1), suggesting a key determinant of disease that could allow the development of more efficient targeted therapeutic strategies.

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental 'hotspots' providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease., Competing Interests: MS, AS, NK, GC, CF, PI, AD, MB, SM, MR, ZH, CC, MD, PJ, DM, AP No competing interests declared, (© 2022, Silver et al.)

Bladder cancer (BC) is the ninth leading cause of cancer death with one of the highest recurrence rates among all cancers. One of the main risks for BC development is exposure to nitrosamines present in tobacco smoke or in other products. Aberrant epigenetic (DNA methylation) changes accompanied by deregulated gene expression are an important element of cancer pathogenesis. Therefore, we aimed to determine DNA methylation signatures and their impacts on gene expression in mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a carcinogen similar to compounds found in tobacco smoke. Following BBN administration mice developed non-invasive or invasive bladder cancers. Surprisingly, muscle- and neuronal-related pathways emerged as the most affected in those tumors. Hypo- and hypermethylation changes were present within non-invasive BC, across CpGs mapping to the genes involved in muscle- and neuronal-related pathways, however, methylation differences were not sufficient to affect the expression of the majority of associated genes. Conversely, invasive tumors displayed hypermethylation changes that were linked with alterations in gene expression profiles. Together, these findings indicate that bladder cancer progression could be revealed through methylation profiling at the pre-invasive cancer stage that could assist monitoring of cancer patients and guide novel therapeutic approaches.

Metformin and weight loss relationships with epigenetic age measures-biological aging biomarkers-remain understudied. We performed a post-hoc analysis of a randomized controlled trial among overweight/obese breast cancer survivors (N = 192) assigned to metformin, placebo, weight loss with metformin, or weight loss with placebo interventions for 6 months. Epigenetic age was correlated with chronological age (r = 0.20-0.86; P < 0.005). However, no significant epigenetic aging associations were observed by intervention arms. Consistent with published reports in non-cancer patients, 6 months of metformin therapy may be inadequate to observe expected epigenetic age deceleration. Longer duration studies are needed to better characterize these relationships.Trial Registration: Registry Name: ClincialTrials.Gov.Registration Number: NCT01302379.Date of Registration: February 2011.URL: https://clinicaltrials.gov/ct2/show/NCT01302379., (© 2021. The Author(s).)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background : DNA methylation is an epigenetic control mechanism that may be altered by environmental exposures. We have previously reported that in utero exposure to the mycotoxin and liver carcinogen aflatoxin B1 from the maternal diet, as measured using biomarkers in the mothers' blood, was associated with differential DNA methylation in white blood cells of 6-month-old infants from The Gambia. Methods : Here we examined aflatoxin B1-associated differential DNA methylation in white blood cells of 24-month-old children from the same population ( n = 244), in relation to the child's dietary exposure assessed using aflatoxin albumin biomarkers in blood samples collected at 6, 12 and 18 months of age. HM450 BeadChip arrays were used to assess DNA methylation, with data compared to aflatoxin albumin adduct levels using two approaches; a continuous model comparing aflatoxin adducts measured in samples collected at 18 months to DNA methylation at 24 months, and a categorical time-dose model that took into account aflatoxin adduct levels at 6, 12 and 18 months, for comparison to DNA methylation at 24 months. Results : Geometric mean (95% confidence intervals) for aflatoxin albumin levels were 3.78 (3.29, 4.34) at 6 months, 25.1 (21.67, 29.13) at 12 months and 49.48 (43.34, 56.49) at 18 months of age. A number of differentially methylated CpG positions and regions were associated with aflatoxin exposure, some of which affected gene expression. Pathway analysis highlighted effects on genes involved with with inflammatory, signalling and growth pathways. Conclusions : This study provides further evidence that exposure to aflatoxin in early childhood may impact on DNA methylation.

HPV oncoproteins can modulate DNMT1 expression and activity, and previous studies have reported both gene-specific and global DNA methylation alterations according to HPV status in head and neck cancer. However, validation of these findings and a more detailed analysis of the transposable elements (TEs) are still missing. Here we performed pyrosequencing to evaluate a 5-CpG methylation signature and Line1 methylation in an oropharyngeal squamous cell carcinoma (OPSCC) cohort. We further evaluated the methylation levels of the TEs, their correlation with gene expression and their impact on overall survival (OS) using the TCGA cohort. In our dataset, the 5-CpG signature distinguished HPV-positive and HPV-negative OPSCC with 66.67% sensitivity and 84.33% specificity. Line1 methylation levels were higher in HPV-positive cases. In the TCGA cohort, Line1, Alu and long terminal repeats (LTRs) showed hypermethylation in a frequency of 60.5%, 58.9% and 92.3%, respectively. ZNF541 and CCNL1 higher expression was observed in HPV-positive OPSCC, correlated with lower methylation levels of promoter-associated Alu and LTR, respectively, and independently associated with better OS. Based on our findings, we may conclude that a 5-CpG methylation signature can discriminate OPSCC according to HPV status with high accuracy and TEs are differentially methylated and may regulate gene expression in HPV-positive OPSCC.

Upper aerodigestive tract (UADT) tumors present different biological behavior and prognosis, suggesting specific molecular mechanisms underlying their development. However, they are rarely considered as single entities (particularly head and neck subsites) and share the most common genetic alterations. Therefore, there is a need for a better understanding of the global DNA methylation differences among UADT tumors. We performed a genome-wide DNA methylation analysis of esophageal (ESCC), laryngeal (LSCC), oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas, and their non-tumor counterparts. The unsupervised analysis showed that non-tumor tissues present markedly distinct DNA methylation profiles, while tumors are highly heterogeneous. Hypomethylation was more frequent in LSCC and OPSCC, while ESCC and OSCC presented mostly hypermethylation, with the latter showing a CpG island overrepresentation. Differentially methylated regions affected genes in 127 signaling pathways, with only 3.1% of these being common among different tumor subsites, but with different genes affected. The WNT signaling pathway, known to be dysregulated in different epithelial tumors, is a frequent hit for DNA methylation and gene expression alterations in ESCC and OPSCC, but mostly for genetic alterations in LSCC and OSCC. UADT tumor subsites present differences in genome-wide methylation regarding their profile, intensity, genomic regions and signaling pathways affected.

Introduction: Lebanon is among the top countries worldwide in combined incidence and mortality of breast cancer, which raises concern about risk factors peculiar to this country. The underlying molecular mechanisms of breast cancer require elucidation, particularly epigenetics, which is recognized as a molecular sensor to environmental exposures., Purpose: We aim to explore whether DNA methylation levels of AHRR (marker of cigarette smoking), SLC1A5 and TXLNA (markers of alcohol consumption), and LINE-1 (a genome-wide repetitive retrotransposon) can act as molecular mediators underlying putative associations between breast cancer risk and pertinent extrinsic (tobacco smoking and alcohol consumption) and intrinsic factors [age and body mass index (BMI)]., Methods: This is a cross-sectional pilot study which includes breast cancer cases (N = 65) and controls (N = 54). DNA methylation levels were measured using bisulfite pyrosequencing on available peripheral blood samples (N = 119), and Multivariate Imputation by Chained Equations (MICE) was used to impute missing DNA methylation values in remaining samples. Multiple mediation analysis was performed to assess direct and indirect (via DNA methylation) effects of intrinsic and extrinsic factors on breast cancer risk., Results: In relation to exposure, AHRR hypo-methylation was associated with cigarette but not waterpipe smoking, suggesting potentially different biomarkers of these two forms of tobacco use; SLC1A5 and TXLNA methylation were not associated with alcohol consumption; LINE-1 methylation was inversely associated with BMI (β-value [95% confidence interval (CI)] = -0.04 [-0.07, -0.02]), which remained significant after adjustment for age, smoking and alcohol consumption. In relation to breast cancer, there was no detectable association between AHRR, SLC1A5 or TXLNA methylation and cancer risk, but LINE-1 methylation was significantly higher in breast cancer cases when compared to controls (mean ± SD: 72.00 ± 0.66 versus 70.89 ± 0.73, P = 4.67 × 10 -14 ). This difference remained significant after adjustment for confounders (odds ratio (OR) [95% CI] = 9.75[3.74, 25.39]). Moreover, LINE-1 hypo-methylation mediated 83% of the inverse effect of BMI on breast cancer risk., Conclusion: This pilot study demonstrates that alterations in blood LINE-1 methylation mediate the inverse effect of BMI on breast cancer risk. This warrants large scale studies and stratification based on clinic-pathological types of breast cancer., (Copyright © 2021. Published by Elsevier Inc.)

Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here, we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissues adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritized by weighting methylation results using RNA-sequencing data from The Cancer Genome Atlas and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR), with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2 , and THSD4 , had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high-incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization. SIGNIFICANCE: This largest genome-wide DNA methylation study on ESCC from high-incidence populations of the world identifies functionally relevant and robust DNAme events that could serve as potential tumor-specific markers. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2612/F1.large.jpg., (©2021 American Association for Cancer Research.)

Coffee and tea are extensively consumed beverages worldwide which have received considerable attention regarding health. Intake of these beverages is consistently linked to, among others, reduced risk of diabetes and liver diseases; however, the mechanisms of action remain elusive. Epigenetics is suggested as a mechanism mediating the effects of dietary and lifestyle factors on disease onset. Here we report the results from epigenome-wide association studies (EWAS) on coffee and tea consumption in 15,789 participants of European and African-American ancestries from 15 cohorts. EWAS meta-analysis of coffee consumption reveals 11 CpGs surpassing the epigenome-wide significance threshold (P-value <1.1×10 -7 ), which annotated to the AHRR, F2RL3, FLJ43663, HDAC4, GFI1 and PHGDH genes. Among them, cg14476101 is significantly associated with expression of the PHGDH and risk of fatty liver disease. Knockdown of PHGDH expression in liver cells shows a correlation with expression levels of genes associated with circulating lipids, suggesting a role of PHGDH in hepatic-lipid metabolism. EWAS meta-analysis on tea consumption reveals no significant association, only two CpGs annotated to CACNA1A and PRDM16 genes show suggestive association (P-value <5.0×10 -6 ). These findings indicate that coffee-associated changes in DNA methylation levels may explain the mechanism of action of coffee consumption in conferring risk of diseases.

Both mucosal and cutaneous Human Papillomaviruses (HPVs) can be detected in the oral cavity, but investigations regarding the epidemiology of cutaneous HPVs at this site are scarce. We assessed mucosal (alpha) and cutaneous (beta and gamma) HPV infection in oral samples of HIV-infected and uninfected men who have sex with men (MSM). Oral rinse-and-gargles were collected from 310 MSM. Alpha HPVs were detected using the Linear Array, whereas beta and gamma HPVs were detected using multiplex PCR and Luminex technology. An amplicon-based next-generation sequencing (NGS) protocol was applied to a subset of samples collected from 30 HIV-uninfected and 30 HIV-infected MSM. Beta HPVs were significantly more common than alpha types (53.8% vs. 23.9% for HIV-infected subjects, p < 0.0001; 50.3% vs. 17.1% for HIV-uninfected subjects, p < 0.0001). Gamma HPVs were also frequently detected (30.8% and 25.9% in HIV-infected and uninfected MSM, respectively). NGS produced 2,620,725 reads representative of 146 known HPVs (16 alpha-PVs, 53 beta-PVs, 76 gamma-PVs, one unclassified) and eight putative new HPVs, taxonomically assigned to the beta genus. The oral cavity contains a wide spectrum of HPVs, with beta types representing the predominant genus. The prevalence of beta and gamma HPVs is high even in immunorestored HIV-infected individuals. NGS confirmed the abundance of cutaneous HPVs and identified some putative novel beta HPVs. This study confirms that cutaneous HPVs are frequently present at mucosal sites and highlights that their pathological role deserves further investigation since it may not be limited to skin lesions.

Actinic keratosis (AK) arises on photo-damaged skin and is considered to be the precursor lesion of cutaneous squamous cell carcinoma (cSCC). Many findings support the involvement of β human papillomaviruses (HPVs) in cSCC, while very little is known on γ HPV types. The objective of this study was to characterize the spectrum of PV types in healthy skin (HS) and AK samples of the same immunocompetent individuals using next generation sequencing (NGS). Viral DNA of 244 AK and 242 HS specimens were amplified by PCR using two different sets of primers (FAP59/64 and FAPM1). Purified amplicons were pooled and sequenced using NGS. The study resulted in the identification of a large number of known β and γ PV types. In addition, 27 putative novel β and 16 γ and 4 unclassified PVs were isolated. HPV types of species γ-1 (e.g. HPV4) appeared to be strongly enriched in AK versus HS. The NGS analysis revealed that a large spectrum of known and novel PVs is present in HS and AK. The evidence that species γ-1 HPV types appears to be enriched in AK in comparison to HS warrants further studies to evaluate their role in development of skin (pre)cancerous lesions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)