A large scale detection of MLH1 methylation is lacking in esophageal cancer. MLH1 is a well-known mismatch repair gene. The mechanism of MLH1 in DNA double strand break (DSB) repair remains unclear. Esophageal cancer cell lines and 1018 cases of primary cancer samples were employed. Methylation specific PCR, Western Blot, and CRISPR/Cas9 knockout technique were utilized. Methylation of MLH1 was detected in 3.93%. MLH1 methylation was significantly associated with tumor differentiation, male gender, smoking, and tumor size (all p < 0.05). The median overall survival (OS) was 24.7 months (95% CI 13.4-36.0) and 51.5 months (95% CI 40.4-62.5) in MLH1 methylated and unmethylated groups, respectively. OS was shorter in MLH1 methylated compared to unmethylated group patients (p < 0.01). Multivariate factor analysis indicated that MLH1 methylation is an independent poor prognosis marker (p < 0.05). MLH1 promotes ataxia telangiectasia mutated (ATM), ataxia telangiectasia and RAD3-related (ATR), and non-homologous end-joining repair (NHEJ), while inhibiting microhomology-mediated end joining (MMEJ) repair signaling pathways. Deletion of MLH1 sensitized esophageal cancer cells to novobiocin. MLH1 plays important roles in DSB repair and deletion of MLH1 sensitizes ESCC cells to Polθ inhibitor.

Background/ObjectivesEpstein-Barr Virus (EBV) is a ubiquitous virus associated with a variety of diseases including cancers. Evidence has emerged that the C promoter is methylated in many EBV-associated malignancies, whereas in free virion DNA it is unmethylated. We have developed and evaluated a methylation-specific PCR assay for the EBV C Promoter (MSPCP) that can be applied to human biological specimens to quantify EBV methylation.MethodsTwo sets of methylation-specific primers were designed to anneal to bisulfite-converted DNA sequences with 3 CpGs in the forward primer binding site, and 2 CpGs in the reverse primer binding site. We evaluated this method in synthetic oligonucleotides, DNA extracted from cell lines, virion supernatants, and a variety of clinical specimens. EBV methylation of Cp, as measured by MSPCP, was validated with two orthogonal methods in select samples.ResultsIn contrived samples, this method had a linear range between 0–100% methylation. Application of this assay to DNA extracted from 11 formalin-fixed paraffin-embedded biopsy specimens showed high-level C promoter methylation in EBV-associated tumors (94–100%) but not in EBV-associated lymphoid hyperplasia. High-level EBV methylation was also detected in cell-free DNA extracted from the plasma of 13 patients with EBV-associated Hodgkin lymphoma. In contrast, EBV methylation was either not-detected, or detected at very low levels, in saliva from 25 adults in a general university population consistent with the presence of virion DNA.ConclusionsMSPCP is a simple, rapid and accurate method that characterizes the methylation status of the EBV C promoter, which may be useful in a variety of research and clinical settings.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13027-025-00702-x.

Cholangiocarcinoma (CC) is a highly lethal malignancy that urgently requires reliable prognostic biomarkers. Although MUC1 expression and promoter methylation have been implicated in CC, the clinical significance of promoter methylation pattern composition, beyond average methylation levels, remains unclear. Here, we investigated the relationship between MUC1 promoter methylation heterogeneity, MUC1 mRNA expression, and prognosis in CC. We analyzed bisulfite amplicon sequencing data and mRNA expression of MUC1, DNA methylation-related enzymes (TET1, TET2, TET3, Dnmt1, and Dnmt3a), and tumor microenvironment stress markers in 131 CC tissues. In the neoplastic region, high MUC1 mRNA expression was associated with poor overall survival (HR = 0.131, 95% CI: 0.02to0.95, p = 0.042) and correlated with the abundance of completely unmethylated promoter patterns (r = 0.386, p < 0.001). Among the enzymes analyzed, only TET3 expression significantly correlated with the abundance of completely unmethylated patterns in the neoplastic region (Cohen's f2 = 0.108, p = 0.009), suggesting a potential region-specific regulatory association. We visualized beta-diversity in methylation pattern composition using t-SNE and classified samples into two groups based on a linear decision boundary in the t-SNE space. This classification stratified prognosis independently of clinical factors (HR = 0.291, 95% CI: 0.06to0.94, p = 0.037; multivariate p = 0.021). These findings propose a novel, composition-based epigenetic stratification framework in CC, revealing that MUC1 promoter methylation pattern structure-rather than average methylation level-has prognostic relevance. Our results highlight the potential of pattern-resolved methylation profiling in the development of clinically applicable epigenetic biomarkers.

Introduction Nasopharyngeal carcinoma (NPC) is a serious cancer with a poor prognosis and a significant risk of metastasis. Although its epigenetic control mechanisms are still unknown, epigallocatechin-3-gallate (EGCG) exhibits strong anticancer properties. Promoter methylation in malignancies often silences PACRG, a putative tumor suppressor gene. This study aimed to determine whether EGCG suppresses cancer by altering PACRG gene expression in NPC cells. Material and methods NPC cells were subjected to EGCG or 5-aza-dC. Cellular functions were assessed by CCK-8, Transwell, and flow cytometry assays. Methylation-specific PCR (MSP) was used to identify PACRG promoter methylation, and gene expression was measured using quantitative real-time PCR (qRT-PCR) and Western blot (WB). The expression of DNA methyltransferase-related genes was evaluated. siRNA targeting PACRG was used to assess its functional involvement in the effects mediated by EGCG. Results EGCG exerted dose- and time-dependent effects on NPC cells by reducing proliferation and migration while inducing apoptosis and G2 phase cell cycle arrest. Mechanistically, EGCG significantly reduced the mRNA expression and enzymatic activity of DNMT1, DNMT3A, and DNMT3B, resulting in decreased PACRG promoter methylation and restored PACRG expression. Functional assays revealed that knockdown of PACRG diminished the inhibitory effect of EGCG on NPC cells, indicating that the epigenetic reactivation of PACRG partially mediates the tumor-suppressive function of EGCG. Conclusions Our results revealed that EGCG inhibits NPC progression by reducing PACRG promoter methylation, highlighting that PACRG demethylation and reactivation are a promising therapeutic strategy.

DNA methylation is a key epigenetic mechanism regulating gene expression during spermatogenesis. This study investigated the effects of experimental amebiasis induced by Entamoeba histolytica and its treatment with metronidazole (MTZ) on the methylation status of the Spermatogenesis Associated 6 (SPATA6) gene and male reproductive function. Twenty-four adult male rats were assigned to control, infected, and MTZ-treated groups. Following treatment, testicular tissues were analyzed for SPATA6 promoter methylation via bisulfite sequencing, infection confirmation by PCR, histopathological changes by hematoxylin and eosin (H&E) staining, Inhibin B and Androgen-Binding Protein (ABP) expression by immunohistochemistry, and sperm quality indices. The infected group exhibited distinct non-CpG methylation at a SPATA6 locus, confirmed reproductive tract infection, severe testicular damage, increased expression of Inhibin B and ABP, and significantly impaired sperm parameters. MTZ treatment successfully cleared the parasite and partially restored testicular architecture and sperm count; however, residual abnormalities in sperm motility, histology, and SPATA6 methylation persisted. While these findings suggest that parasitic infection and its treatment may induce epigenetic dysregulation in the testis, the direct functional link between the observed methylation change and reproductive outcomes remains inconclusive due to the limited scope of analysis. These results underscore the need for genome-wide methylation and transcriptomic profiling to better characterize the molecular basis of infection- and treatment-related reproductive effects. The study provides initial insights into infection-associated epigenetic modulation in male reproduction, with potential implications for fertility and reproductive health.

The prognosis and resistance to temozolomide in glioblastoma have been evaluated based on O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation using bulk tumor samples resected via surgery. However, it remains unclear whether MGMT promoter methylation differs between glioblastoma stem cells and the overall tumor within the patient's brain. Chemoresistance of glioblastoma stem cells is a critical factor in understanding the refractory nature of the disease. This study aimed to assess MGMT promoter methylation in glioblastoma stem cells and compare it with that in differentiated tumor cells. Glioblastoma stem-like cells (GSCs) and differentiated GSCs (dGSCs) were cultured from surgically resected tumors. Following DNA extraction and bisulfite conversion, real-time methylation-specific PCR was performed to quantify MGMT promoter methylation in three tumor cell types: GSCs, dGSCs, and surgically resected tumor samples. MGMT protein and CD133 expression levels were assessed via flow cytometry. Based on CD133 expression and microscopic evaluation, dGSCs exhibited a more differentiated phenotype than GSCs. No significant differences were observed in ΔCt values or MGMT promoter methylation ratios between GSCs and dGSCs. Similarly, ΔCt values and methylation ratios showed negligible differences between GSCs and surgically resected tumors. MGMT was highly expressed in both GSCs and dGSCs, consistent with an unmethylated MGMT promoter. MGMT promoter methylation status in glioblastoma stem cells remained unchanged during differentiation and was comparable to that in surgically resected tumors. As temozolomide is presumed to be ineffective against glioblastoma stem cells with unmethylated MGMT promoters, MGMT blockade may be a beneficial therapeutic strategy.

This study investigated the expression, methylation patterns, and clinicopathological implications of ARHGAP40 in renal cell carcinoma (RCC), the most common urinary malignancy. A total of 60 clear cell renal cell carcinomas (ccRCC), 30 papillary renal cell carcinomas (pRCC), 30 chromophobe renal cell carcinomas (chRCC), and 13 other RCC subtypes were enrolled. ARHGAP40 expression was analyzed in both RCC tissues and matched paracancerous normal tissues using immunohistochemistry (IHC). The methylation status of the ARHGAP40 promoter region was assessed in both normal and tumor samples by bisulfite sequencing PCR (BSP). Circulating tumor DNA (ctDNA) extracted from peripheral blood samples of RCC patients (20), patients with benign renal tumors (1), and healthy controls (14) was quantitatively analyzed for methylation using quantitative methylation-specific PCR (qMSP). ARHGAP40 expression was significantly downregulated in RCC compared to matched normal tissues (P < 0.001). This reduced expression correlated with tumor necrosis (P = 0.009) but showed no significant association with age, gender, tumor location, tumor diameter, TNM stage, or vascular invasion. In the ccRCC subtype, ARHGAP40 expression exhibited a progressive decrease with larger tumor diameter (P = 0.045), advancing histological grade (P = 0.032), and tumor necrosis (P = 0.011). The methylation status of ARHGAP40 was consistent with its expression level in both tumor and adjacent normal tissues. Methylated ARHGAP40 DNA was detectable only in RCC patient ctDNA samples. ARHGAP40 is epigenetically silenced in RCC through methylation-mediated downregulation, which correlates with tumor necrosis and grade. The detection of methylated ARHGAP40 in ctDNA holds promise as a potential biomarker for early RCC diagnosis.

Background/PurposeSjögren's syndrome (SS) is a chronic systemic autoimmune disease characterized by lymphocytic infiltration and formation of lymphoepithelial lesions (LEL) in exocrine glands, leading to secretory dysfunction. DNA methylation, a dynamically regulated epigenetic mark, has been increasingly recognized as a key regulatory mechanism in the pathogenesis of autoimmune diseases including SS, and holds promise for identifying novel diagnostic and therapeutic strategies.MethodsReduced representation bisulfite sequencing (RRBS) was performed on 4 cases of SS and 3 controls to profile genome-wide DNA methylation patterns. Differentially methylated regions (DMRs) and associated differentially methylated genes (DMGs) were detected, followed by functional enrichment analysis. Integration with transcriptomic data (GSE40611) was performed to identify overlapping epigenetic and transcriptional changes.ResultsA total of 29,462 DMRs were detected, with 24,116 hypermethylated and 5,346 hypomethylated regions, indicating an overall increase in methylation levels of SS, and DMGs located in gene promoter regions were significantly enriched in pathways related to immune response, transcriptional regulation, and inflammation. Nine hub genes (LCP2, BTK, LAPTM5, ARHGAP9, IKZF1, WDFY4, CSF2RB, ARHGAP25, DOCK8) were identified, which displayed promoter hyper—or hypomethylation, indicating the complex epigenetic regulatory mechanisms.ConclusionThis study reveals extensive DNA methylation alterations in SS, providing new insights into the epigenetic mechanisms underlying pathogenesis. Moreover, these findings suggest potential biomarkers or therapeutic targets for further investigation to elucidate detailed molecular mechanisms of SS.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13072-025-00637-y.

Protein restriction reprograms the multi-organ proteomic landscape of mouse aging.

IntroductionHemophilia is a rare X-linked bleeding disorder leading to recurrent hemarthroses, hemophilic arthropathy, and impaired quality of life. A chronic lifelong disease, hemophilia might be associated with accelerated biological aging. Here, we investigated whether biological age derived from epigenetic age estimators differs in hemophilia.Patients/MethodsWe collected blood samples from men with severe (<1 IU/dL; PWSH,n = 20) or mild hemophilia (≥5 IU/dL; PWMH,n = 20), and age-matched healthy male controls (n = 20). DNA methylation of cytosine–phosphate–guanine (CpG) dinucleotides at five genes (ASPA,ITGA2B,PDE4C,FHL2,CCDC10SB) was measured by bisulfite pyrosequencing. Biological age was estimated using two epigenetic aging signatures, each including three CpGs. We investigated differences in biological age and the rate of biological aging between study groups using separate linear regressions on chronological age and study group without and with an interaction, respectively.ResultsDeviations of epigenetic from chronological age were high for both 3-CpG age estimators, with results suggesting systematic overprediction. In both linear regressions using the two 3-CpG estimates, respectively, evidence for a different rate of biological aging in severe hemophilia was weak. The rate of biological aging in PWSH was 0.24 (95% CI, 0.01–0.48) and 0.21 (0.04–0.37) higher compared with PWMH, and 0.05 (−0.19–0.29) and 0.17 (−0.00–0.34) higher compared with healthy controls, respectively. Hemophilic arthropathy was associated with an increased rate of biological aging.ConclusionEvidence for a difference in epigenetic aging as reflected by two 3-CpG estimators in severe compared with mild hemophilia or healthy controls was weak.

Disclosure: L. Zoff: None. F.M. Garcia: None. G. Veneruzzo: None. G. Aschettino: None. M.C. Mattone: None. E.B. Berensztein: None. C. Terada: None. N. Perez Garrido: None. R.M. Marino: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None.Being born SGA in 46, XY DSD patients without molecular diagnosis and with no specific disorders of undermasculinization have a higher prevalence than expected for the general population. Numerous links have been made between infant growth restriction and specific epigenetic alterations, including changes in DNA methylation, thereby implicating such regulation in appropriate growth and development. Moreover, several studies have suggested that SGA children present with permanent alterations in the epigenome due to the programming of fetal tissues. The Growth hormone (GH)/ insulin-like growth factor type 1 (IGF1) and Insulin axis, crucial in human growth and development, is involved in adrenogonadal differentiation in mice. We hypothesized that epigenetic changes in the GH/IGFs and Insulin axis, could mediate the association between being born SGA and with DSD in humans. Our aim was to evaluate both the levels and patterns of methylation across the regulatory regions of GHR, IGF1R, and INSR genes in foreskin (FT) from 46,XY DSD children born either AGA (AGADSD-FT, n=7; 0.8-7.37y) or SGA (SGADSD-FT, n=7; 1.3-7.3y) vs age-matched control children (C-FT; n=7, 0.9-7y) using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG-rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The average CpG methylation levels of the SP1-responsive IGF1R proximal promoter [GRCh38:15:98648387: 98648592:1 (-152 to +53)] and the 5´UTR regulatory region [GRCh38:15:98649214: 98649496:1 (+676 to +958)] were significantly higher in SGADSD-FT than in C-FT and AGADSD-FT (Kruskal Wallis test, p<0.05). In locus-by-locus comparative analyses, higher methylation levels of CpG-23, CpG-17, CpG-15, CpG-13, and CpG-11 within the unique Sp1-dependent initiator motif of the IGF1R gene were detected in SGADSD-FT compared with C-FT and AGADSD-FT, p<0.05. The mean methylation levels of INSR promoter regions [GRCh38:19:7294796:7295022:-1 (-608 to -381) and GRCh38:19:7294410:7294702:1 (-288 to +5)], covering the binding site of activating transcription factors, were higher in DSD 46, XY patients, both in SGADSD-FT and in AGADSD-FT than in C-FT (p<0.05). There were no significant differences in GHR promoter methylation among the three FT groups. Promoter DNA methylation is a fundamental epigenetic mark associated with transcriptional repression during development. According to our findings, it could be speculated that epigenetic repression, mainly of IGF1R, might contribute to mediate the association between being born SGA and 46XYDSD in humans. Nevertheless, whether or not the epigenetic changes observed in postnatal foreskin are present at the time of sexual differentiation during intrauterine life must be determined.Presentation: Saturday, July 12, 2025

Disclosure: S.E. Khoury: None. E. Morales-Grahl: None. L. Thompson: None. A.C. Gore: None. N.Y. Eldiraoui: None.This study aims to evaluate how early life exposure of rats to endocrine-disrupting chemicals (EDCs) affects development and sexual differentiation of the brain through molecular and epigenetic programming. Our lab and others have shown that EDC exposure affects the hypothalamus in a sex, time, and exposure dependent manner. These effects lead to changes in gene expression related to gonadal steroid hormone signaling in the brain, shifts in pubertal timing, and altered DNA methylation patterns in hypothalamic regions. Current work is addressing effects of EDC exposure on mating behaviors in rats and whether epigenetic modification in brain regions involved in these behaviors underlie any observed physiological changes. In our lab we use NeuroMix, an EDC mixture containing 9 common industrial chemicals at environmentally relevant doses. We expose rats orally to NeuroMix in early postnatal life through feeding of rat dams, during hormone sensitive periods of brain development in the offspring. Brain tissues are collected at postnatal day 1, adolescence, and during early adulthood and bed nucleus of the stria terminalis and preoptic area of the hypothalamus are dissected for Tag-Seq to look at mRNA expression and whole genome bisulfite sequence (WGBS) to assess changes to gene expression and DNA methylation associated with sex differences at these time points. In adulthood, a subset of early adult males are observed in a natural mating environment and number of and latency to mounts, intromission, and ejaculation are counted. A subset of early adult females are tested and recorded in a paced mating paradigm to uncover differences in female typical sexual behaviors due to NeuroMix treatment. The results of these experiments will provide novel insights into the effects of NeuroMix treatment on sexually dimorphic behaviors and brain epigenetics. Supported by NIH R35 ES035024Presentation: Saturday, July 12, 2025

Disclosure: L. Zoff: None. F.M. Garcia: None. G. Veneruzzo: None. G. Aschettino: None. M. Valdora: None. D. Rodriguez: None. M.C. Mattone: None. N. Perez Garrido: None. R.M. Marino: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None.About 11% of all children born annually in the world are defined as SGA. Approximately 15% of them will continue to present with low height for their ages, associated with a non-catch-up growth phenotype. However, the pathophysiological mechanism underlying these findings is unknown. Several studies have suggested that the embryonic environmental characteristics of patients SGA could induce changes at the epigenetic level that affect gene transcription and would be stable throughout life. The GH/IGF1 axis and insulin are crucial stimulators of growth, cell proliferation, and metabolism. This study aimed to compare the methylation status of GHR, IGF1R, and INSR genes in peripheral blood in a cohort of boys with idiopathic short stature without GH-treatment who were born SGA (birthweight <10th percentile for the gestation, n=14; 4-13yr) and in age-matched control boys (CTRL, n=19; 4-15yr)using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG-rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The PCA score plot showed a clear separation between the CTRL and SGA groups. The two principal components explained 92.5% of the variance. The mean CpG methylation levels of the SP1-responsive IGF1R proximal promoter [GRCh38:15:98648387: 98648592:1 (-152 to +53)] were significantly higher in SGA than in CTRL (Mann-Whitney-Wilcoxon test, p<0.05). Compared with CNTRL, the CpG methylation levels of the TATA-less V2 proximal promoter as well as within the V2, V3, and V9 alternative exons in the regulatory 5´-UTR of the GHR gene [GRCh38:5:42423379:42423768:1 (-59 to +330), GRCh38:5:42424357:42424668:1 (+919 a +1230) and GRCh38:5:42424672:42424968:1 (+1234 a +1530)] were lower in SGA (p<0.05). Promoter DNA methylation is a fundamental epigenetic mark associated with transcriptional repression during development. According to our findings, it could be speculated that epigenetic repression of IGF1R might contribute to the lack of catch-up growth in short SGA patients. Finally, the human GHR gene is a complex transcription unit, and the impact or functional relevance of our findings and its relation to lessen activation of IGF1R pathways remains to be elusive.Presentation: Saturday, July 12, 2025

Disclosure: A. Hattori: None. A. Seki: None. Y. Naiki: None. A. Nakamura: None. T. Michigami: None. Y. Katoh-Fukui: None. T. Ogata: None. M. Kagami: None. M. Fukami: None.Introduction: Male-specific growth genes, alongside gonadal sex steroids, have been proposed as contributors to sex differences in height; however, no such gene has been identified. SHOX, a key growth gene located in the pseudoautosomal region on the short arm (PAR1) of the sex chromosomes, influences height in a dose-dependent manner. Conventionally, genes on PAR1, including SHOX, were thought to escape X-chromosome inactivation (XCI), thus being expressed equally in men and women. In 2017, Tukiainen et al. reported that most genes on PAR1 are expressed in a male-dominant manner. Nevertheless, sex differences in SHOX expression remain largely unexplored due to its highly tissue-specific expression. Objective: To explore the role of SHOX in sex differences in height. Methods: We compared male and female cartilage and/or chondrocyte samples using the following methods: (i) microarray analysis for X chromosome gene expression (n = 16), (ii) RT-qPCR for SHOX expression (n = 36), (iii) reduced representation bisulfite sequencing (RRBS) for X chromosome DNA methylation (n = 4), and (iv) pyrosequencing for DNA methylation of SHOX-flanking regions (n = 22). Furthermore, we established fibroblast clones derived from a boy and a girl with polydactyly, which enabled us to compare DNA methylation patterns around SHOX among the active and inactive X chromosomes and the Y chromosome using long-read sequencing. Results: Microarray analysis showed male-dominant expression of PAR1 genes, including SHOX. RT-qPCR confirmed male-dominant SHOX expression. RRBS revealed that the X chromosome exhibited female-dominant DNA methylation, which was pronounced in the X-specific region and mild in PAR1. RRBS also showed that SHOX-flanking regions exhibited sexually dimorphic DNA methylation. Specifically, male-dominant DNA methylation was observed in a region upstream of SHOX and a region spanning intron 5-exon 6a, whereas female-dominant DNA methylation was detected in intron 2. These findings were confirmed by pyrosequencing. In silico analysis suggested that the region with female-dominant DNA methylation in intron 2 functions as an enhancer of SHOX. Long-read sequencing of fibroblast clones revealed hypomethylation of intron 2 in the active X and Y chromosomes and hypermethylation in the inactive X chromosome. Discussion: Male-dominant SHOX expression in cartilage tissue indicates a role of SHOX in sex differences in height. Sexually dimorphic DNA methylation around SHOX implies that XCI represses SHOX expression in women. In particular, allele-specific DNA methylation in intron 2 (hypomethylation of the active X and hypermethylation of the inactive X) suggests that this region functions as an enhancer involved in the repression of SHOX expression due to XCI. Conclusion: Male-dominant SHOX expression, driven by the spreading of XCI to SHOX, likely contributes to sex differences in height.Presentation: Saturday, July 12, 2025

Endometrial carcinoma (EC) with deficient DNA mismatch repair (dMMR) is a specific molecular entity with unique clinicopathological features. Herein, we depicted the mutation profile of dMMR ECs and explored the molecular heterogeneity among dMMR subgroups with different etiologies. Next-generation sequencing (NGS) based on a 1021-gene panel was applied to 74 dMMR ECs and 43 proficient DNA mismatch repair (pMMR) ECs. In addition, methylation-specific Polymerase Chain Reaction (PCR) was applied for accessing MLH1 promoter hypermethylation (MLH1me+) in dMMR cases. The mutation rates of PTEN, ARID1A, KRAS, and MSH2 were significantly higher in dMMR group, while the CTNNB1 and MSH3 mutations were more commonly observed in pMMR group (p<0.05). Compared to pMMR ECs, dMMR ECs had significantly higher alteration frequencies in WNT, NOTCH, Cell Cycle and MMR, HRR and BER pathway (p<0.05). Remarkably, the interaction patterns within and across pathways were different between dMMR and pMMR groups. Intriguingly, no CTNNB1 mutation were found in dMMR ECs, while half of the WNT-activated pMMR ECs were CTNNB1 mutated, which were generally mutually exclusive with other WNT pathway key genes. The median tumor mutational burden (TMB) of dMMR ECs was significantly higher than pMMR ECs. However, ultra-high TMB value was related to pathogenic POLE mutation both in dMMR and pMMR ECs. As for dMMR subgroups (MLH1me+, Lynch and Lynch-like), KEAP1 and FBXW7 mutations, which may have potential predictive effect of immunotherapy, were enriched in the Lynch subgroup. dMMR ECs has distinctive genomic profile with molecular heterogeneity, which may have potential prognostic and therapeutic implications.

DNA methylation is a key epigenetic modification underlying cellular identity. Conventional methods based on CpG site-level data often lack sensitivity in detecting low-frequency methylation signals. Here, we present Alpha, a novel method combining unbiased segmentation with robust read-level identification of low frequency cell-type-specific methylation signals. Methylation markers identified by Alpha exhibited significant enrichment in regulatory genomic elements such as enhancers, active promoters, and transcription factor binding sites. In simulated cell-type admixtures, Alpha-derived markers demonstrated improved deconvolution performance, exhibiting lower error metrics compared to beta-value based methods (DSS), even with limited marker numbers (N < 50). We combined Alpha with a non-negative least squares approach (Alpha-NNLS) to enable sensitive detection of circulating tumor DNA (ctDNA) in simulated cell-free DNA from breast and colon cancers, outperforming existing read-level methylation-based tumor fraction estimation methods (CelFEER and UXM). We applied Alpha-NNLS to targeted bisulfite sequencing data from early-stage colon cancer plasma samples and demonstrated strong concordance with existing approaches (R2 = 0.98), supporting its potential for sensitive detection of ctDNA.

HighlightsWhat are the main findings?Radiation-acquired HeLaR cervical cancer cells exhibit significant afatinib resistance.EZH2 epigenetically silences PTEN, leading to sustained AKT activation.What is the implication of the main findings?Pharmacologic inhibition of EZH2 or PI3K/AKT restores afatinib sensitivity.In vivo, combination therapy with an EZH2 inhibitor and afatinib suppresses tumor growth without toxicity.The EZH2–PTEN–AKT axis represents a potential therapeutic target in recurrent and radioresistant cervical cancer.Background: Cervical cancer remains a major global health burden, and treatment failure due to radioresistance and secondary drug resistance severely limits clinical outcomes. Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator implicated in tumor progression. This study aimed to determine whether EZH2-mediated PTEN silencing drives afatinib resistance via AKT activation in radiation-resistant cervical cancer cells. Methods: A radioresistant cervical cancer cell line (HeLaR) was established following cumulative irradiation (70 Gy). Cell viability, clonogenic survival, methylation-specific PCR (MSP), chromatin immunoprecipitation (ChIP), and Western blot analyses were conducted. EZH2 (Dznep; tazemetostat), PI3K, and AKT inhibitors were tested in combination with afatinib. A xenograft mouse model was used for in vivo validation. Results: HeLaR cells exhibited upregulation of EZH2 and H3K27me3, downregulation of PTEN, and sustained AKT activation. EZH2 inhibition restored PTEN expression, attenuated AKT phosphorylation, and re-sensitized cells to afatinib. MSP and ChIP confirmed EZH2-mediated PTEN promoter silencing. PI3K inhibition reproduced these effects, whereas ERK inhibition had minimal impact. In xenograft models, combined treatment with Dznep and afatinib significantly suppressed tumor growth compared to single agents. Conclusions: EZH2-driven PTEN suppression promotes AKT-dependent afatinib resistance in radiation-resistant cervical cancer. Targeting the EZH2–PTEN–AKT axis may provide a potential therapeutic approach to mitigate combined radioresistance and chemoresistance in recurrent cervical cancer, although further preclinical and clinical validation is required.

Most T1 gastric cancer (GC) harbor lymph node metastasis (LNM) at a rate of <20%; however, owing to the difficulty in accurately diagnosing LNM preoperatively, many patients with T1 GC undergo unnecessary invasive radical gastrectomy with lymphadenectomy. In the present study, we established an epigenetic liquid biopsy assay for the preoperative diagnosis of LNM in T1 GC. A comprehensive biomarker discovery was performed by analyzing genome-wide DNA methylation profiling. We obtained 277 clinical specimens, including 177 surgical tissues and 100 pre-operative plasmas. DNA methylation biomarkers were trained and validated using quantitative methylation-specific polymerase chain reaction (qMSP) assays. We identified six novel differentially methylated regions, including at least two differentially methylated CpG probes (|Delta-beta| >0.12 and p<0.05) within 100 bp, through genome-wide biomarker discovery. A DNA methylation panel was generated using qMSP assays in clinical tissue specimens, with an area under the curve (AUC) of 0.80. This panel was validated in an independent clinical cohort, and a combined model, which integrated the DNA methylation model with preoperative computed tomography -based findings, was established through multivariate logistic regression analyses (AUC: 0.84). Finally, we translated this model into a liquid biopsy, and this cell-free DNA (cfDNA) methylation model exhibited robust performance for LNM identification in T1 GC (AUC: 0.86) and allowed 44% of patients to avoid unnecessary invasive operations, without missing any LNM-positive patients. We have successfully developed a cfDNA methylation signature-based liquid biopsy diagnostic assay that allows for robust and less-invasive LNM detection in patients with T1 GC.

Through targeted next-generation sequencing of 83 non-small cell lung cancer (NSCLC) patients with first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) resistance, we detected 11% TET2 mutations in the T790M-negative subgroup. To explore the molecular mechanism of TET2 in EGFR TKI resistance, reduced representation bisulfite sequencing (RRBS) was adopted to analyze the global genomic methylation profiles and detect the differentially methylated genes in the TET2-knockdown (KD) PC9 and control PC9 cell lines, following bioinformatics analysis of gene ontology (GO) functions and kyoto encyclopedia of genes and genomes (KEGG) signaling to screen for genes associated with drug resistance. TET2 KD attenuated gefitinib-induced apoptosis and decreased the sensitivity of EGFR-mutant lung cancer cells to gefitinib. Forty-three drug resistance genes with hypermethylated promoter regions were identified via RRBS and bioinformatic analysis in PC9TET2 KD cells. Then, 10 candidate genes were screened for further validation. RT‒PCR demonstrated that the expression of AXIN2 and CSK was significantly lower in PC9TET2 KD cells than in control cells. Furthermore, AXIN2 KD attenuated gefitinib-induced apoptosis and decreased the sensitivity of PC9 cells to gefitinib. Importantly, we found that the demethylation drug decitabine (DCA) could reverse gefitinib resistance in PC9TET2 KD cells and mouse models. These results indicate that the methylation of AXIN2 induced by TET2 repression is a novel resistance mechanism of EGFR TKIs in EGFR-mutant NSCLC. Demethylation drugs have the potential to overcome EGFR TKI resistance induced by loss-of-function TET2 mutations.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20263-8.

BackgroundDNA methylation is a critical epigenetic modification that functions in diverse biological processes. High-throughput sequencing has revealed substantial variations in DNA methylation patterns across species. While extensively studied in vertebrates, knowledge of invertebrate DNA methylation remains limited.ResultsWe examined the DNA methylation landscape of the ancient-looking marine mollusk, Nautilus pompilius, a “living fossil”. We identified key DNA methyltransferase genes (DNMT1 and DNMT3) by homologous searches against the N. pompilius genome and employed whole-genome bisulfite sequencing (WGBS) to characterize methylation patterns. Despite generally similar gene body methylation (gbM) and genome-wide methylation levels to other invertebrates, the N. pompilius exhibits a characteristic low methylation bias specifically in the promoter and first exon regions.ConclusionsN. pompilius has a typical gene body methylation pattern. Both promoter and gene body methylation may work together to regulate gene expression in N. pompilius. Our research provides valuable information to the ever-expanding methylation map of invertebrates.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-11907-0.