Tumor recurrence and metastasis remain the principal causes of mortality in patients with head and neck squamous cell carcinoma (HNSCC). However, the key molecular drivers underlying HNSCC progression and their regulatory mechanisms have not yet been fully elucidated. In this study, we investigated the biological role of the long noncoding RNA MIR4435-2HG in HNSCC. Integrated analyses across multiple public databases demonstrated that MIR4435-2HG is significantly upregulated in HNSCC and is closely associated with pathological T stage, clinical stage, and histological grade. Through further bioinformatic screening and functional validation, upstream transcription factor 1 (USF1) was identified as a critical downstream effector of MIR4435-2HG. Mechanistically, MIR4435-2HG directly interacts with USF1 and promotes its protein stability by facilitating USF1 succinylation, thereby enhancing tumor cell migration and invasion through the induction of epithelial - mesenchymal transition. Moreover, dual-luciferase reporter assays and RT-qPCR analyses revealed that CREB1 transcriptionally upregulates MIR4435-2HG expression, establishing a positive feedback regulatory circuit. Collectively, our findings demonstrate that MIR4435-2HG promotes USF1 succinylation and stabilization, leading to the formation of a MIR4435-2HG - USF1-AKT - CREB1 signalling loop that drives epithelial - mesenchymal transition and facilitates the invasive and metastatic potential of HNSCC cells.
Cutaneous melanoma (CM) is highly invasive with limited prognostic tools, and the prognostic relevance of lactylation, a novel post-translational modification, remains unexplored. We developed a lactylation-related prognostic model using relative methylation orderings from The Cancer Genome Atlas (TCGA) melanoma cohort (n = 458), which was validated in two dependent cohorts (n = 241). Comprehensive analyses of immune infiltration, therapy efficacy, drug sensitivity, and tumour mutation burden were conducted between high- and low-risk groups. Results showed that 284 lactylation-related genes collected classified TCGA melanoma patients into two clusters with significant survival differences (Kaplan-Meier log-rank test, p = 0.046). Between the two clusters, 279 differentially methylated loci were identified (false discovery rate < 0.05 and |Δβ| >0.1). Based on these loci, 1,743 locus pairs were significantly associated with overall survival (Univariate Cox regression, false discovery rate < 0.05). From these significant pairs, the CM 11 locus-pair prognostic model (CM-LP11) consisting of 11 pairs was constructed, which could effectively stratify patients into high- and low-risk groups with significantly different overall survival across all datasets (all p < 0.05, log-rank test). The high-risk group exhibited an immunosuppressive microenvironment with reduced CD8+ T cells and B cells and increased monocytes (p < 0.05, Wilcoxon test). Functional analysis of methylation differences between risk groups showed significant immune response-related pathways such as antigen processing and presentation. The high- and low-risk patients also exhibited distinct sensitivity to cytotoxic chemotherapy, targeted therapy, and immunotherapy, with the low-risk group showing higher immunophenoscore, cytolytic activity, and tumor mutational burden. CM-LP11 represents a robust lactylation-related prognostic biomarker that predictive survival outcomes and therapeutic responses in CM patients, proving insights for personalized treatment strategies.
Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Although epigenetic alterations are common in CRC, the epigenetic changes that occur during colorectal carcinogenesis remain unclear. Thus, we sought to elucidate the role of NOS1 methylation in colorectal carcinogenesis, which is essential for understanding disease pathology. We used the UCSC Xena database to comparatively analyze NOS1 expression and methylation status between tumour and adjacent normal tissues across 33 cancer types. Low expression and hypermethylation of NOS1 have been identified in 12 cancer types, with CRC demonstrating this characteristic epigenetic regulation. NOS1 promoter methylation status was examined using genomic DNA extraction and MassARRAY EpiTYPER methylation analysis. NOS1 hypermethylation was confirmed among CRCs in in-house dataset 1 (p < 0.001), and among CRCs and advanced adenomas in in-house dataset 2 (p < 0.001). An upward trend in methylation changes was identified from non-advanced adenoma to advanced adenoma to CRC (p for trend < 0.001). Quantitative real-time PCR was used to analyze NOS1 expression in in-house Dataset 3, and significantly low NOS1 expression was also identified in CRCs (p < 0.001). Kaplan-Meier estimator and Cox proportional hazard models were used to assess the prognostic and predictive roles of NOS1. NOS1 hypermethylation was significantly associated with better disease-specific survival (DSS) in CRC patients. NOS1 hypermethylation is an epigenetic driver of colorectal tumorigenesis and is associated with better survival.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, yet current surveillance with alpha-fetoprotein (AFP) and ultrasound (US) lacks sufficient accuracy, particularly for early-stage disease. This study aimed to develop and clinically validate a blood-based digital PCR (dPCR) assay targeting novel methylation biomarkers for HCC detection. Genome-wide methylation profiles from HCC, normal, and other cancer types were analysed to identify candidates, which were then screened and verified in cancer cell lines, primary tumour tissues, and plasma specimens. A total of 186 plasma samples were evaluated, including 66 healthy controls, 60 patients with chronic liver disease (CLD) without HCC, and 60 patients with HCC. FAR1 methylation emerged as an HCC-specific biomarker, showing significantly higher levels in liver cancer cell lines and HCC tumour tissues compared with controls. A dPCR assay targeting FAR1 achieved 70.0% sensitivity (42/60; 95% CI, 56.8%-81.2%) and 96.8% specificity (122/126; 95% CI, 92.1-99.1%), with an area under the curve (AUC) of 0.90 (95% CI, 0.85-0.96). Combining FAR1 with AFP improved sensitivity to 88.3% (53/60; 95% CI, 77.4%-95.2%) while maintaining 96.8% specificity. Notably, early-stage HCC (Barcelona Clinic Liver Cancer [BCLC] stage 0-A) was detected with 85.4% sensitivity (35/41; 95% CI, 77.4%-94.4%). This blood-based dPCR assay demonstrated high diagnostic performance, underscoring its potential as a noninvasive tool to enhance early detection and clinical management of HCC in high-risk populations.
Epigenetic aging prior to midlife is gaining interest as an intervenable period to address health and cognitive aging. Epigenetic changes may index DNA methylation aging rates, but methylation profiles may not be substitutable across tissues. We compared DNA methylation clocks and age acceleration in saliva, buffy coat (BC), and peripheral blood mononuclear cells (PBMC) collected in 91 individuals (7 unpaired, 20 siblings, 64 twins; 18 monozygotic (MZ), 14 dizygotic (DZ) pairs) from the Colorado Adoption/Twin Study of Lifespan behavioral development and cognitive aging (CATSLife1; Mean age = 30.90 years [range = 28.07-41.13]; 50.5% female). Across 15 DNA methylation clocks, chronological age and DNA methylation ages were moderately associated (mean Spearman correlations: r = 0.37, Saliva; r = 0.40, BC; r = 0.34, PBMC). In mixed-effects models, saliva showed higher DNA methylation ages (B = 3.77-19.72 years vs BC, p < 0.001), whereas PBMC and BC were comparable (B = -0.06-0.39 years vs BC, p ≥ 0.436). The exception was the next-generation clock DunedinPace showing comparability (p = 0.486). Similar patterns were observed for age acceleration estimates. Altogether, MZ pairs (meta-analytic r = 0.49, 95%CI = 0.32,0.66) and DZ pairs (meta-analytic r = 0.38, 95%CI = 0.25,0.52) were moderately correlated (Spearman), but MZ pairs showed heterogeneity across tissues (p < 0.034): saliva was lower (mean r = 0.33, SD = 0.25) than BC (mean r = 0.64, SD = 0.10) and PBMC (mean r = 0.49, SD = 0.18). Next-generation PCGrimAge and DunedinPace clocks showed consistent zygosity correlations across tissues, while multi-tissue clocks (e.g. ZhangQ) showed comparable MZ-DZ correlations. While saliva-based DNA methylation is not a direct substitute for blood-based DNA methylation, BC and PBMC show comparability; nevertheless, all tissue types may be appropriate for DNA methylation aging studies when compared within tissues.
