Background/Objectives: Triple-negative breast cancer (TNBC) is an aggressive subtype lacking ER, PR, and HER2 expression, with limited targeted therapies and poor outcomes. Epigenetic dysregulation, particularly aberrant DNA methylation, is a key driver. Decitabine, a DNA methyltransferase inhibitor (DNMTi), shows promise by reactivating silenced tumor suppressor genes and modulating immune responses. This systematic review evaluates preclinical and clinical evidence on decitabine’s efficacy, mechanisms, and translational potential in TNBC. Methods: A PRISMA-2020 compliant search of PubMed, EBSCO, Web of Science, and Semantic Scholar was conducted up to April 2025. Included studies assessed decitabine alone or in combination in TNBC preclinical or clinical settings. Risk of bias was assessed using QUIPS and RoB 2.0 tools. Results: Twenty-five studies were included. In vitro, decitabine-induced growth inhibition, apoptosis, and re-expression of silenced genes (such as BRCA1 and CDH1). In vivo, it reduced tumor burden and enhanced anti-tumor immunity through MHC-I, PD-L1, and STING pathway upregulation. Synergy was noted with anti-PD-1, HDAC inhibitors, and chemotherapy. Resistance mechanisms included persistent DNMT activity, low DCK, and miRNA-driven escape (miR-155–TSPAN5). Conclusions: Decitabine demonstrates strong preclinical and early clinical potential in TNBC via epigenetic reprogramming and immune activation. Future strategies should focus on biomarker-based selection and resistance mitigation.

Objectives: Breast cancer (BC) subtypes such as HR+, HER2+, and triple-negative (TNBC) show distinct molecular features, treatment responses, and outcomes. DNA methylation is a key, targetable epigenetic regulator in BC. This study examined whether the DNA methyltransferase inhibitor decitabine (DAC) produces subtype-specific epigenomic and transcriptional effects in breast cancer cell lines representing distinct molecular subtypes. Methods: Gene expression and DNA methylation data from DAC-treated and untreated T-47D (Luminal-A) and JIMT-1 (HER2-amplified, trastuzumab-resistant with a TNBC-like phenotype) breast cancer cell lines were obtained from a published dataset. Differential expressions were assessed using limma , and methylation changes were defined using β-value thresholds. Integrated epigenomic–transcriptional analysis, functional enrichment, Horvath clock CpG evaluation, and survival analysis were performed in the METABRIC and TCGA cohorts. Results: In JIMT-1, DAC caused hypomethylation at 1195 CpG sites and upregulation of 187 genes, including TFAP2E , an age-associated locus selectively hypomethylated after DAC. In T-47D, DAC induced hypomethylation at 1937 CpGs and upregulated 248 genes. Amongst these, KRT20 was upregulated despite promoter hypermethylation, indicating a subtype-specific regulatory architecture. DAC-responsive genes in JIMT-1 were enriched for cytokine signaling and piRNA-mediated epigenetic silencing, whereas T-47D showed enrichment for extracellular matrix organization, collagen dynamics, and piRNA processing pathways. Horvath clock CpG analysis showed selective perturbation of age-associated sites. Survival analysis identified 114 DAC-responsive genes associated with overall survival in ER/PR-positive BC and 8 in the JIMT-1-derived gene set. Conclusion: DAC induces subtype-dependent epigenomic and transcriptional remodeling, selectively disrupts age-associated regulatory programs, and underscores the need for subtype-stratified evaluation of epigenetic therapies in breast cancer.