Abstract
Background: Breast cancer (BC) continues to be a predominant cause of cancer-related deaths among women globally, highlighting the complexity of the disease, its propensity for metastasis, and resistance to conventional therapies. The discovery of clinical biomarkers and therapeutic targets is crucial for advancing BC treatment strategies. Methods: We conducted a Transcriptome-Wide Association Study (TWAS) and Weighted Gene Co-expression Network Analysis (WGCNA) to pinpoint genes associated with BC, with GSTM4 emerging as a candidate tumor suppressor. To further elucidate GSTM4's role, we analyzed its mRNA and protein expression using multiple databases. The prognostic significance of GSTM4 was evaluated using KM-Plotter tools, while its epigenetic profile was examined through GSCA. Protein-Protein Interaction (PPI) networks were constructed with GeneMANIA and STRING to explore GSTM4's functional interactions. The ssGSEA algorithm and TIMER were employed to link GSTM4 with the immune context of BC. Single-cell RNA sequencing from GSE148673 was analyzed to investigate GSTM4's influence on the tumor immune microenvironment. In vitro experiments were designed to assess GSTM4's impact on BC cell behavior. Results: GSTM4 expression is diminished in BC tissues and is positively linked to better patient outcomes. Epigenetic studies indicate that GSTM4 silencing may partly result from promoter deletion mutation. The PPI network and enrichment analysis are consistent with GSTM4's multifaceted role in glutathione metabolism, detoxification, antioxidant defense, and oxidoreductase reactions. GSTM4 expression shows a negative correlation with immune checkpoint gene expression and is associated with an enhanced antitumor immune response in BC as well as increased sensitivity to anticancer drugs. Single-cell RNA sequencing analysis suggests a association of GSTM4 with the prevention of epithelial transformation, the mitigation of BC cell malignancy, and the promotion of tumor microenvironment interactions. In vitro studies confirm GSTM4's inhibitory effects on BC cell proliferation, invasion, and stemness. Conclusion: Integrated analyses identify GSTM4 as a potential tumor suppressor and prognostic biomarker in BC, suggesting its promise for advancing therapies.