Breast cancer is a highly heterogeneous disease characterized by complex molecular interactions involving multiple biological pathways associated with tumor progression, therapeutic resistance, immune regulation, and genomic instability. Conventional functional enrichment approaches, such as over-representation analysis (ORA), are widely used to interpret high-throughput genomic datasets; however, these methods often analyze genes as independent entities and may therefore generate broad and overlapping biological categories that are difDicult to interpret in complex diseases. The aim of the present study was to investigate whether network-based annotation enrichment using the NetAn framework could improve the biological speciDicity, sensitivity, and interpretability of breast cancer associated gene sets compared with conventional enrichment approaches.
A curated dataset consisting of 997 breast cancer associated genes was analyzed using conventional enrichment methods implemented in g:ProDiler and clusterProDiler, followed by network-based enrichment analysis using the NetAn workDlow. Protein–protein interaction data from STRINGdb v12.0 were used to construct interaction networks, perform network guided gene expansion, and identify interaction deDined functional clusters. Functional enrichment analysis was subsequently performed on both the complete gene set and the individual network clusters. Cytoscape was used for network visualization and topological analysis.
Conventional enrichment analyses identiDied signiDicantly enriched pathways associated with PI3K-Akt signaling, MAPK signaling, estrogen signaling, homologous recombination, Fanconi anemia pathway, immune signaling, and DNA repair. Network analysis successfully mapped 709 genes to the STRING interaction network and generated a densely connected protein–protein interaction network containing 2,010 high-conDidence interactions. Network-based clustering identiDied biologically distinct functional modules associated with oncogenic signaling, epithelial differentiation, immune-inDlammatory signaling, and genome stability pathways. Comparative analyses further demonstrated that the clustered NetAn workDlow identiDied the highest number of tumor-relevant annotations while improving functional stratiDication and reducing enrichment redundancy relative to conventional ORA approaches.
Collectively, the Dindings of this study demonstrate that incorporation of protein–protein interaction topology can improve interpretation of heterogeneous breast cancer associated gene sets by partitioning broad enrichment proDiles into biologically coherent interaction-deDined functional modules. These results support the value of network-based annotation enrichment approaches in systems biology, cancer genomics, and future precision oncology research.