Functional and mechanistic inactivation of immune hubs orchestrates tumor development and progression in lung adenocarcinoma

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer (NSCLC) and remains a leading cause of cancer-related deaths worldwide. Despite advances in targeted therapies and immune checkpoint inhibitors, survival outcomes remain poor, underscoring the need for novel immune-related biomarkers to improve diagnosis, prognosis, and therapeutic strategies. METHODOLOGY: We retrieved 1,284 immune-related genes from the ImmPort database and constructed a protein-protein interaction (PPI) network using STRING, with hub genes identified via Cytoscape. Expression validation was performed using RT-qPCR with TaqMan assays in LUAD and normal lung cell lines, and extended validation was carried out through GSCA (TCGA datasets), HPA, OncoDB, cBioPortal, KM Plotter, TISIDB, and TargetScan. Functional assays including proliferation, colony formation, wound-healing, and xenografts were performed in LUAD cell lines (A549, H1299) following overexpression of hub genes. RESULTS: Four hub genes, IFNG, TNF, CD4, and CD8A, were identified as central immune regulators. All were significantly downregulated in LUAD tissues and cell lines, with ROC analysis confirming diagnostic potential. TCGA and HPA datasets validated reduced expression at both mRNA and protein levels, while promoter methylation and CNV alterations were linked to transcriptional repression. Kaplan-Meier analysis revealed that low expression predicted poor overall survival. TargetScan identified four candidate miRNAs (miR-130b-3p, miR-326, miR-4319, and miR-4644), validated as upregulated in LUAD, with ROC curves showing strong diagnostic accuracy. Immune subtype and infiltration analysis confirmed strong associations of hub genes with T-cell-mediated immunity and immune checkpoint molecules, while drug sensitivity analysis linked their expression to responses to chemotherapy and targeted agents. Functional assays demonstrated that hub gene overexpression suppressed proliferation, migration, and invasion in vitro and inhibited tumor growth in xenograft models. CONCLUSION: This integrative study establishes IFNG, TNF, CD4, and CD8A as novel immune-related biomarkers in LUAD, regulated through genetic, epigenetic, and post-transcriptional mechanisms. Their diagnostic, prognostic, and therapeutic significance highlights their potential as targets for precision oncology in LUAD.