MAP3K8 is associated with an immune-active tumor microenvironment and suppresses malignant phenotypes of NSCLC cells in vitro

MAP3K8, also known as Tpl2/Cot, participates in MAPK- and NF-κB-related signaling and plays important roles in inflammation and immune regulation. However, its expression pattern, prognostic value, and immune relevance in non-small cell lung cancer (NSCLC) remain unclear. Transcriptomic, clinical, pan-cancer, single-cell RNA-sequencing, and cell-line datasets were integrated to characterize MAP3K8 expression and its clinical and immune associations in NSCLC, particularly lung adenocarcinoma (LUAD). Survival, immune-infiltration, functional-enrichment, molecular-subtype, N6-methyladenosine (m6A)-related, Tumor Immune Dysfunction and Exclusion (TIDE), and exploratory computational drug-response analyses were performed. An independent institutional cohort of 114 patients with NSCLC was additionally analyzed to validate the association between MAP3K8 protein expression and overall survival. MAP3K8 protein expression and subcellular localization were evaluated by Western blotting, immunohistochemistry, and immunofluorescence. To investigate the tumor-cell-intrinsic effects of MAP3K8, MAP3K8 was overexpressed in A549 cells and depleted in H1975 cells, followed by proliferation, migration, and invasion assays. MAP3K8 expression was reduced in LUAD tissues, and higher expression was associated with favorable overall survival. This association was further supported in an independent institutional cohort of 114 patients with NSCLC, in which high MAP3K8 protein expression was associated with significantly longer overall survival (log-rank P < 0.001) and remained significantly associated with survival after multivariable adjustment for the clinicopathological variables included in the model. Single-cell analysis showed that MAP3K8 was predominantly expressed in dendritic cells, monocytes/macrophages, and other immune-cell populations rather than malignant epithelial cells. Higher MAP3K8 expression was associated with an immune-active tumor microenvironment. Functional gain- and loss-of-function experiments further showed reciprocal effects of MAP3K8 manipulation on NSCLC cell proliferation, migration, and invasion. MAP3K8 expression is associated with favorable prognosis and an immune-active tumor microenvironment in LUAD/NSCLC. The prognostic association observed in public transcriptomic data was further supported by an independent institutional cohort. Complementary gain- and loss-of-function experiments indicate that MAP3K8 can negatively regulate malignant phenotypes of NSCLC cells in vitro. However, because MAP3K8 expression in primary tumors is strongly influenced by immune-cell composition, further studies incorporating tumor purity, immune-cell fractions, and independent treatment-annotated cohorts are required to establish its independent prognostic and mechanistic significance.