An entosis-informed prognostic index for survival stratification and molecular characterization in lung adenocarcinoma

Abstract

Entosis is an actomyosin-dependent cell-in-cell process that may culminate in lysosome-mediated entotic cell death and is implicated in tumor evolution. Its clinical relevance in lung adenocarcinoma (LUAD) remains unclear. To construct an Entosis-related Prognostic Prediction Index (EPPI), this study integrated TCGA-LUAD transcriptomic, clinical, and somatic mutation data. Three independent GEO cohorts provided external validation. The initial candidate gene set covered components driving cell-in-cell formation and the intracellular degradation of internalized cells. For subgroup comparison, the analytical approach incorporated survival analysis, functional enrichment, tumor microenvironment profiling, mutation analysis, immunotherapy assessment, and drug-sensitivity prediction. Tissue and cellular distributions of the EPPI genes were mapped using spatial transcriptomics and single-cell RNA sequencing. Molecular docking assessed predicted drug-target interactions. Finally, in vitro RAC1 knockdown experiments evaluated the cellular function of a representative EPPI gene. Comprising RND3, RAC1, KRAS, CTNNA1, AURKA, and ACTB, the final EPPI consistently stratified overall survival across all cohorts and showed moderate prognostic discrimination. The high-EPPI group had greater tumor purity and mutational burden, with enhanced cell-cycle, DNA-repair, epithelial–mesenchymal transition, and NF-κB programs, whereas the low-EPPI group showed greater immune and stromal infiltration. TIDE and immunophenoscores also differed between subgroups. Drug-response models predicted greater sensitivity to cisplatin and paclitaxel in the high-EPPI group and to several targeted agents in the low-EPPI group. Molecular docking predicted RAC1–EHT-1864 and AURKA–alisertib interactions. Spatial and single-cell analyses showed heterogeneous EPPI-gene expression, with ACTB and RAC1 broadly distributed and AURKA restricted to selected populations. RAC1 depletion suppressed LUAD-cell proliferation, DNA synthesis, migration, and invasion. The EPPI is an entosis-informed prognostic index for survival stratification in LUAD. Based on this index, patients can be separated into subgroups with distinct proliferative features, genomic alterations, tissue compositions, and predicted drug sensitivities. These findings associate entosis-related molecular features with distinct cellular and clinical characteristics of LUAD.