SUBSTRATE STIFFNESS DRIVES A TRANSCRIPTIONAL PROGRAM LINKED TO POOR PROGNOSIS IN LUNG ADENOCARCINOMA

To identify stiffness-associated transcriptional changes in lung cancer cells and evaluate their prognostic relevance in adenocarcinoma cohorts. A549 cell were cultured on soft (2kPa) and stiff (32kPa) substrates. Proliferation was measured by MTT at days 3, 5 and 7, and RNA-seq was performed in parallel. Differentially expressed genes at 7 days (padj < 0.05, |log2FC| > 1) were tested for survival association in the GSE68465 (n=439) using multivariate Cox regression. Genes both stiffness-regulated and survival-associated defined a 78-gene rigidity signature. Prognostic value was evaluated with in Cox models and validated in an independent cohort (GSE72094, n=393). Functional enrichment analysis identified related biological processes. Proliferation increased significantly at 7 days on stiff matrices. Transcriptomic analysis identified 2585 differentially expressed genes, 235 associated with survival and 78 forming the rigidity signature In the discovery cohort, this signature predicted worse survival (HR per SD = 1.7 CI 1.45-1.9, concordance = 0.74, p<0.001). Validation in an independent cohort confirmed its prognostic power (HR per SD = 1.7 CI 1.4-2.0, concordance = 0.71, p<0.001). Enrichment analysis revealed pathways related to positive regulation of locomotion and cell migration. Substrate stiffness modulates cancer cell behaviour, inducing a transcriptional phenotype associated with increased mortality in lung adenocarcinoma.