Loss of protein phosphatase 1 regulator TIMAP protein triggers EMT in A549 cells.

backgroundEpithelial–mesenchymal transition (EMT) is a cellular reprogramming process that enables epithelial cells to acquire mesenchymal traits, leading to enhanced motility, invasiveness, and resistance to therapy. Transforming growth factor-β-inhibited, membrane-associated protein (TIMAP) is a regulatory subunit of protein phosphatase 1. Altered TIMAP expression has been implicated in tumor progression, suggesting a potential role in EMT.

methodsHuman A549 lung adenocarcinoma cells were subjected to stable lentiviral shRNA-mediated knockdown of TIMAP. Phenotypic, molecular, and functional consequences of TIMAP depletion were assessed using Western blotting, RT-qPCR, high-content imaging, electric cell–substrate impedance sensing, RNA sequencing with pathway enrichment, cytokine array profiling, 3D spheroid assays, trans-endothelial migration assays and metabolic activity measurements. EMT-associated signalling pathways, including TGF-β/SMAD, Wnt/β-catenin, and PI3K/Akt, were examined. Expression and survival correlations were evaluated using publicly available lung adenocarcinoma datasets.

resultsTIMAP knockdown was confirmed at both transcript and protein levels and induced pronounced morphological changes toward a mesenchymal-like phenotype. shTIMAP cells exhibited enhanced attachment, accelerated wound closure, and increased transendothelial migration. Transcriptomic profiling revealed enrichment of EMT-associated pathways. shTIMAP cells displayed reduced epithelial protein markers such as E-cadherin and ZO-1 and increased mesenchymal markers like N-cadherin, Snail, and Slug expression. Activation of EMT-related signalling, including elevated phosphorylation of SMAD2/3, β-catenin, and Akt was also observed. Re-expression of TIMAP or pharmacological inhibition of the TGF-β pathway reversed EMT phenotype. TIMAP depletion altered the secretory profile toward pro-inflammatory, pro-invasive cytokines and disrupted 3D spheroid architecture, promoting spheroid disintegration and invasive outgrowth. TIMAP depletion shifted cellular metabolism from oxidative phosphorylation towards glycolysis and induced cancer stem cell phenotype, with upregulation of CD44, CD133 and downregulation of CD24. Clinically, TIMAP expression is significantly downregulated in lung adenocarcinoma already in early-stage disease, and low tumoral TIMAP levels predict poor survival.

conclusionLoss of TIMAP promotes EMT, enhances motility and invasion, and induces pro-tumorigenic signalling and secretory changes in A549 lung adenocarcinoma cells. These effects likely contribute to the association between low TIMAP expression and adverse patient outcomes, highlighting TIMAP as a potential prognostic biomarker and therapeutic target in lung cancer.