Loss of Calpain-2 attenuates TGF-β-driven Epithelial-Mesenchymal transition and invasive behavior in A549 lung cancer cells.

Lung cancer accounts for the highest number of cancer-related deaths globally, largely due to its high propensity for early and widespread metastasis. Calpains, a family of calcium-dependent cysteine proteases, are involved in various physiological processes, including cytoskeletal remodeling, migration, and invasion. Among them, calpain-2 has been implicated in promoting cancer cell invasiveness and metastatic behavior, although its precise role in non-small cell lung cancer (NSCLC) remains poorly understood. In this study, calpain-2-deficient cells were generated from the A549 lung adenocarcinoma cell line using CRISPR/Cas9 technology, and the effects of calpain-2 loss on cell migration, invasion, and epithelial-mesenchymal transition (EMT) were evaluated in comparison with control cells. We also evaluated the effects of transforming growth factor-β (TGF-β), a cytokine known to regulate diverse cellular processes, including proliferation, migration, and differentiation. Our results demonstrated that calpain-2 deficiency significantly reduced cell migratory capacity, EMT progression, and invasive potential following TGF-β stimulation. These effects were mediated by multiple molecular mechanisms, including impaired actin filament remodeling due to calpain-2 deficiency, reduced activation of Matrix Metalloproteinase (MMP)-2 and MMP-9, and decreased expression of EMT-related transcription factors such as Snail, Slug, and ZEB1. Collectively, these findings suggest that calpain-2 plays a central role in TGF-β-mediated cancer cell migration, invasion, and EMT progression, and that targeting calpain-2 function may represent a potential therapeutic strategy for inhibiting lung cancer metastasis.