Pharmacological inhibition of JNK-MAPK disrupts cigarette smoke-induced RUNX2/Galectin-3 driven EMT and cancer stemness in lung adenocarcinoma.

Abstract

Cigarette smoke (CS), a major driver of lung cancer (LC), promotes epithelial-mesenchymal transition (EMT) and stemness resulting in metastasis, therapy resistance, and recurrence, but the precise mechanism is elusive. Building on our earlier identification of Runt related transcription factor-2 (RUNX-2) and Galectin-3 (Gal-3) as mediators of CS-induced EMT, in this study, we aim to identify a potential molecular mechanism and delineate the upstream regulators of RUNX-2 using A549 lung adenocarcinoma cells and human small airway epithelial cells (SAECs) cultured at the air-liquid interface (ALI). CSE exposure markedly elevated intracellular reactive oxygen species (ROS), assessed via DCFH-DA assay, and promoted invasive behavior (Boyden chamber assay), spheroid formation, and colony formation, the hallmarks of cancer stemness. Expression analysis via RT-qPCR, immunoblotting, and immunocytochemistry revealed that CSE upregulated EMT and stemness-associated markers, notably via upregulating RUNX2 and Galectin-3, at both transcriptional and translational levels through the involvement of c-Jun N-terminal kinase- Mitogen-Activated Protein Kinase (JNK-MAPK) pathways. A specific pharmacological inhibitor of JNK (SP600125) significantly attenuated CSE-induced RUNX2 and Galectin-3 expression, and also reversed CSE-driven EMT marker alterations, suppressed transcriptional EMT perturbations, and reduced proinflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). In conclusion, this study identifies that ROS/JNK/RUNX2/Gal-3 axis drives CS-induced oncogenic plasticity, suggesting that targeted inhibition of this pathway could be an effective strategy for mitigating CS-related LC progression.