Abstract
<title>Abstract</title> <p>Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases and is a major cause of cancer-related mortality, largely owing to metastasis. Anoikis resistance is essential for tumor cell survival during metastatic dissemination, yet its molecular regulation in NSCLC remains unclear. In this study, we identify the deubiquitinase USP3 as a key mediator of anoikis resistance and metastasis in NSCLC. USP3 enhanced tumor cell survival under non-adherent conditions and promoted metastatic colonization in vitro and in vivo. Mechanistically, mass spectrometry and co-immunoprecipitation identified DNMT1 as a USP3-interacting protein. USP3 deubiquitinated and stabilized DNMT1, thereby maintaining DNMT1 protein levels and supporting anoikis resistance and metastatic capacity. qPCR and ELISA further showed that TGF-β1 expression was regulated downstream of this axis. Clinically, USP3/DNMT1/TGF-β1 co-expression in NSCLC tumor cells was more frequently detected in patients with lymph node metastasis. These findings define a USP3–DNMT1 regulatory axis that promotes anoikis resistance and metastasis in NSCLC and may provide potential therapeutic targets for metastatic disease.</p>