Autophagy-Associated Regulation of MTOR, BECN1, and SQSTM1 Expression in NSCLC Cells

Background: Autophagy is implicated in cancer progression and therapeutic response, but the effects of the DNA-demethylating agent decitabine (5-aza-dC) on autophagy-related pathways in non-small-cell lung cancer (NSCLC) remain incompletely defined. Objective: This study investigated treatment-associated changes in BECN1, SQSTM1, and MTOR using integrated bioinformatic and experimental approaches. Method: GEPIA2 and STRING were used to assess gene expression, clinical associations, correlations, and functional interactions in LUAD and LUSC. A549 and NCI-H460 cells were treated with 2.5 or 5 µM 5-aza-dC for 72 h, followed by RT-qPCR, Western blotting, and acridine-orange flow cytometry. Results: Bioinformatic analysis revealed subtype-dependent expression patterns and positive correlations of BECN1 with MTOR and SQSTM1, although none of the three genes was significantly associated with overall survival. Experimentally, 5-aza-dC reduced MTOR mRNA in both cell lines, whereas BECN1 and SQSTM1 showed cell-dependent responses. Protein analysis demonstrated increased Beclin-1 and SQSTM1/p62 abundance, while total mTOR was significantly reduced only in H460 cells at 5 µM. Acridine-orange fluorescence increased after treatment, particularly in H460 cells at 5 µM. Conclusions: 5-aza-dC induces cell-line-dependent changes in autophagy-related transcripts and proteins in NSCLC cells. However, these findings do not establish mTORC1 activity, autophagic flux, or direct promoter demethylation.