AbstractAutophagy is a cellular mechanism by which cells degrade intracellular components in lysosomes, maintaining cellular homeostasis. It has been hypothesized that autophagy could have a role in cancer prevention through the elimination of damaged proteins and organelles; this could explain epidemiological evidence showing the chemopreventive properties of the autophagy-inducer metformin. In this study, we analyzed the autophagy-related effect of metformin in both cancer initiation and progression in non-tumorigenic cells. We also analyzed the induction of tumorigenesis in autophagy-deficient cells, and its correlation with the ER stress. Our results showed that metformin induced massive cell death in preneoplastic JB6 Cl 41-5a cells treated with tumor promoter (phorbol) and in NIH/3T3 treated with H₂O₂. Inhibiting autophagy with wortmannin or ATG7 silencing, the effect of metformin decreased, indicating an autophagy-related cytotoxic activity under stress conditions. We also found an induction of tumorigenesis in ATG7-silenced NIH/3T3 cell clone (3T3-619C3 cells), but not in wild-type and in scrambled transfected cells, and an upregulation of unfolded protein response (UPR) markers in 3T3-619C3 cells treated with H₂O₂. These findings suggest that autophagic cell death could be considered as a new mechanism by which eliminate damaged cells, representing an attractive strategy to eliminate potential tumorigenic cells.