Abstract: Background and purpose: Ethanol has been reported to stimulate progression of breast cancer, yet the underlying mechanism is not fully understood. This study aimed to investigate effects of ethyl alcohol (EtOH) on the calcium-activated neutral protease (CANP)-cyclin E/focal adhesion kinase (FAK) signaling and cell migration in breast cancer cells, as well as the role of epidermal growth factor receptor (EGFR) in the EtOH-stimulated effects, in order to assess the signaling mechanism(s) underlying how EtOH enhances cancer progression. Methods: Human breast cancer cell line MCF-7 was employed as a model system, with MCF-10A mammary epithelial cells as control. In vitro wound healing assay was carried out to evaluate EtOH-induced cell migration. The effects of EtOH or epidermal growth factor on the proteolysis of cyclin E/FAK were detected by Western blot. EGFR inhibitor (EGFR-I) and a specific inhibitor for CANP, Calpeptin, were applied to pretreat cultured cells to explore their influences on the cell migration and cyclin E/FAK proteolysis triggered by EtOH. Results: Treatment of model cells with EtOH (0.3%) stimulated significant proteolysis of cyclin E/FAK in a dose-/time-dependent manner and increased migration (+47.30%, P<0.05) in MCF-7 breast cancer cells, but had no significant effect on migration in MCF-10A cells. Pretreatment with Calpeptin (10 μmol/L) significantly reduced EtOH (0.3%)- or EGFR (10 ng/mL)-induced cyclin E/FAK truncation. EGFR-I (3 μmol/L) profoundly reduced EtOH-indcued CANP dependent proteolysis of CANP1 and cyclin E/FAK as well as cell migration (-53.00%, P<0.01). Conclusion: EtOH significantly stimulates activation of CANP via EGFR pathway, resulting in proteolysis of cyclin E/FAK and migration in MCF-7 breast cancer cells, suggesting EGFR-CANP signaling to be a potential target for suppression of metastasis in breast cancer.

Key words: Ethanol, Epidermal growth factor receptor, Calcium-activated neutral protease, Migration, Breast cancer