In this study, we report the identification of a novel role of SIRT6 inboth epirubicin and paclitaxel resistance in breast cancer. We foundthat SIRT6 protein levels are elevated in paclitaxel- and epirubicinresistantMCF-7 cells compared with the parental sensitive cells. SIRT6 knockout and depletion sensitized cells to both paclitaxel andepirubicin treatment, whereas SIRT6 ectopic overexpression led toincreased resistance to paclitaxel and epirubicin. Moreover, ourdata suggest that SIRT6 could be mediating epirubicin resistancethrough enhancing the DNA repair response to epirubicin-inducedDNA damage. Clonogenic assays also revealed that mouse embryonicfibroblasts (MEFs) lacking SIRT6 have decreased long-termviability in response to epirubicin. The tumour suppressor FOXO3aincreases its levels of acetylation in MEFs depleted of SIRT6,whereas its induction by epirubicin is attenuated in breast cancercells overexpressing SIRT6. Further cell viability studies demonstratethat deletion of FOXO1/3/4 in MEFs can confer sensitivity toboth paclitaxel and epirubicin, suggesting that SIRT6 reduces paclitaxeland epirubicin sensitivity, at least in part, through modulatingFOXO acetylation and expression. Consistently, immunohistochemicalanalysis of 118 breast cancer patient samples revealed thathigh SIRT6 nuclear staining is significantly associated with pooreroverall survival (P = 0.018; Kaplan-Meier analysis). MultivariateCox analysis demonstrated that nuclear SIRT6 staining remainedassociated with death after correcting for tumour stage and lymphnodeinvolvement (P = 0.033). Collectively, our data suggest thatSIRT6 has a role in paclitaxel and epirubicin sensitivity via targetingFOXO proteins and that SIRT6 could be a useful biomarker andtherapeutic target for paclitaxel- and epirubicin-resistant cancer.