INTRODUCTION AND OBJECTIVES: Breast cancer is the leading cause of cancer deaths among women in the world. Recently, our group has demonstrated that FOXK2 transcription factor mediates drug sensitivity in breast cancer cells through transcriptional activation of FOXO3a. Conversely, drug-resistant cells and poor outcome patients show constitutively high protein levels of FOXK2, suggesting that post-translational modifications might be inactivating FOXK2 functions, contributing to drug resistance. An in silico analyses revealed the oncogenic kinase AKT (isoform 1) as the main possible regulator of FOXK2. Thus, our objective is to investigate the role of AKT phosphorylation in the regulation of FOXK2 expression and evaluate the impact of AKT-FOXK2 regulatory axis on doxorubicin resistance in breast cancer. MATERIALS AND METHODS: The MTT and clonogenic assays were performed to assess drug-induced cytotoxicity. For AKT1 knockdown, cells were transfected with non-silencing control (NSC) and AKT1 siRNA using Lipofectamine RNAimax. The LY294002 PI3K inhibitor and the MK2206 AKT inhibitor were used for pharmacological inhibition of AKT phosphorylation. For FOXK2 overexpression, cells were transfected with the empty vector (pCMV5) and the pCMV5-FOXK2 vector using Lipofectamine2000. For inducible constitutively active AKT overexpression, cells were transfected with pBABE-(myr)Akt:ER vector and treated with 200 nM of 4-hydroxytamoxifen. The NE-PER kit was used for cytoplasmic and nuclear extractions. Protein expression in whole and fractioned lysates were examined by Western Blotting. RESULTS AND CONCLUSIONS: Our data indicate that higher protein levels of FOXK2 are associated with increased levels of AKT phosphorylation and with drug resistance in breast cancer cells. Also, FOXK2 and phosphorylated AKT expression was found localized predominantly in the nucleus of drug-resistant MDA-MB-231 cells. Following genetic and MK2206 pharmacological inhibition of AKT, MDA-MB-231 cells showed decreased cell viability, increased sensitivity to doxorubicin treatment and reduced FOXK2 protein levels, particularly of slower mobility bands. Following LY294002 treatment, FOXK2 protein levels were slightly altered and there was no change in FOXK2 subcellular localization, suggesting that AKT activation is not involved in FOXK2 nuclear-cytoplasmic shuttling in our model. Co-transfection experiments revealed that AKT1 inhibition not only decreased endogenous levels, but also exogenous expression of FOXK2. Consistently, overexpression of constitutively active AKT increased FOXK2 protein levels either in MCF-7 and MDA-MB-231 cells. Altogether, our results suggest that AKT regulates FOXK2 expression in chemoresistant breast cancer cells, providing novel insights into molecular mechanisms of drug resistance. Future experiments involving treatment with lambda phosphatase and co-immunoprecipitation will allow a better understanding of AKT-FOXK2 axis.