AKT-MEDIATED REGULATION OF FOXK2 TRANSCRIPTION FACTOR: MOLECULAR MECHANISMS AND POTENTIAL ROLE IN BREAST CANCER DRUG RESISTANCE

INTRODUCTION AND OBJECTIVES: FOXK2 is a transcription factor which modulates drug sensitivity in breast cancer cells. In contrast, drug-resistant cell lines exhibit constitutively high FOXK2 protein levels, suggesting that post-translational modifications might impair its functions. An in silico analysis revealed AKT oncogenic kinase as the main putative regulator of FOXK2 by phosphorylation. Then, the objective of this project is to evaluate the effect of AKT modulation in the regulation of FOXK2 protein levels and the impact of the AKT-FOXK2 axis on drug resistance in breast cancer. MATERIALS AND METHODS: The MTT and clonogenic assays were performed to assess drug-induced cytotoxicity in MCF-7 (chemosensitive) and MDA-MB-231 (chemoresistant) cell lines. AKT1 and GSK3 knockdown was done by RNA interference. LY294002 and MK-2206 were used for pharmacological inhibition of AKT. Overexpression of FOXK2 and constitutively active AKT was performed by transient transfections. The NE-PER kit was used for cytoplasmic and nuclear extractions. Protein levels in whole and fractioned lysates were examined by Western Blotting following electrophoresis in SDS-PAGE and Phos-tag gels. RNA levels were examined by real-time PCR (SYBR Green Master Mix method). RESULTS AND CONCLUSION: Inhibition of AKT, but not GSK3β, resulted in a reduction in endogenous and exogenous FOXK2 protein levels, particularly in the lower mobility bands. Interestingly, FOXK2 RNA levels are increased in AKT-inhibited cells, suggesting that modulation of FOXK2 protein levels is possibly dependent on post-translational modifications. Differently from MCF-7, FOXK2 and phosphorylated AKT are predominantly found in the nucleus of MDA-MB-231 cells. Also, FOXK2 subcellular localization is not modulated following AKT inhibition in MDA-MB-231 cells. Experiments involving lambda-phosphatase treatment and Phos-tag gels suggest that FOXK2 mobility is altered in AKT-modulated cell models. Notably, an in silico analysis identified higher levels of phosphorylated FOXK2 at different sites in breast tumor samples. Finally, inhibition and overexpression of AKT impact drug response, which is associated with modulation of FOXK2 protein bands. In summary, our results suggest that AKT can regulate FOXK2 protein levels in drug-resistant breast cancer cells. Future investigations will determine whether AKT directly phosphorylates FOXK2, as well as identify the crucial sites for AKT-FOXK2 interaction and biological functions.