Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) 
that causes growth delay in cancer cell lines and human tumor xenografts 
expressing high levels of EGFR. An understanding of the downstream cellular 
targets of gefitinib will allow the discovery of biomarkers for predicting 
outcomes and monitoring anti-EGFR therapies and provide information for key 
targets for therapeutic intervention. In this study, we investigated the role of 
FOXO3a in gefitinib action and resistance. Using two gefitinib-sensitive (i.e., 
BT474 and SKBR3) as well as three other resistant breast carcinoma cell lines 
(i.e., MCF-7, MDA-MB-231, and MDA-MB-453), we showed that gefitinib targets the 
transcription factor FOXO3a to mediate cell cycle arrest and cell death in 
sensitive breast cancer cells. In the sensitive cells, gefitinib treatment 
causes cell cycle arrest predominantly at the G(0)-G(1) phase and apoptosis, 
which is associated with FOXO3a dephosphorylation at Akt sites and nuclear 
translocation, whereas in the resistant cells, FOXO3a stays phosphorylated and 
remains in the cytoplasm. The nuclear accumulation of FOXO3a in response to 
gefitinib was confirmed in tumor tissue sections from breast cancer patients 
presurgically treated with gefitinib as monotherapy. We also showed that 
knockdown of FOXO3a expression using small interfering RNA (siRNA) can rescue 
sensitive BT474 cells from gefitinib-induced cell-proliferative arrest, whereas 
reintroduction of active FOXO3a in resistant MDA-MB-231 cells can at least 
partially restore cell-proliferative arrest and sensitivity to gefitinib. These 
results suggest that the FOXO3a dephosphorylation and nuclear localization have 
a direct role in mediating the gefitinib-induced proliferative arrest and in 
determining sensitivity to gefitinib.