Critical role of FoxO3a transcription factor in response to therapy with EGFR inhibitors, gefitinib and lapatinib, in breast cancer

Abstract

21018 Background: Inhibition of the EGF-receptor is a potential therapeutic strategy for breast cancer. The PI3-K/PKB signalling pathway plays an important role in cell proliferation, survival and malignant transformation. The Forkhead box group O transcriptional factor, FoxO3a, is a direct downstream target of PKB. Here we studied the role of the FoxO3a in response to the EGFR inhibitors Gefitinib and Lapatinib. Methods: Response of breast cancer cell lines to treatment with Gefitinib and Lapatinib was evaluated by proliferation assay and FACS analysis. Expression of FoxO3a and its downstream targets was analysed using Western blotting. Sub-cellular localisation of FoxO3a was analysed by confocal microscopy. FoxO3a mRNA level was measured by RTq-PCR. siRNA transfections were performed using Oligofectamine reagent. Immunohistological staining was performed on patient samples to validate our findings. Results: Treatment of a panel of breast cancer cell lines with both inhibitors resulted in decreased proliferation due to G1-arrest and apoptosis in two sensitive cell-lines, BT474 and SKBR3. Western blot analysis revealed that response to the treatment was associated with a decrease in PKB and FoxO3a phosphorylation and thus the nuclear relocalisation of FoxO3a. Confocal microscopy confirmed that treatment induced nuclear translocation of FoxO3a only in sensitive but not resistant cells. This relocation of FoxO3a was accompanied by an induction of FoxO3a mRNA and its principal targets, p27kip1 and Bim. Transfection of the sensitive BT474 cells with FoxO3a specific siRNA resulted in a significant reduction in FoxO3a protein expression and Gefitinib-induced cell death. Comparison of immunohistochemical staining of biopsies from breast cancer patients before and after Gefitinib treatment revealed that therapy resulted in a significant increase in nuclear FoxO3a staining. Conclusion: Our results demonstrate for the first time that the EGFR-family inhibitors, Gefitinib and Lapatinib, specifically target FoxO3a to induce cell cycle arrest and apoptosis. This finding helps to define the mechanism of action of Gefitinib and Lapatinib and may provide novel insights into the molecular basis for resistance to these inhibitors. No significant financial relationships to disclose.