FoxO3a Transcriptional Regulation of Bim Controls Apoptosis in Paclitaxel-treated Breast Cancer Cell Lines

Andrew Sunters,Silvia Fernández De Mattos,Marie Stahl,Jan J Brosens,Georgia Zoumpoulidou,Catherine A Saunders,Paul J Coffer,René H Medema,R Charles Coombes,Eric W.-F Lam

doi:10.1074/jbc.m309523200

Abstract

Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Consequently, we have studied the role of the FoxO transcription factors in determining cellular response to paclitaxel. Western blotting revealed that in a panel of nine breast cancer cell lines expression of FoxO1a and FoxO3a correlated with the expression of the pro-apoptotic FoxO target Bim, which was associated with paclitaxel-induced apoptosis. In MCF-7 cells, which were paclitaxel-sensitive, the already high basal levels of FoxO3a and Bim protein increased dramatically after drug treatment, as did Bim mRNA, which correlated with apoptosis induction. This was not observed in MDA-231 cells, which expressed low levels of FoxOs and Bim. Gene reporter experiments demonstrated that in MCF-7 cells maximal induction of Bim promoter was dependent on a FoxO binding site, suggesting that FoxO3a is responsible for the transcriptional up-regulation of Bim. Gene silencing experiments showed that small interference RNA (siRNA) specific for FoxO3a reduced the levels of FoxO3a and Bim protein as well as inhibited apoptosis in paclitaxel-treated MCF-7 cells. Furthermore, siRNA specific for Bim reduced the levels of Bim protein and inhibited apoptosis in paclitaxel-treated MCF-7 cells. This is the first demonstration that up-regulation of FoxO3a by paclitaxel can result in increased levels of Bim mRNA and protein, which can be a direct cause of apoptosis in breast cancer cells.

Highlights

Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood
Gene silencing experiments showed that small interference RNA specific for FoxO3a reduced the levels of FoxO3a and Bim protein as well as inhibited apoptosis in paclitaxel-treated MCF-7 cells
Apoptosis Induced by Paclitaxel on a Panel of Breast Carcinoma Cell Lines—To delineate the mechanisms involved in paclitaxel-induced apoptosis in breast cancer, a panel of breast cancer cell lines were treated with 10 nM paclitaxel for 48 h, and the level of apoptosis was measured using annexin V staining

Summary

Introduction

Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Breast cancer is one of the most common malignancies affecting women in the western world and arises following the accumulation of a series of somatic changes that serve to increase the rate of cellular proliferation and/or reduce the levels of apoptosis These changes are often found to involve deregulation of key signal transduction pathways. One key FoxO target gene is the cyclin-dependent kinase inhibitor (CKI) p27Kip1 [10, 11], which serves to inhibit cell cycle progression by inhibiting the kinase activity of cyclin-CDK holoenzymes, thereby reducing cell proliferation Another important target gene is Bim, a BH3 domain protein that is capable of inducing apoptosis [11,12,13]. Overexpression of PKB [15] and inactivation of the PI3K/PKB pathway inhibitor, PTEN (phosphatase and tensin homologue deleted on chromosome TEN), are frequently observed in breast cancer [16, 17], indicating a potential role for FoxOs in modulating both cell cycle and apoptosis during tumorigenesis and treatment

Methods

Results

Conclusion