FOXO3a regulates expression of ER target genes and CDK inhibitors, and induces apoptosis in MCF-7

Ectopic expression of Forkhead box class O (FOXO)3a reduces the expression of some estrogen receptor (ER)-regulated genes and enhances the expression of cyclin-dependent kinase (CDK) inhibitors in MCF7-FO cells in the presence of 17β-estradiol (E2). Immunoblotting (IB) analyses for HA-FOXO3a, endogenous FOXO3a, p27Kip1, p21Cip1, p57Kip2, cyclin D, cyclin E, cathepsin D, progesterone receptor (PgR), ER-α, and ER-β protein expression in MCF7-FO33 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 and MCF7-C5) cells were performed with specific antibodies, as indicated. Equal loading was confirmed by the same IB analysis with antibodies against β-actin or β-tubulin. MDA-MB-453 (MDA-453, ER-negative) cells were transfected with either FOXO3a or an empty pCDNA3.1 vector (control), as indicated. Total lysates of the transfected cells were prepared and subjected to SDS-PAGE followed by IB analysis with the indicated antibodies.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

ER-α and ER-β bind to the amino-terminal and carboxyl-terminal domains of FOXO3a, respectively

Glutathione-S-transferase (GST) – pull down assays. Whole cell lysates from 293T cells were incubated with the GST-forkhead box class O (FOXO)3a (GST-FO [amino acids 1 to 300] and GST-FO [amino acids 301 to 673]) fusion proteins as indicated and GST alone (negative control), and analyzed by SDS-PAGE and immunoblotting with an antibody (Ab) against estrogen receptor (ER)-α or ER-β (upper panels) and an anti-GST Ab (lower panel) as protein controls. FOXO3a downregulates FOXM1. Immunoblotting (IB) analyses for endogenous FOXO3a, forkhead box M1 (FOXM1), and β-actin (loading control) protein expression in MCF7-FO10 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 wt and MCF7-C12) cells were performed with specific antibodies antibodies as indicated. MCF7-d8_pa cells (pooled clones of MCF-7 FOXO3a-knockdown derivatives) were established with retroviruses expressing small hairpin RNA against human FOXO3a. The expression levels of FOXO3a and p27Kip1 in MCF-7 wild-type (wt) and MCF7-d8_pa cells were determined by IB with specific Abs against FOXO3a or p27Kip1 or β-actin (loading control). Silencing endogenous FOXO3a in MCF-7 cells promoted tumorigenesis . The tumor growth rates of control group MCF-7 wt and knockdown group MCF7-d8-pa were determined after injection of cells (2 × 10cells/mouse) as indicated into the mammary fat pads of female athymic mice not given supplemental 17β-estradiol (E2; indicated as – E2). Data are expressed as means and standard deviations from two experiments with five mice in each group. *< 0.01 between MCF7-d8-pa group versus control MCF-7 wt group.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

FOXO3a and FOXO1a inhibit the transactivation activities of ER-α and ER-β

293T cells were co-transfected with estrogen receptor (ER)-responsive element (ERE)-luc (firefly luciferase [luc] reporter containing EREs), pRL-TK (renilla luc as a transfection control for normalization), ER-α (panel a) or ER-β (panel b), and forkhead box class O (FOXO)3a plus IκB kinase (IKK)-β or an empty vector (control) as indicated. Total lysates of the transfected cells were prepared and subjected to luc assays. Total lysates of 293T cells were co-transfected with ERE-luc, pRL-TK, ER-α (panel c) or ER-β (panel d), and FOXO1a plus IKK-β or an empty vector as indicated and subjected to luc assays. All cells were cultured in the presence of 17β-estradiol (E2). The relative reporter luc activity was normalized with pRL-TK. Data are expressed means and standard deviations from three repeated experiments, which were performed independently. *< 0.05 between FOXO (FOXO3a or FOXO1a) minus IKK-β (lane 3) versus FOXO plus IKK-β (lane 4).<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

Ectopic expression of FOXO3a in estrogen-dependent breast cancer cells suppresses breast tumor development

Forkhead box class O (FOXO)3a suppresses estrogen receptor (ER)-positive breast tumor development in a mouse model of breast cancer. The MCF7-FO pooled cell lines and the control MCF7-C pooled cell lines were injected (2 × 10cells/mouse) into the mammary fat pads of female athymic mice given supplementary 17β-estradiol (E2), as described in Materials and methods. Growth curves of tumor size are the means of the MCF7-FO pooled cell lines (designated MCF7-FO) and the control MCF7-C pooled cell lines (designated MCF7-C); error bars indicate standard deviation from three experiments. *< 0.05 between control MCF7-C group versus MCF7-FO group.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

Ectopic expression of FOXO3a in estrogen-dependent breast cancer cells suppresses cell proliferation in cell culture

Growth of forkhead box class O (FOXO)3a over-expressing MCF7-FO (MCF7-FO10, MCF7-FO33, and MCF7-FO41) cells and control (MCF7-C4, MCF7-C5, and MCF7-C12) cells in the absence of 17β-estradiol (E2) was determined by counting trypan-blue stained cells with a hemocytometer. Growth curves are the means of the three MCF7-FO cell lines (MCF7-FO10, MCF7-FO33, and MCF7-FO41) and the three control cell lines (MCF7-C4, MCF7-C5, and MCF7-C12); error bars indicate standard deviation from three experiments. Growth of the same sets of cells in the presence of E2 (1 nmol/l). Growth curves are the means of the three MCF7-FO cell lines (designated MCF7-FO average) and the three control cell lines (designated MCF7-C average); error bars indicate standard deviaton from three experiments. *< 0.05 between control MCF7-C group versus MCF7-FO group.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p