FoxO3a Transcriptional Regulation of Bim Controls Apoptosis in Paclitaxel-treated Breast Cancer Cell Lines
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m309523200
ISSN1083-351X
AutoresAndrew Sunters, Silvia Fernández de Mattos, Marie Ståhl, Jan J. Brosens, Georgia Zoumpoulidou, Catherine Saunders, Paul J. Coffer, René H. Medema, R. Charles Coombes, Eric W.‐F. Lam,
Tópico(s)Cancer Mechanisms and Therapy
ResumoPaclitaxel 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. 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. 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. Signal transduction pathways within the cell transmit the extracellular signals to transcription factors, resulting in changes in gene expression. These changes in gene expression are often cell type-specific and lead to cell growth, differentiation, or apoptosis, thereby regulating normal tissue structure and function. One key signal transduction pathway highly conserved in eukaryotes is the phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3Kphosphatidylinositol 3-kinasePKBprotein kinase BCKIcyclin-dependent kinase inhibitorPBSphosphate-buffered salineGAPDHglyceraldehyde-3-phosphate dehydrogenasesiRNAsmall interference RNA. pathway (1.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar), which is stimulated by a number of growth factors, including insulin and insulin-like growth factor 1 (1.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). Once a receptor tyrosine kinase has become activated by binding a specific ligand, it becomes autophosphorylated and binds PI3K heterodimers either directly, or indirectly via insulin receptor substrate (IRS) adaptor proteins (1.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar, 2.Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. 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Phosphatidylinositol (3,4,5)-triphosphate binds to protein kinase B (PKB) also called AKT, a serine/threonine kinase, via its pleckstrin homology domain and recruits it to the inner surface of the cell membrane where it becomes activated by another pleckstrin homology domain-containing protein, PDK1 (1.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar, 2.Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell. Biol. 1999; 1: 69-71Crossref PubMed Scopus (201) Google Scholar, 3.Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (833) Google Scholar). PKB phosphorylates a number of key substrates, including glycogen synthase kinase 3β and mammalian target of rapamycin, which is involved in cell growth and translation, and the transcription factor NFκB (1.Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4657) Google Scholar). phosphatidylinositol 3-kinase protein kinase B cyclin-dependent kinase inhibitor phosphate-buffered saline glyceraldehyde-3-phosphate dehydrogenase small interference RNA. Genetic studies on the nematode Caenorhabditis elegans identified the DAF16 protein as a key downstream substrate of the insulin/PI3K/PKB signaling pathway, and showed that DAF16 is inactivated by PKB phosphorylation (4.Birkenkamp K.U. Coffer P.J. J. Immunol. 2003; 171: 1623-1629Crossref PubMed Scopus (136) Google Scholar, 5Tran, H., Brunet, A., Griffith, E. C., and Greenberg, M. E. (2003) Science's STKE, http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/53/re5.Google Scholar). The mammalian orthologues of the DAF16 protein are the FoxO class of the forkhead family of winged helix transcription factors, FoxO1a, FoxO3a, and FoxO4 (formally known as FKHR, FKHR-L1, and AFX, respectively) (4.Birkenkamp K.U. Coffer P.J. J. Immunol. 2003; 171: 1623-1629Crossref PubMed Scopus (136) Google Scholar, 5Tran, H., Brunet, A., Griffith, E. C., and Greenberg, M. E. (2003) Science's STKE, http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/53/re5.Google Scholar). In the absence of growth factor stimulation, FoxOs are localized in the nucleus, where they function as transcription factors. Upon stimulation of the PI3K signaling cascade, FoxOs become phosphorylated by PKB on highly conserved serine and threonine residues (6.Engstrom M. Karlsson R. Jonsson J.I. Exp. Hematol. 2003; 31: 316-323Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 8.Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). These phosphorylations result in impairment of DNA binding ability and increased binding affinity for the 14-3-3 protein (6.Engstrom M. Karlsson R. Jonsson J.I. Exp. Hematol. 2003; 31: 316-323Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 8.Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). Newly formed 14-3-3-FoxO complexes are then exported from the nucleus (9.Biggs 3rd, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (942) Google Scholar), thereby inhibiting FoxO-dependent transcription. One key FoxO target gene is the cyclin-dependent kinase inhibitor (CKI) p27Kip1 (10.Medema R. Kops G.J.P.L. Bos J.L. Burgering B.M.T. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1226) Google Scholar, 11.Dijkers P.F. Medema R.H. Pals C. Banerji L. Thomas N.S. Lam E.W. Burgering B.M. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Mol. Cell. Biol. 2000; 20: 9138-9148Crossref PubMed Scopus (542) Google Scholar), 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.Dijkers P.F. Medema R.H. Pals C. Banerji L. Thomas N.S. Lam E.W. Burgering B.M. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Mol. Cell. Biol. 2000; 20: 9138-9148Crossref PubMed Scopus (542) Google Scholar, 12.Dijkers P.F. Medemadagger R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar, 13.Gilley J. Coffer P.J. Ham J. J. Cell Biol. 2003; 162: 613-622Crossref PubMed Scopus (552) Google Scholar). Consequently, activation of the PI3K pathway serves to repress FoxO-mediated growth arrest and apoptosis. However, regulation of FoxO target genes is multifactorial, and therefore other transcription factors and post-translation regulatory events will also influence the final level of protein expression. It has been demonstrated that some promoters lacking the FoxO consensus site are still capable of being activated by FoxOs. Furthermore, there is evidence that FoxOs can interact strongly with other transcription factors such as the estrogen receptor-α (14.Schuur E.R. Loktev A.V. Sharma M. Sun Z. Roth R.A. Weigel R.J. J. Biol. Chem. 2001; 276: 33554-33560Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), suggesting that the FoxOs can also modulate transcription via interaction with other transcription factors (14.Schuur E.R. Loktev A.V. Sharma M. Sun Z. Roth R.A. Weigel R.J. J. Biol. Chem. 2001; 276: 33554-33560Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). These results suggest that changes in gene expression resulting from the FoxO arm of the PI3K/PKB signaling pathway are likely to be cell type-dependent. Interestingly, overexpression of PKB (15.Razzini G. Berrie C.P. Vignati S. Broggini M. Mascetta G. Brancaccio A. Falasca M. FASEB J. 2000; 14: 1179-1187Crossref PubMed Scopus (74) Google Scholar) and inactivation of the PI3K/PKB pathway inhibitor, PTEN (phosphatase and tensin homologue deleted on chromosome TEN), are frequently observed in breast cancer (16.Lu Y. Lin Y.Z. LaPushin R. Cuevas B. Fang X. Yu S.X. Davies M.A. Khan H. Furui T. Mao M. Zinner R. Hung M.C. Steck P. Siminovitch K. Mills G.B. Oncogene. 1999; 18: 7034-7045Crossref PubMed Scopus (267) Google Scholar, 17.Mills G.B. Lu Y. Fang X. Wang H. Eder A. Mao M. Swaby R. Cheng K.W. Stokoe D. Siminovitch K. Jaffe R. Gray J. Semin. Oncol. 2001; 28: 125-141Crossref PubMed Google Scholar), indicating a potential role for FoxOs in modulating both cell cycle and apoptosis during tumorigenesis and treatment. Typically, treatment of breast cancer involves surgery and chemotherapy using drugs that target the estrogen receptor, such as tamoxifen (18.Ali S. Coombes R.C. Nat. Rev. Cancer. 2002; 2: 101-112Crossref PubMed Scopus (704) Google Scholar). Although anti-estrogen therapy can be effective in treating estrogen-dependent tumors, some tumors exhibit intrinsic or acquired resistance to these modalities, and alternative treatments are needed (18.Ali S. Coombes R.C. Nat. Rev. Cancer. 2002; 2: 101-112Crossref PubMed Scopus (704) Google Scholar). Of these, the taxanes are one of the most frequently used classes of drugs, which include paclitaxel and its derivatives (19.Symmans F.W. Drug Resist. Updat. 2001; 4: 297-302Crossref PubMed Scopus (14) Google Scholar, 20.Blagosklonny M.V. Fojo T. Int. J. Cancer. 1999; 83: 151-156Crossref PubMed Scopus (335) Google Scholar). These drugs act by interacting with the cellular microtubules, and in particular, with the microtubules associated with the spindle apparatus during mitosis (19.Symmans F.W. Drug Resist. Updat. 2001; 4: 297-302Crossref PubMed Scopus (14) Google Scholar, 20.Blagosklonny M.V. Fojo T. Int. J. Cancer. 1999; 83: 151-156Crossref PubMed Scopus (335) Google Scholar). Although the primary cellular targets of the taxanes are microtubules, our understanding of the signal transduction pathways involved in the induction of apoptosis by taxanes in breast cancer cells is not complete. Consequently, we have studied the role of the PI3K/PKB pathway, and chiefly the FoxO family of transcription factors, in mediating the apoptosis and survival of breast cancer cell lines treated with paclitaxel. In this report, we demonstrate that in a drug-sensitive cell line accumulation of hypophosphorylated FoxO3a is responsible for the up-regulation of the pro-apoptotic protein Bim, whereas in a less sensitive cell line there is a reduction in hypophosphorylated FoxO3a, and significantly less Bim expression and apoptosis. We also propose that the regulation of FoxO family members by the PI3K/PKB pathway may be of prognostic value in the prediction of paclitaxel sensitivity in breast cancer. Cell Culture—A panel of human breast carcinoma cell lines HMT 3522, MCF-7, 734 B, ZR-75-1, T47-D, CAL-51, CAMA-1, MDA-MB-231, and SKBR-7 were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, and 100 units/ml penicillin/streptomycin, in a humidified incubator in an atmosphere of 10% CO2 at 37 °C. Paclitaxel was obtained from Sigma chemical company (Poole Dorset) and dissolved in Me2SO. Transfections and Gene Reporter Assays—Based on the published mouse Bim promoter sequence (21.Bouillet P. Zhang L.C. Huang D.C. Webb G.C. Bottema C.D. Shore P. Eyre H.J. Sutherland G.R. Adams J.M. Mamm. Genome. 2001; 12: 163-168Crossref PubMed Scopus (132) Google Scholar), a 790-bp fragment of the human Bim promoter, containing a single inverse Forkhead box TAAACAC (nucleotide–266/–259 relative to the 3′terminus of the promoter) was amplified from genomic DNA by PCR using the following primers: 5′-AAGCTTCCCGCCCTCACCCGGGA-3′ and 5′-GAGCTCCAACAAACTGCAGACC-3′, cloned in pGEM-T easy vector (Promega, Southampton, UK) and validated by sequencing. The human Bim promoter was then mutated to introduce a single G to C substitution in the core of the Forkhead box (GTAAACAC) by site-directed point mutagenesis using the following primers: 5′-TTACTCCGGTAAAGACGCCAGGGA-3′ and 5′-TCCCTGGCGTCTTTACCGGAGTAA-3′. Subsequently, a double PCR was performed using the human Bim promoter primers described before. The pGL2-hBim and pGL2-mutant hBim plasmids were then created by cloning the respective Klenow-blunted EcoRI promoter fragment into the SmaI-linearized pGL2Basic vector (Promega, UK). MCF-7 and MDA-MB-231 cells were transfected using the calcium phosphate co-precipitation method as described previously (22.Lam E.W.-F. Watson R.J. EMBO J. 1993; 12: 2705-2713Crossref PubMed Scopus (317) Google Scholar). Briefly, calcium phosphate precipitates containing 1 μg of wild type Bim promoter firefly-luciferase reporter plasmid (pGL2-hBim) or mutant plasmid (pGL2-mutant-hBim) together with 0.2 μg of a Renilla luciferase transfection control (pRL-TK; Promega) were incubated overnight with subconfluent cell cultures in each well of a 24-well plate. The cells were then washed twice in PBS, treated with paclitaxel, and harvested for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Apoptosis Determination Using Annexin V Staining—To determine the extent of apoptosis, Annexin V staining was used according to the manufacturer's instructions (R&D Systems). Briefly, paclitaxel-treated cells were trypsinized and washed in PBS containing 2% bovine serum albumin, before being stained in fluorescein isothiocyanate-labeled annexin V and propidium iodide (R&D Systems). Cells were analyzed by flow cytometry using a FACScan (BD Biosciences), and data were processed using CellQuest software (BD Biosciences). Cell Cycle Analysis—Cell cycle analysis was performed using propidium iodide staining as described previously (23.Williams C.D. Linch D.C. Watts M.J. Thomas N.S. Blood. 1997; 90: 194-203Crossref PubMed Google Scholar). Briefly, cells were trypsinized, washed in PBS, then fixed in 90% ethanol. Fixed cells were then washed twice in PBS and stained in 50 μm propidium iodide containing 5 μg/ml DNase-free RNase for 1 h, then analyzed by flow cytometry using a FACScan (BD Biosciences) and analyzed using Cell Quest software (BD Biosciences). Western Blotting—Western blotting was performed on whole cell extracts prepared by lysing cells in Nonidet P-40 lysis buffer (1% Nonidet P-40, 100 mm NaCl, 20 mm Tris-HCl, pH 7.4, 10 mm NaF, 1 mm sodium orthovanadate, 30 mm Naβ-glycerophosphate, and protease inhibitors (“Complete” protease inhibitor mixture, as instructed by the manufacturer, Roche Applied Science)) on ice for 15 min. Insoluble material was removed by centrifugation, and protein concentration was determined by Bio-Rad Dc protein assay (Bio-Rad). 20 μg of protein was size-fractionated using SDS-PAGE, and electro-transferred onto Protran nitrocellulose membranes (Schleicher and Schuell). Membranes were incubated with specific antibodies recognizing FKHR/FoxO1a (9462s, Cell Signaling Technologies), FKHR/FoxO1a phosphorylated at Ser-253 (9461s, Cell Signaling Technologies), FKHR-L1/FoxO3a (Upstate 06951), FKHR-L1/FOXO3a phosphorylated at Ser-253 (Upstate 06953), BIM (Calbiochem, 202000), and β-tubulin (BD Pharmingen 556321). A mouse monoclonal antibody recognizing human p27Kip1 was generated by Dr. Eric Lam. Primary antibodies were detected using horseradish peroxidase-linked anti-mouse or anti-rabbit conjugates as appropriate (DAKO), and visualized using the ECL detection system (Amersham Biosciences). Northern Blotting Analysis—Total RNA was isolated from treated cells using the RNeasy kit (Qiagen) and quantitated using UV spectrophotometry, and gene expression was analyzed by Northern blotting as detailed previously (22.Lam E.W.-F. Watson R.J. EMBO J. 1993; 12: 2705-2713Crossref PubMed Scopus (317) Google Scholar). Briefly, 40 μg of RNA was size-fractionated using 1.5% (w/v) formaldehyde-agarose gel electrophoresis. Following electrophoresis, RNA was transferred onto a Hybond N+ nitrocellulose membrane (Amersham Biosciences) using capillary transfer Northern blotting as described previously (22.Lam E.W.-F. Watson R.J. EMBO J. 1993; 12: 2705-2713Crossref PubMed Scopus (317) Google Scholar). Bim, p27Kip1, and GAPDH mRNAs were detected using 32P-labeled human cDNA probes described previously (24.Banerji L. Glassford J. Lea N.C. Thomas N.S. Klaus G.G. Lam E.W.-F. Oncogene. 2001; 20: 7352-7367Crossref PubMed Scopus (51) Google Scholar, 25.Dijkers P.F. Birkenkamp K.U. Lam E.W.-F. Thomas N.S. Lammers J.W. Koenderman L. Coffer P.J. J. Cell Biol. 2002; 156: 531-542Crossref PubMed Scopus (318) Google Scholar). Real-time Quantitative RT-PCR—Total RNA was isolated as before and DNase I-treated. Equal amounts of total RNA (2 μg) was reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen), and the resulting first strand cDNA was diluted and used as template in the real-time quantitative-PCR analysis. All measurements were performed in triplicate. The mRNAs analyzed were Bim and GAPDH, which served as internal control and was used to normalize for variances in input cDNA. The following gene-specific primer pairs were designed using the ABI Primer Express software: GAPDH-sense (5′-ATTTGGTCGTATTGGGCGCCTGGTCACC-3′) and GAPDH-antisense (5′-GAAGATGGTGATGGGATTTC-3′); Bim-sense (5′-CACAAAACCCCAAGTCCTCCTT-3′) and Bim-antisense (5′-TTCAGCCTGCCTCATGGAA-3′). Specificity of each primer was determined using NCBI BLAST module. Detection of Bim expression was performed with SYBR Green (Applied Biosystems) and an ABI PRISM 7700 Sequence Detection System (Applied Biosystems), using the relative standard curve method. Gene Silencing with Small Interfering RNAs—The siRNA oligonucleotides were purchased from Dharmacon Research Inc. (Lafayette, CO). MCF-7 and MDA-MB-231 cells were cultured in six-well plates until 60% confluent. Cells in 2 ml of culture medium were transfected with 4 μg of annealed oligonucleotides using LipofectAMINE (Invitrogen, UK) according to manufacturer's instructions. 24 h after transfection the cells were treated with 10 nm paclitaxel or Me2SO vehicle. After 48 h of treatment, both the suspension and the adherent cells were collected for either Western blot analysis, or Annexin V/propidium iodide staining. The FoxO3a siRNA sequence corresponds to the coding region 46–64 relative to the first nucleotide of the start codon (GenBank™ accession number BC020227) and does not match any other genomic sequence, except for that of the pseudogene FoxO3b in a Blast search from the NCBI website (www.ncbi.nlm.nih.gov). FoxO3a sense 5′-ACUCCGGGUCCAGCUCCAC(dTdT)-3′; FoxO3a antisense 5′-GUGGAGCUGGACCCGGAGU(dTdT)-3′. The Bim siRNA sequence corresponds to the coding region 39–57 relative to the first nucleotide of the start codon of the published the BimEL cDNA (GenBank™ accession number NM_138621). This sequence is present in all known Bim mRNA splice forms, but does not match any other sequence, in the GenBank™. Bim sense 5′-CAAUUGUCUACCUUCUCGG(dTdT)-3′; Bim antisense 5′-CCGAGAAGGUAGACAAUUG(dTdT)-3′; control sense 5′-UUCUCCGAACGUGUCACGU(dTdT)-3′; and control antisense 5′-ACGUGACACGUUCGGAGAA(dTdT)-3′. 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. The data shown in Fig. 1A clearly demonstrate that there is extensive heterogeneity in the extent of apoptosis induction in the panel of cell lines. The least paclitaxel-sensitive cell lines were MDA-MB-231 and T47D, which displayed an increase in apoptosis of less than 5%. Intermediate sensitivity (an increase of between 5 and 10%) was observed in the ZR-75-1 and CAL-51 cell lines. The most drug-sensitive cell lines were HMT3552, MCF-7, 734B, CAMA-1, and SKBR-7, which all exhibited increases in apoptosis of greater than 10% following paclitaxel treatment. Consequently, Western blotting experiments were performed to identify differences in expression of potential apoptotic regulators that could explain the heterogeneous apoptotic response. The Protein Expression of FoxO Family Members and FoxO Targets Is Heterogeneous in a Panel of Breast Carcinoma Cell Lines—It has been shown that the FoxO family of transcription factors can induce the pro-apoptotic BH3-only protein Bim in hematopoietic cells, thereby causing cell death (11.Dijkers P.F. Medema R.H. Pals C. Banerji L. Thomas N.S. Lam E.W. Burgering B.M. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Mol. Cell. Biol. 2000; 20: 9138-9148Crossref PubMed Scopus (542) Google Scholar, 25.Dijkers P.F. Birkenkamp K.U. Lam E.W.-F. Thomas N.S. Lammers J.W. Koenderman L. Coffer P.J. J. Cell Biol. 2002; 156: 531-542Crossref PubMed Scopus (318) Google Scholar). Furthermore, because the PI3K/PKB pathway is often deregulated in breast cancer (26.Page C. Huang M. Jin X. Cho K. Lilja J. Reynolds R.K. Lin J. Int. J. Oncol. 2000; 17: 23-28PubMed Google Scholar, 27.Perez-Tenorio G. Stal O. Br. J. Cancer. 2002; 86: 540-545Crossref PubMed Scopus (360) Google Scholar), and the FoxO family of transcription factors is subject to inhibitory phosphorylation by PKB, we sought to examine the expression levels of FoxO3a and FoxO1a to determine whether its expression correlated with the levels of apoptosis induced by paclitaxel in a panel of breast cancer cell lines. The expression pattern of total FoxO3a shows that expression is highest in MCF-7 and 734B and T47D, with intermediate levels of expression in HMT3522 and ZR-75-1 (Fig. 1B). The expression of FoxO3a is lowest in CAL-51, CAMA-1, MDA-MB-231, and SKBR-7. In the case of FoxO1a, the levels of total protein and the phosphorylated form are highest in MCF-7 and 734B. The expression levels in the remaining cell lines are much lower. Western blotting experiments demonstrated that the level of the FoxO target p27Kip1 was high in MCF-7, 734B, ZR-75-1, T47D, and CAL-51, with expression in HMT3552, CAMA-1, MDA-MB-231, and SKBR-7 being much lower. The expression of Bim also shows heterogeneity, its expression being high in HMT3552, MCF-7, 734B, and ZR-75-1, and very low in comparison with the remaining cell lines. The expression of β-tubulin was determined as a control for equal loading of the gels, and showed little variation. Taken together, these data suggest that in the MCF-7, 734B, T47D, and ZR-75-1, and to a lesser extent, the HMT3552 cell lines, there is a correlation between the expression of FoxO1a and -3a, and the expression of one or other of the known FoxO targets p27Kip1 and Bim. What is clear from the data is that the high level of expression of Bim in HMT3552, MCF-7, and 734B correlates with the induction of apoptosis by paclitaxel. Conversely, the low levels of Bim expression in T47D and MDA-MB-231 also correlated with the lack of apoptosis induction. The exceptions to this are ZR-75-1, which expresses high levels of Bim, but does not undergo apoptosis readily, and SKBR-7 and CAMA-1, which display high levels of apoptosis despite low levels of Bim, suggesting other mechanisms are responsible for regulating apoptosis in these cells. Paclitaxel Induces Cell Death in a Drug-sensitive Cell Line, but G2/M Arrest in a Less sensitive Cell Line—To investigate in more detail the role of FoxO family members and Bim in paclitaxel-induced apoptosis, we chose to perform further experiments in two cell lines: MCF-7, which expressed high levels of FoxOs and Bim and underwent apoptosis readily, and MDA-MB-231 cells, which expressed low levels of FoxOs and Bim and was refractory to paclitaxel-induced apoptosis. The cell-cycle phase distribution in these two cell lines was measured using flow cytometry of ethanol-fixed propidium iodide-stained cells after treatment with 10 nm paclitaxel. The data shown in Fig. 2 clearly shows that in the MCF-7 cell line there is an increase in the fraction of cells in the G2/M phase of the cell cycle for the first 24 h of treatment. After 24 h, there was a gradual increase in the number of dead and dying cells containing <2N DNA content. Although the determination of apoptosis in Fig. 1A was made using annexin V staining, we have found that the number of cells in the <2N fraction and the number of annexin V-positive cells are very similar following paclitaxel treatment. These results would suggest that the MCF-7 cells arrest in G2/M phase before death. In contrast, the MDA-MB-231 cells accumulate in the G2/M phase for the first 24 h and then become polyploid but do not undergo extensive cell death. Paclitaxel Induces Increased Protein Expression of FoxO3a, Bim, and p27Kip1 in a Drug-sensitive Breast Cancer Cell Line—To study the role of FoxOs in determining the apoptotic response to paclitaxel in MCF-7 and MDA-MB-231 cells, we compared the changes in protein expression following treatment with paclitaxel at either 10 or 75 nm. Whole cell protein extracts were prepared 24, 48, and 72 h after drug treatment, which from the previous experiment were the times when apoptosis was initiated (24 h) and then was observed to be maximal (48 and 72 h). The protein expression was then analyzed by Western blotting. The data clearly demonstrated that in both cell lines that there was an increase in the levels of both phospho- and total FoxO1a 48 h after 10 nm paclitaxel treatment (Fig. 3A). Following treatment with 75 nm paclitaxel, the induction is still observable in the MDA-MB-231 cells, and is less pronounced in the MCF-7 cells, which underwent extensive cell death (data not shown). What is most striking is the observation that expression of both phosphorylated and total FoxO3a dramatically increased 48 h after 10 nm paclitaxel treatment in the MCF-7 cells but not in the MDA-MB-231 cells. Interestingly, this expression pattern is also observed in the 75 nm MCF-7 treatment group, but to a lesser extent. These data clearly demonstrate that FoxO3a is induced in the paclitaxel-sensitive MCF-7, but to
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