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RUNX3 Cooperates with FoxO3a to Induce Apoptosis in Gastric Cancer Cells

2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês

10.1074/jbc.m512151200

ISSN

1083-351X

Autores

Yasuko Yamamura, Wei Lin Lee, Ken‐ichi Inoue, Hiroshi Ida, Yoshiaki Ito,

Tópico(s)

Cancer Mechanisms and Therapy

Resumo

The transcription factor RUNX3, which mediates apoptosis and cell growth inhibition in gastric epithelial cells, is a candidate tumor suppressor that is frequently lost in gastric cancer cells. Here, we found that restoration of RUNX3 expression in the cell line not expressing RUNX3 induced apoptosis and that it physically interacted with the Forkhead transcription factor FoxO3a/FKHRL1, known to be an important regulator of apoptosis and the cell cycle. Active unphosphorylated FoxO3a/FKHRL1 was expressed in the gastric cancer cell lines. RUNX3-induced apoptosis depended on the expression of Bim, a proapoptotic BH3-only protein, and both RUNX3 and FoxO3a/FKHRL1 were required for induction of Bim expression. Furthermore, we showed that interaction of RUNX3 and FoxO3a/FKHRL1 was also indispensable for Bim expression and apoptosis in mouse embryonic fibroblasts. In the Bim promoter, RUNX3 bound to two conserved RUNX-binding elements (RBE1 and RBE2), with RBE1 being immediately downstream of a FoxO-binding element. The physical interaction of RUNX3 and FoxO3a/FKHRL1 on the Bim promoter activated transcription of Bim. These findings show that RUNX3 cooperates with FoxO3a/FKHRL1 to participate in the induction of apoptosis by activating Bim and may play an important role in tumor suppression in gastric cancer. The transcription factor RUNX3, which mediates apoptosis and cell growth inhibition in gastric epithelial cells, is a candidate tumor suppressor that is frequently lost in gastric cancer cells. Here, we found that restoration of RUNX3 expression in the cell line not expressing RUNX3 induced apoptosis and that it physically interacted with the Forkhead transcription factor FoxO3a/FKHRL1, known to be an important regulator of apoptosis and the cell cycle. Active unphosphorylated FoxO3a/FKHRL1 was expressed in the gastric cancer cell lines. RUNX3-induced apoptosis depended on the expression of Bim, a proapoptotic BH3-only protein, and both RUNX3 and FoxO3a/FKHRL1 were required for induction of Bim expression. Furthermore, we showed that interaction of RUNX3 and FoxO3a/FKHRL1 was also indispensable for Bim expression and apoptosis in mouse embryonic fibroblasts. In the Bim promoter, RUNX3 bound to two conserved RUNX-binding elements (RBE1 and RBE2), with RBE1 being immediately downstream of a FoxO-binding element. The physical interaction of RUNX3 and FoxO3a/FKHRL1 on the Bim promoter activated transcription of Bim. These findings show that RUNX3 cooperates with FoxO3a/FKHRL1 to participate in the induction of apoptosis by activating Bim and may play an important role in tumor suppression in gastric cancer. Runx transcription factors are α subunits of the polyomavirus enhancer-binding protein 2 (PEBP2) 2The abbreviations used are: PEBP2, polyomavirus enhancer-binding protein 2; TGF-β, transforming growth factor-β; PKB, protein kinase B; PI3K, phosphoinositide 3-kinase; FasL, Fas ligand; NGF, nerve growth factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; MEF, mouse embryonic fibroblast; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; siRNA, small interfering RNA; HA, hemagglutinin; FBE, FoxO-binding element. 2The abbreviations used are: PEBP2, polyomavirus enhancer-binding protein 2; TGF-β, transforming growth factor-β; PKB, protein kinase B; PI3K, phosphoinositide 3-kinase; FasL, Fas ligand; NGF, nerve growth factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; MEF, mouse embryonic fibroblast; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; siRNA, small interfering RNA; HA, hemagglutinin; FBE, FoxO-binding element./core-binding factor (CBF), which consists of α and β subunits. They have a highly conserved Runt domain responsible for DNA binding and heterodimer formation with the β subunit (PEBP2β/CBFβ) (1Ito Y. Miyazono K. Curr. Opin. Genet. Dev. 2003; 13: 43-47Crossref PubMed Scopus (274) Google Scholar). Three Runx transcription factors, Runx1, Runx2, and Runx3, have been identified in mammals. Although all these α subunits have closely related structures and biochemical properties and bind the same DNA-binding motifs (1Ito Y. Miyazono K. Curr. Opin. Genet. Dev. 2003; 13: 43-47Crossref PubMed Scopus (274) Google Scholar), they possess distinct biological functions in vivo. Runx1 is essential for definitive hematopoiesis, Runx2 plays critical roles in osteoblast maturation and osteogenesis, and Runx3 is ubiquitously expressed and involved in a variety of biological activities including development of gastrointestinal tract, neurogenesis, and lineage specification of thymocytes (1Ito Y. Miyazono K. Curr. Opin. Genet. Dev. 2003; 13: 43-47Crossref PubMed Scopus (274) Google Scholar). Abnormalities in human Runx (RUNX) genes have been linked with some diseases (1Ito Y. Miyazono K. Curr. Opin. Genet. Dev. 2003; 13: 43-47Crossref PubMed Scopus (274) Google Scholar, 2Balmain A. Nature. 2002; 417: 235-237Crossref PubMed Scopus (37) Google Scholar). RUNX1/AML1 is an important translocation break-point in acute leukemias and heterozygous loss of RUNX2 causes cleidocranial dysplasia syndrome. RUNX3 is implicated as a tumor suppressor gene in gastric cancer. Runx3-deficient mice exhibit hyperplasias in gastric mucosa due to reduced apoptosis and stimulated proliferation of gastric epithelial cells. Gastric epithelial cells are less sensitive to the proapoptotic and growth inhibitory effects of transforming growth factor-β (TGF-β) (3Li Q.L. Ito K. Sakakura C. Fukamachi H. Inoue K. Chi X.Z. Lee K.Y. Nomura S. Lee C.W. Han S.B. Kim H.M. Kim W.J. Yamamoto H. Yamashita N. Yano T. Ikeda T. Itohara S. Inazawa J. Abe T. Hagiwara A. Yamagishi H. Ooe A. Kaneda A. Sugimura T. Ushijima T. Bae S.C. Ito Y. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar). Furthermore, RUNX3 is inactivated in 40% of early stage and in nearly 90% of advanced stage gastric carcinomas by hemizygous deletion and hypermethylation of its promoter (3Li Q.L. Ito K. Sakakura C. Fukamachi H. Inoue K. Chi X.Z. Lee K.Y. Nomura S. Lee C.W. Han S.B. Kim H.M. Kim W.J. Yamamoto H. Yamashita N. Yano T. Ikeda T. Itohara S. Inazawa J. Abe T. Hagiwara A. Yamagishi H. Ooe A. Kaneda A. Sugimura T. Ushijima T. Bae S.C. Ito Y. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar). Among the three Runx transcription factors, Runx3 is predominantly induced by TGF-β to bind and activate the germ line Ig α promoter, thereby directing Ig class switching to IgA in splenic B cells and the surface IgM+ B cell line I.29μ (4Shi M.J. Stavnezer J. J. Immunol. 1998; 161: 6751-6760PubMed Google Scholar). RUNX3 interacts physically with Smad transcription factors (Smads), which are signal transducers of TGF-β, and induces TGF-β-dependent transcription of the germ line Ig α promoter in a cooperative manner (5Hanai J. Chen L.F. Kanno T. Ohtani-Fujita N. Kim W.Y. Guo W.H. Imamura T. Ishidou Y. Fukuchi M. Shi M.J. Stavnezer J. Kawabata M. Miyazono K. Ito Y. J. Biol. Chem. 1999; 274: 31577-31582Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 6Zhang Y. Derynck R. J. Biol. Chem. 2000; 275: 16979-16985Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Although these findings suggest that RUNX3 cooperates with its putative cofactor to exert its role as a tumor suppressor by activating proapoptotic genes, little is known about the molecular mechanisms whereby RUNX3 induces apoptosis. Members of the FoxO subfamily of Forkhead transcription factors represent the mammalian orthologs of DAF-16, which regulates longevity in the nematode Caenorhabditis elegans. In mammals, FoxO subfamily members regulate diverse cell functions, such as apoptosis, cell cycle progression, and DNA repair (7Accili D. Arden K.C. Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). The serine/threonine kinase Akt/PKB (protein kinase B) regulates the activities of three FoxO proteins, FoxO1/FKHR, FoxO3a/FKHRL1, and FoxO4/AFX (8Brunet 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 (5410) Google Scholar). Akt/PKB rapidly phosphorylates FoxO proteins following its activation by phosphoinositide 3-kinase (PI3K). In turn, the phosphorylated FoxO proteins associate with 14-3-3 protein, which functions as a scaffold within the cytoplasm, and are sequestered within the cytosol, rendering them unable to bind to the promoters of their target genes in the nucleus to regulate their transcription. Recently, IκB kinase has also been shown to phosphorylate and inactivate FoxO3a in breast cancer cells (9Hu M.C. Lee D.F. Xia W. Golfman L.S. Ou-Yang F. Yang J.Y. Zou Y. Bao S. Hanada N. Saso H. Kobayashi R. Hung M.C. Cell. 2004; 117: 225-237Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). In contrast, unphosphorylated FoxO proteins are active forms and are located in the nucleus where they bind to the promoters of their target genes. FoxO3a has been shown to mediate apoptosis by activating proapoptotic genes in a variety of cells. Overexpression of constitutively active FoxO3a induces apoptosis by activating the Fas ligand (FasL) expression and the Fas-FasL apoptotic pathway in the human leukemia T cell line Jurkat (8Brunet 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 (5410) Google Scholar). FoxO3a is responsible for Bim-mediated apoptosis in the interleukin (IL)-3-dependent pre-B cell line Ba/F3 and the IL-2-dependent T cell line CTLL-2 following the withdrawal of IL-3 and IL-2 from their cultures, respectively (10Dijkers P.F. Birkenkamp K.U. Lam E.W. Thomas N.S. Lammers J.W. Koenderman L. Coffer P.J. J. Cell Biol. 2002; 156: 531-542Crossref PubMed Scopus (318) Google Scholar, 11Stahl M. Dijkers P.F. Kops G.J. Lens S.M. Coffer P.J. Burgering B.M. Medema R.H. J. Immunol. 2002; 168: 5024-5031Crossref PubMed Scopus (518) Google Scholar). FoxO3a also contributes to the nerve growth factor (NGF) deprivation-induced apoptosis of developing sympathetic neurons by activating Bim (12Gilley J. Coffer P.J. Ham J. J. Cell Biol. 2003; 162: 613-622Crossref PubMed Scopus (547) Google Scholar). The anti-cancer drug paclitaxel also induces Bim-dependent apoptosis by activating FoxO3a in MCF-7 breast cancer cells (13Sunters A. Fernández de Mattos S. Stahl M. Brosens J.J. Zoumpoulidou G. Saunders C.A. Coffer P.J. Medema R.H. Coombes R.C. Lam E.W. J. Biol. Chem. 2003; 278: 49795-49805Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Moreover, FoxO3a increases expression of TRAIL (tumor necrosis factor-related apoptosis inducing ligand), which is a proapoptotic member of the TNF family, and induces apoptosis downstream of the PTEN tumor suppressor in prostate cancer (14Modur V. Nagarajan R. Evers B.M. Milbrandt J. J. Biol. Chem. 2002; 277: 47928-47937Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Thus, FoxO3a has generally been considered an inducer of apoptosis. However, expression of the constitutively active FoxO3a protects pheochromocytoma PC12 and the colon carcinoma DLD-1 cells from apoptosis induced by oxidative stress (15Nemoto S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (736) Google Scholar, 16Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1266) Google Scholar), as well as neutrophils from apoptosis induced by the Fas-FasL signaling in inflammation (17Jonsson H. Allen P. Peng S.L. Nat. Med. 2005; 11: 666-671Crossref PubMed Scopus (174) Google Scholar). The biological consequences of FoxO3a depend on cell type and stimuli surrounding cells. The transcriptional activity of FoxO3a is modulated through interaction with its cofactors. Here, we address the mechanism through which RUNX3 exerts its tumor suppressor activity by inducing apoptosis in gastric cancer cells. RUNX3 is shown to physically interact with FoxO3a expressed in gastric cancer cell lines to activate Bim and induce apoptosis. The same interaction is also demonstrated in mouse embryonic fibroblasts, suggesting that Runx3 is involved in Bim-dependent apoptosis, which is transcriptionally regulated by FoxO3a in a variety of cell types. Cell Culture, Transfection, and Reagents—COS7, AGS, AGS-derived transfectant clone, and NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. MKN28, MKN45, KATO III, and SNU16 cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum. Primary mouse embryonic fibroblasts (MEFs) were prepared from embryos produced from intercrosses of Runx3+/- mice (3Li Q.L. Ito K. Sakakura C. Fukamachi H. Inoue K. Chi X.Z. Lee K.Y. Nomura S. Lee C.W. Han S.B. Kim H.M. Kim W.J. Yamamoto H. Yamashita N. Yano T. Ikeda T. Itohara S. Inazawa J. Abe T. Hagiwara A. Yamagishi H. Ooe A. Kaneda A. Sugimura T. Ushijima T. Bae S.C. Ito Y. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar) at 14.5 days post-coitus and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of cDNA was performed using FuGENE 6 (Roche Applied Science) or Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. Human recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Plasmid Construction—A FLAG epitope was introduced by PCR into the N terminus of RUNX3 cDNA obtained from pEFBOS-T7-RUNX3 (18Bae S.C. Takahashi E. Zhang Y.W. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene (Amst.). 1995; 159: 245-248Crossref PubMed Scopus (152) Google Scholar), and the resulting cDNA was fully sequenced. The cDNA was further subcloned into pcDNA3.1(+) and pIND(SP1)/Hygro vectors (Invitrogen). The FoxO3a cDNA obtained from FOXO3a-Myc (9Hu M.C. Lee D.F. Xia W. Golfman L.S. Ou-Yang F. Yang J.Y. Zou Y. Bao S. Hanada N. Saso H. Kobayashi R. Hung M.C. Cell. 2004; 117: 225-237Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar) was subcloned into pcDNA3.1(+). Mutagenesis of the Bim promoter (19Bouillet 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) was performed using a QuikChange multi-site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the mutations confirmed by sequencing the DNA. Ecdysone-inducible Transient Expression of FLAG-RUNX3—Cells were cotransfected with pVgRXR (Invitrogen) and recombinant pIND(SP1)/Hygro containing the FLAG-RUNX3 cDNA. Stable transfectant clones were selected with zeocin (Invitrogen) and hygromycin-B (Invitrogen). Transient expression of FLAG-RUNX3 was induced in the transfectant clone cells by treatment with ponasterone A (Invitrogen). Apoptosis Detection—Apoptosis was quantified using an annexin V-fluorescein isothiocyanate apoptosis detection kit II (BD Biosciences) according to the manufacturer's instructions. Briefly, cells were trypsinized, washed, stained with fluorescein isothiocyanate-conjugated annexin V and propidium iodide, and then analyzed by flow cytometry using a FACS vantage (BD Biosciences). The data were analyzed using the Cell Quest software (BD Biosciences). Apoptosis was also examined using cell death detection ELISA PLUS assays (Roche Applied Science), which measure the presence of cytoplasmic histone-associated mono- and oligonucleosomes as a result of apoptosis, in accord with to the manufacturer's instructions. Semiquantitative RT-PCR Analysis and Real-time Quantitative RT-PCR Analysis—Total RNA was isolated using an RNeasy mini kit (Qiagen, Hilden, Germany) and treated with DNase I using an RNase-free DNase set (Qiagen). Equal amounts of total RNA were reverse-transcribed using an Omniscript reverse transcriptase kit (Qiagen). The first-strand cDNA was used as a template. The primers used for semi-quantitative RT-PCR analyses of human FasL (5′-CTCTGGAATGGGAAGACACC-3′ and 5′-ACCAGAGAGAGCTCAGATACG-3′) (20Viard-Leveugle I. Bullani R.R. Meda P. Micheau O. Limat A. Saurat J.H. Tschopp J. French L.E. J. Biol. Chem. 2003; 278: 16183-16188Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), human Bim (5′-GAGAAGGTAGACAATTGCAG-3′ and 5′-GACAATGTAACGTAACAGTCG-3′) (21Abrams M.T. Robertson N.M. Yoon K. Wickstrom E. J. Biol. Chem. 2004; 279: 55809-55817Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), human TRAIL (5′-GACCTGCGTGCTGATCGTG-3′ and 5′-TGTCCTGCATCTGCTTCAGCT-3′) (14Modur V. Nagarajan R. Evers B.M. Milbrandt J. J. Biol. Chem. 2002; 277: 47928-47937Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar), and human GAPDH (5′-CACCCATGGCAAATTCCATG-3′ and 5′-TCTAGACGGCAGGTCAGGT-3′) (21Abrams M.T. Robertson N.M. Yoon K. Wickstrom E. J. Biol. Chem. 2004; 279: 55809-55817Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) were described previously. The primers used for real-time quantitative RT-PCR analyses of RUNX3, human Bim, and human GAPDH were Assays-on-Demand gene expression products (Applied Biosystems, Foster City, CA). The primers used for real-time quantitative RT-PCR analyses of mouse BimEL (5′-GTCCTCCAGTGGGTATTTCT-3′ and 5′-TCCTCCTTGGACTTCTAGAC-3′) and mouse GAPDH (5′-GTATGTCGTGGAGTCTACTGGTGT-3′ and 5′-CCGGATGTACCGGAGGTTCCTCAT-3′) were described previously (22Akiyama T. Bouillet P. Miyazaki T. Kadono Y. Chikuda H. Chung U.I. Fukuda A. Hikita A. Seto H. Okada T. Inaba T. Sanjay A. Baron R. Kawaguchi H. Oda H. Nakamura K. Strasser A. Tanaka S. EMBO J. 2003; 22: 6653-6664Crossref PubMed Scopus (223) Google Scholar). The primers for GAPDH served as an internal control for normalization of the results. Small Interfering RNA—Control non-targeting siRNA and siRNA duplexes targeting human Bim were purchased from Dharmacon (Lafayette, CO). Cells were transfected with the siRNA duplexes using Lipofectamine 2000 (Invitrogen) and subsequently subjected to Western blot and apoptosis detection analyses at 72 h after transfection. Luciferase Assay—Cells were transiently transfected with luciferase reporter constructs, mixtures of expression plasmids encoding RUNX3 (18Bae S.C. Takahashi E. Zhang Y.W. Ogawa E. Shigesada K. Namba Y. Satake M. Ito Y. Gene (Amst.). 1995; 159: 245-248Crossref PubMed Scopus (152) Google Scholar), PEBP2β2 (23Ogawa E. Inuzuka M. Maruyama M. Satake M. Naito-Fujimoto M. Ito Y. Shigesada K. Virology. 1993; 194: 314-331Crossref PubMed Scopus (438) Google Scholar), or FoxO3a-TM (8Brunet 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 (5410) Google Scholar), and an internal control pRL-TK vector (Promega, Madison, WI). Luciferase activity was measured after 20 h using the dual-luciferase reporter assay system (Promega) in an LB 960 Microplate Luminometer Centro (Berthold Technologies, Bad Wildbad, Germany). Luciferase activity was normalized for the transfection efficiency using the Renilla luciferase activity from pRL-TK. Electrophoretic Mobility Shift Assay—COS7 cells were transfected with the indicated cDNA expression plasmids, and whole-cell lysates were prepared. Complementary oligonucleotides corresponding to FBE-RBE1 (5′-GGGAAACAAACATATAAACCACAACA-3′) and RBE2 (5′-GCTCAACTACCGCAGAGTCTCAAGAGC-3′)ofthe Bim promoter were biotinylated at their 3′ ends, annealed, and used as probes. Binding reactions were performed and analyzed in a nondenaturing gel as described previously with minor modifications (5Hanai J. Chen L.F. Kanno T. Ohtani-Fujita N. Kim W.Y. Guo W.H. Imamura T. Ishidou Y. Fukuchi M. Shi M.J. Stavnezer J. Kawabata M. Miyazono K. Ito Y. J. Biol. Chem. 1999; 274: 31577-31582Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 24Yamamura Y. Hua X. Bergelson S. Lodish H.F. J. Biol. Chem. 2000; 275: 36295-36302Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). For supershift assays, whole-cell lysates were preincubated with an anti-Runt domain (anti-RUNX) 6B4 monoclonal antibody (25Afzal F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. Physiol. 2005; 204: 63-72Crossref PubMed Scopus (127) Google Scholar) or anti-HA monoclonal antibody (12CA5, Roche Applied Science), and the DNA-protein complexes were visualized using a LightShift chemiluminescent EMSA kit (Pierce). For competition experiments, whole-cell lysates were preincubated with a 200-fold molar excess of unlabeled complementary oligonucleotide probes. Western Blot Analysis and Immunoprecipitation—After lysing cells on ice, whole-cell lysates were resolved by SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA). The membranes were sequentially probed with an appropriate primary antibody and a horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Buckinghamshire, UK), and immunocomplexes were detected using ECL Western blotting detection reagents (Amersham Biosciences). For immunoprecipitation, whole-cell lysates were incubated overnight with anti-FLAG M2 affinity gel Freezer-Safe (Sigma), and the immune complexes were analyzed by Western blot analysis. Anti-FLAG M2 and anti-β-actin (AC-15) monoclonal antibodies were purchased from Sigma. An anti-Bim monoclonal antibody (14A8) was purchased from Chemicon (Boronia, Victoria, Australia). Anti-FKHRL1, anti-phospho-FKHRL1(Thr32), and anti-phospho-FKHRL1(Ser253) monoclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). An anti-AU1 monoclonal antibody was purchased from Covance (Berkeley, CA). An anti-Smad3 monoclonal antibody (FL-425) was purchased from Santa Cruz Biotechnology. Restored Expression of RUNX3 Induces Apoptosis in the AGS Gastric Cancer Cell Line in the Absence of TGF-β—Runx3-deficient gastric epithelial cells are less sensitive to the proapoptotic effects of TGF-β (3Li Q.L. Ito K. Sakakura C. Fukamachi H. Inoue K. Chi X.Z. Lee K.Y. Nomura S. Lee C.W. Han S.B. Kim H.M. Kim W.J. Yamamoto H. Yamashita N. Yano T. Ikeda T. Itohara S. Inazawa J. Abe T. Hagiwara A. Yamagishi H. Ooe A. Kaneda A. Sugimura T. Ushijima T. Bae S.C. Ito Y. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar). In addition, human gastric cancer cell lines including AGS, MKN28, and KATO III lose endogenous RUNX3 expression (3Li Q.L. Ito K. Sakakura C. Fukamachi H. Inoue K. Chi X.Z. Lee K.Y. Nomura S. Lee C.W. Han S.B. Kim H.M. Kim W.J. Yamamoto H. Yamashita N. Yano T. Ikeda T. Itohara S. Inazawa J. Abe T. Hagiwara A. Yamagishi H. Ooe A. Kaneda A. Sugimura T. Ushijima T. Bae S.C. Ito Y. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar) and fail to respond to TGF-β to undergo apoptosis, while they express endogenous RUNX1 and PEBP2β2. 3H. Ida and Y. Ito, unpublished data. To assess the proapoptotic role of RUNX3 in TGF-β-induced apoptosis, we generated an AGS-derived transfectant clone (AGS/RUNX3) that expressed FLAG-tagged RUNX3 in an ecdysone-inducible manner. AGS and AGS/RUNX3 cells were stimulated with an ecdysone analog, ponasterone A, in the presence or absence of TGF-β for 48 h and then analyzed for the expression of FLAG-RUNX3 and the induction of apoptosis. Real-time quantitative RT-PCR analyses showed that treatment with ponasterone A induced RUNX3 mRNA in AGS/RUNX3 cells but not AGS cells (Fig. 1A). The mRNA level of RUNX3 induced in AGS/RUNX3 cells was similar to that in the SNU16 human gastric cancer cell line that expresses endogenous RUNX3 (3Li Q.L. Ito K. Sakakura C. Fukamachi H. Inoue K. Chi X.Z. Lee K.Y. Nomura S. Lee C.W. Han S.B. Kim H.M. Kim W.J. Yamamoto H. Yamashita N. Yano T. Ikeda T. Itohara S. Inazawa J. Abe T. Hagiwara A. Yamagishi H. Ooe A. Kaneda A. Sugimura T. Ushijima T. Bae S.C. Ito Y. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar) and responds to TGF-β to undergo apoptosis (26Kim S.G. Kim S.N. Jong H.S. Kim N.K. Hong S.H. Kim S.J. Bang Y.J. Oncogene. 2001; 20: 1254-1265Crossref PubMed Scopus (52) Google Scholar, 27Ohgushi M. Kuroki S. Fukamachi H. O'reilly L.A. Kuida K. Strasser A. Yonehara S. Mol. Cell. Biol. 2005; 25: 10017-10028Crossref PubMed Scopus (80) Google Scholar). Western blot analysis confirmed ponasterone A-induced expression of FLAG-RUNX3 in AGS/RUNX3 cells but not AGS cells (Fig. 1B). TGF-β did not affect the level of RUNX3 expression. The level of apoptosis was analyzed using annexin V staining. In the absence of TGF-β, the percentage of early apoptotic cells that were positively stained with annexin V but not with propidium iodide was increased from 3.67 to 27.7% following FLAG-RUNX3 expression in AGS/RUNX3 cells but was not increased in AGS cells (Fig. 1C). Cell death detection ELISA assays also showed that apoptosis was induced by ponasterone A but not by TGF-β in AGS/RUNX3 cells (Fig. 1D). The TGF-β stimulation did not increase the percentage of the apoptotic cells. It is important to note that in AGS/RUNX3 cells the induction of apoptosis was dependent on ponasterone A but not on TGF-β.FIGURE 1Restored RUNX3 induces apoptosis in the AGS gastric cancer cell line. A-D, AGS, AGS/RUNX3, SNU16, and MKN45 cells were unstimulated or stimulated with ponasterone A and/or TGF-β. A, RUNX3 mRNA levels were analyzed by real-time quantitative RT-PCR using primers for RUNX3 and human GAPDH. B, whole-cell lysates were separated by SDS-PAGE and analyzed by Western blot with an anti-FLAG or anti-β-actin monoclonal antibody. C, cells were stained with annexin V and propidium iodide (PI) and analyzed for the induction of apoptosis by flow cytometry. D, induction of apoptosis was assessed by cell death detection ELISA assays. E, AGS/RUNX3 cells were transfected with a (CAGA)12MLP-Luc reporter construct and then left unstimulated or stimulated with ponasterone A and/or TGF-β. Relative luciferase activity was measured in cell lysates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the involvement of Smad transcription factors (Smads) that are signal transducers of TGF-β in RUNX3-induced apoptosis, we examined the transcriptional activities of Smads in AGS/RUNX3 cells. Cells were transfected with a luciferase reporter construct, (CAGA)12MLP-Luc (28Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1580) Google Scholar), that is responsive to the binding of activated Smads. Luciferase assays were performed in the presence or absence of ponasterone A and/or TGF-β. The transcriptional activity of (CAGA)12MLP-Luc was not detected in the absence of TGF-β and was greatly increased in the presence of TGF-β (Fig. 1E). The ponasterone A stimulation did not increase the TGF-β-induced transcriptional activity of (CAGA)12MLP-Luc. These results indicated that Smads did not play a critical role in RUNX3-induced apoptosis in AGS/RUNX3 cells. RUNX3 and FoxO3a Cooperate to Mediate Apoptosis in Gastric Cancer Cell Lines—FoxO3a is a critical inducer of apoptosis in PTEN-mediated tumor suppression of prostate cancer (14Modur V. Nagarajan R. Evers B.M. Milbrandt J. J. Biol. Chem. 2002; 277: 47928-47937Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 29Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Crossref PubMed Scopus (494) Google Scholar) and in paclitaxel-mediated tumor suppression of breast cancer cells (13Sunters A. Fernández de Mattos S. Stahl M. Brosens J.J. Zoumpoulidou G. Saunders C.A. Coffer P.J. Medema R.H. Coombes R.C. Lam E.W. J. Biol. Chem. 2003; 278: 49795-49805Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Expression of proapoptotic Bim that mediates TGF-β-dependent apoptosis in B lymphocytes (30Wildey G.M. Patil S. Howe P.H. J. Biol. Chem. 2003; 278: 18069-18077Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) is transcriptionally up-regulated by FoxO3a, resulting in induction of apoptosis in breast cancer cells (13Sunters A. Fernández de Mattos S. Stahl M. Brosens J.J. Zoumpoulidou G. Saunders C.A. Coffer P.J. Medema R.H. Coombes R.C. Lam E.W. J. Biol. Chem. 2003; 278: 49795-49805Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). To assess the involvement of FoxO3a in RUNX3-induced apoptosis of gastric cancer cells, we examined the activity of FoxO3a in AGS, MKN28, KATO III, and SNU16 gastric cancer cell lines. Western blot analysis showed that active unphosphorylated FoxO3a, but not inactive phosphorylated (Thr32, Ser253) FoxO3a, was constitutively expressed in all these cell lines (Fig. 2). We next investigated whether FoxO3a is indispensable for RUNX3-induced apoptosis in AGS/RUNX3 cells. FoxG1/Qin is a member of the FoxO subfamily and inhibits the transcriptional activities of FoxO1, FoxO3a, and FoxO4 (31Seoane J. Le H.V. Shen L. Anderson S.A. Massagué J. Cell. 2004; 117: 211-223Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar, 32Aoki M. Jiang H. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13613-13617Crossref PubMed Scopus (177) Google Scholar). AGS/RUNX3 cells were transfected with the AU1-tagged FoxG1 cDNA, stimulated with ponasterone A, and examined for the AU1-FoxG1 expression and the ind

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