Title: Bone Morphogenetic Protein-2 and -4 Play Tumor Suppressive Roles in Human Diffuse-Type Gastric Carcinoma
Abstract: A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma. A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma. Gastric cancer is the fourth most common cancer and the second most common cause of death from cancer in the world.1Yaghoobi M. Bijarchi R. Narod S.A. Family history and the risk of gastric cancer.Br J Cancer. 2010; 102: 237-242Crossref PubMed Scopus (134) Google Scholar According to the Lauren classification, gastric cancer is divided mainly into intestinal and diffuse pathological types.2Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma An attempt at a histo-clinical classification.Acta Pathol Microbiol Scand. 1965; 64: 31-49Crossref PubMed Scopus (5052) Google Scholar A major decline has been reported in the incidence and mortality of intestinal-type gastric carcinoma. Eradication of Helicobacter pylori infection, which is the most important environmental risk factor of intestinal-type gastric carcinoma, prevents the development of intestinal-type gastric carcinoma and so contributes to the decrease in its incidence.3Fukase K. Kato M. Kikuchi S. Inoue K. Uemura N. Okamoto S. Terao S. Amagai K. Hayashi S. Asaka M. Japan Gast Study GroupEffect of eradication of Helicobacter pylori on incidence of metachronous gastric carcinoma after endoscopic resection of early gastric cancer: an open-label, randomised controlled trial.Lancet. 2008; 372: 392-397Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar On the other hand, the number of patients with diffuse-type gastric carcinoma has been increasing, particularly in the West.4Henson D.E. Dittus C. Younes M. Nguyen H. Albores-Saavedra J. Differential trends in the intestinal and diffuse types of gastric carcinoma in the United States, 1973–2000: increase in the signet ring cell type.Arch Pathol Lab Med. 2004; 128: 765-770PubMed Google Scholar In a majority of cases, diffuse-type gastric carcinoma is diagnosed in advanced stages, with rapid progression and poor prognosis. Transforming growth factor beta (TGF)-β, a multifunctional cytokine, exerts growth-inhibitory effects on many types of cells, and is well known as a tumor suppressor during the early stages of carcinogenesis.5Ikushima H. Miyazono K. TGFbeta signalling: a complex web in cancer progression.Nat Rev Cancer. 2010; 10: 415-424Crossref PubMed Scopus (913) Google Scholar Resistance to the growth-inhibitory activity of TGF-β often results in cancer development.6Bierie B. Moses H.L. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer.Nat Rev Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1097) Google Scholar In certain types of cancers, including colon cancer, pancreatic cancer, and gastric cancer, defects are observed in the TGF-β signal transduction pathways.5Ikushima H. Miyazono K. TGFbeta signalling: a complex web in cancer progression.Nat Rev Cancer. 2010; 10: 415-424Crossref PubMed Scopus (913) Google Scholar, 6Bierie B. Moses H.L. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer.Nat Rev Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1097) Google Scholar The role of bone morphogenetic proteins (BMPs) in cancer development and progression remains controversial. Although BMPs were originally identified as molecules that induce ectopic bone formation, BMPs exhibit a broad spectrum of biological activities in various tissues.7Suzuki Y. Montagne K. Nishihara A. Watabe T. Miyazono K. BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling.J Biochem. 2008; 143: 199-206Crossref PubMed Scopus (102) Google Scholar, 8Miyazono K. Kamiya Y. Morikawa M. Bone morphogenetic protein receptors and signal transduction.J Biochem. 2010; 147: 35-51Crossref PubMed Scopus (784) Google Scholar BMPs promote progression of breast and lung cancers,9Katsuno Y. Hanyu A. Kanda H. Ishikawa Y. Akiyama F. Iwase T. Ogata E. Ehata S. Miyazono K. Imamura T. Bone morphogenetic protein signaling enhances invasion and bone metastasis of breast cancer cells through Smad pathway.Oncogene. 2008; 27: 6322-6333Crossref PubMed Scopus (188) Google Scholar, 10Langenfeld E.M. Kong Y. Langenfeld J. Bone morphogenetic protein 2 stimulation of tumor growth involves the activation of Smad-1/5.Oncogene. 2006; 25: 685-692Crossref PubMed Scopus (115) Google Scholar and in this context specific inhibitors of BMP signaling, such as dorsomorphin, may be useful.11Yu P.B. Hong C.C. Sachidanandan C. Babitt J.L. Deng D.Y. Hoyng S.A. Lin H.Y. Bloch K.D. Peterson R.T. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.Nat Chem Biol. 2008; 4: 33-41Crossref PubMed Scopus (822) Google Scholar In contrast, however, the findings of several studies indicate that BMP acts as a tumor suppressor in some types of cancers (eg, brain cancer, prostate cancer, and colorectal cancer).12Piccirillo S.G. Reynolds B.A. Zanetti N. Lamorte G. Binda E. Broggi G. Brem H. Olivi A. Dimeco F. Vescovi A.L. Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.Nature. 2006; 444: 761-765Crossref PubMed Scopus (979) Google Scholar, 13Miyazaki H. Watabe T. Kitamura T. Miyazono K. BMP signals inhibit proliferation and in vivo tumor growth of androgen-insensitive prostate carcinoma cells.Oncogene. 2004; 23: 9326-9335Crossref PubMed Scopus (83) Google Scholar, 14Hardwick J.C. Kodach L.L. Offerhaus G.J. van den Brink G.R. Bone morphogenetic protein signalling in colorectal cancer.Nat Rev Cancer. 2008; 8: 806-812Crossref PubMed Scopus (156) Google Scholar Recent studies have revealed that BMP signaling contributes to the suppression of hamartoma and adenoma formation in the gastric epithelium.15Bleuming S.A. He X.C. Kodach L.L. Hardwick J.C. Koopman F.A. Ten Kate F.J. van Deventer S.J. Hommes D.W. Peppelenbosch M.P. Offerhaus G.J. Li L. van den Brink G.R. Bone morphogenetic protein signaling suppresses tumorigenesis at gastric epithelial transition zones in mice.Cancer Res. 2007; 67: 8149-8155Crossref PubMed Scopus (97) Google Scholar, 16Oshima H. Itadani H. Kotani H. Taketo M.M. Oshima M. Induction of prostaglandin E2 pathway promotes gastric hamartoma development with suppression of bone morphogenetic protein signaling.Cancer Res. 2009; 69: 2729-2733Crossref PubMed Scopus (24) Google Scholar However, the role of BMP signaling in the development and progression of diffuse-type gastric carcinoma has not been fully investigated. BMPs can be classified into several subgroups: the BMP-2/4 group, the osteogenic protein 1 (OP-1) group (BMP-5/6/7/8), the growth and differentiation factor 5, 6, and 7 group (GDF-5/6/7), and the BMP-9/10 group.8Miyazono K. Kamiya Y. Morikawa M. Bone morphogenetic protein receptors and signal transduction.J Biochem. 2010; 147: 35-51Crossref PubMed Scopus (784) Google Scholar BMPs bind to two different types of serine-threonine kinase receptors, type I and type II receptors. Activin receptor-like kinases ALK-1, ALK-2, ALK-3, and ALK-6 function as BMP type I receptors; the activin receptors ACTR-IIA and ACTR-IIB and the BMP receptor type 2 (BMPR-II) serve as BMP type II receptors. BMP-2 and BMP-4 bind preferentially to ALK-3 and ALK-6, whereas BMP-6 and BMP-7 bind strongly to ALK-2 and weakly to ALK-6. BMP-9 and BMP-10 bind to ALK-1 and ALK-2. On ligand binding, two type I receptors and two type II receptors form a heteromeric complex, which, in turn, transduces intracellular signals by phosphorylating BMP-specific receptor-regulated SMADs (R-SMADs), SMAD1/5/8. Phosphorylated BMP-specific R-SMADs form a heteromeric SMAD complex with common-partner SMAD (co-SMAD), SMAD4. This SMAD complex translocates into the nucleus and regulates transcription of various target genes. In addition to the SMAD pathway, non-SMAD pathways, including mitogen-activated protein kinase (MAPK) pathways, are activated by BMPs and may play important roles in cell proliferation and differentiation.17Moustakas A. Heldin C.H. Non-Smad TGF-beta signals.J Cell Sci. 2005; 118: 3573-3584Crossref PubMed Scopus (908) Google Scholar Kim et al18Kim J.Y. Park D.Y. Kim G.H. Choi K.U. Lee C.H. Huh G.Y. Sol M.Y. Song G.A. Jeon T.Y. Kim D.H. Sim M.S. Smad4 expression in gastric adenoma and adenocarcinoma: frequent loss of expression in diffuse type of gastric adenocarcinoma.Histol Histopathol. 2005; 20: 543-549PubMed Google Scholar reported that loss of expression of SMAD4 is frequently found in diffuse-type gastric carcinoma. Because SMAD4 is shared by TGF-β and BMP signaling pathways, loss of SMAD4 expression leads to perturbation of both pathways. The role of TGF-β signaling in diffuse-type gastric carcinoma has been well characterized,19Komuro A. Yashiro M. Iwata C. Morishita Y. Johansson E. Matsumoto Y. Watanabe A. Aburatani H. Miyoshi H. Kiyono K. Shirai Y.T. Suzuki H.I. Hirakawa K. Kano M.R. Miyazono K. Diffuse-type gastric carcinoma: progression, angiogenesis, and transforming growth factor beta signaling.J Natl Cancer Inst. 2009; 101: 592-604Crossref PubMed Scopus (60) Google Scholar, 20Johansson E. Komuro A. Iwata C. Hagiwara A. Fuse Y. Watanabe A. Morishita Y. Aburatani H. Funa K. Kano M.R. Miyazono K. Exogenous introduction of tissue inhibitor of metalloproteinase 2 reduces accelerated growth of TGF-beta-disrupted diffuse-type gastric carcinoma.Cancer Sci. 2010; 101: 2398-2403Crossref PubMed Scopus (15) Google Scholar, 21Ehata S. Johansson E. Katayama R. Koike S. Watanabe A. Hoshino Y. Katsuno Y. Komuro A. Koinuma D. Kano M.R. Yashiro M. Hirakawa K. Aburatani H. Fujita N. Miyazono K. Transforming growth factor-beta decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.Oncogene. 2011; 30: 1693-1705Crossref PubMed Scopus (64) Google Scholar and it is worth examining whether perturbation of BMP signaling also contributes to the development of diffuse-type gastric carcinoma. We investigated the role of BMP signaling in the progression of diffuse-type gastric carcinoma using human gastric cancer cells established from signet-ring cell carcinoma and from poorly differentiated adenocarcinoma. We present here, for the first time, evidence that BMP-2 and BMP-4 suppress proliferation of diffuse-type gastric carcinoma cells through induction of p21 (p21WAF1/CIP1) and function as potent tumor suppressors in this type of gastric carcinoma. Human diffuse-type gastric carcinoma OCUM-12 and OCUM-2MLN cells were established as described previously.22Fujihara T. Sawada T. Hirakawa K. Chung Y.S. Yashiro M. Inoue T. Sowa M. Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis.Clin Exp Metastasis. 1998; 16: 389-398Crossref PubMed Scopus (53) Google Scholar, 23Kato Y. Yashiro M. Noda S. Tendo M. Kashiwagi S. Doi Y. Nishii T. Matsuoka J. Fuyuhiro Y. Shinto O. Sawada T. Ohira M. Hirakawa K. Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma.Br J Cancer. 2010; 102: 898-907Crossref PubMed Scopus (57) Google Scholar OCUM-2MLN cells were cultured as described previously,24Nagano Y. Koinuma D. Miyazawa K. Miyazono K. Context-dependent regulation of the expression of c-Ski protein by Arkadia in human cancer cells.J Biochem. 2010; 147: 545-554Crossref PubMed Scopus (29) Google Scholar and OCUM-12 cells were cultured under the same conditions. Human diffuse-type gastric carcinoma HSC-39 cells were established as described previously.25Yanagihara K. Seyama T. Tsumuraya M. Kamada N. Yokoro K. Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene.Cancer Res. 1991; 51: 381-386PubMed Google Scholar HSC-39 cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL). All cells were grown in a 5% CO2 atmosphere at 37°C. BMP-4, BMP-6, and BMP-9 (R&D Systems, Minneapolis, MN) were used at a concentration of 30 ng/mL. TGF-β1 (R&D Systems) was used at a concentration of 1 ng/mL. Dorsomorphin (Sigma-Aldrich, St. Louis, MO) was dissolved in dimethyl sulfoxide and used at a concentration of 3 μmol/L. Doxycycline was obtained from Clontech (Mountain View, CA). We used a lentiviral vector system to establish diffuse-type gastric carcinoma cells stably expressing green fluorescent protein (GFP), the dominant-negative form of ALK-3 (dnALK3), and the constitutively active form of ALK-3 (caALK3). A lentiviral vector encoding GFP (CS-CDF-CG-PRE; a gift from Dr. Hiroyuki Miyoshi, RIKEN) was used as the control. cDNAs encoding ALK-3 that lacks the intracellular domain with a carboxyl-terminal HA (influenza hemagglutinin) epitope tag or ALK3QD with a carboxyl-terminal FLAG epitope tag were inserted into the lentiviral vector CSII-EF-RfA. cDNAs encoding caALK3 with a carboxyl-terminal HA epitope tag or Aequorea coerulescens GFP (AcGFP) were inserted into a Tet-ON lentivector (CSIV-TRE-RfA-CMV-KT; a gift from Dr. Hiroyuki Miyoshi). Lentivirus was produced basically as described previously26Ehata S. Hanyu A. Fujime M. Katsuno Y. Fukunaga E. Goto K. Ishikawa Y. Nomura K. Yokoo H. Shimizu T. Ogata E. Miyazono K. Shimizu K. Imamura T. Ki26894, a novel transforming growth factor-beta type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line.Cancer Sci. 2007; 98: 127-133Crossref PubMed Scopus (154) Google Scholar and was concentrated using Lenti-X concentrator (Clontech) to infect OCUM-12 and HSC-39 cells. HSC-39-Tc-AcGFP or HSC-39-Tc-caALK3 cells were established by isolating Kusabira Orange-expressing cells with semi-limiting dilution. Total RNAs were extracted using Isogen reagent (Nippon Gene, Tokyo, Japan) or an RNeasy mini kit (Qiagen, Valencia, CA). First-strand cDNA synthesis, semi-quantitative RT-PCR, and quantitative real-time RT-PCR were performed as described previously,27Hoshino Y. Katsuno Y. Ehata S. Miyazono K. Autocrine TGF-beta protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim.J Biochem. 2011; 149: 55-65Crossref PubMed Scopus (46) Google Scholar with primer sequences as listed in Table 1. Semi-quantitative RT-PCR conditions were as follows: 25 to 40 cycles of 94°C (15 s), 55 to 60°C (30 s), and 68°C (1 minute). Values obtained in quantitative real-time RT-PCR were normalized to ACTB (encoding β-actin).Table 1Primers Used in RT-PCRGeneForward primerReverse primerSemi-quantitative RT-PCR ACVRL15′-CTCTACGACTTTCTGCAGAG-3′5′-CCACTTGTAGGACTCAAAGC-3′ ACVR15′-ATGTCTTTTAGCCTGCCTGCTG-3′5′-ATCAAGCTGATTGGTGCTCTGG-3′ BMPR1A5′-TGATTTGGAACAGGATGAAGC-3′5′-TGTAGCACATTTCAGGAAGTC-3′ TGFBR15′-TCGCCCTTTTATTTCAGAGGGTACT-3′5′-ACAGCAAGTTCCATTCTTCTTTACC-3′ BMPR1B5′-GCAGCACAGACGGATATTGT-3′5′-TTTCATGCCTCATCAACACT-3′ ACVR2A5′-GCAAAATGAATACGAAGTCTA-3′5′-GCACCCTCTAATACCTCTGGA-3′ ACVR2B5′-ACACGGGAGTGCATCTACTACAACG-3′5′-TCATGAGCTGGGCCTTCCAGA-3′ BMPR25′-CTGCACAGTGTGCTGAGGAAAG-3′5′-TGAACTGCCCTGTTACTGCCA-3′ TGFBR25′-ATAAGGCCAAGCTGAAGCAG-3′5′-CTTCTGGAGCCATGTATCTTG-3′ SMAD15′-TGCCACTCAACGCCACTTTT-3′5′-TCATAAGCAACCGCCTGAACAT-3′SMAD25′-CCCATCGAAAAGGATTGCCACA-3′5′-TGCATGGAAGGTTTCTCCAACC-3′ SMAD35′-GGACGACTACAGCCATTCCA-3′5′-TTCCGATGTGTCTCCGTGTCA-3′ SMAD45′-CTTTGAAATGGATGTTCAG-3′5′-CATCCTGATAAGGTTAAGGG-3′ SMAD55′-ACGTCAATGGCCAGCTTGTT-3′5′-TCCAACGGCTTTAGCTCATGA-3′ SMAD85′-ATCTTTGTGCAGAGCCGGAA-3′5′-TCCTGGCGATGATACTCAGCA-3′ BMP25′-CCAGAAACGAGTGGGAAAAC-3′5′-AAGTCCACGTACAAAGGGTG-3′ BMP45′-ACTGGTCCACCACAATGTGACACG-3′5′-GCTGAAGTCCACATAGAGCGAGTG-3′ ACTB5′-TCACCCACACTGTGCCCATCTACGA-3′5′-CAGCGGAACCGCTCATTGCCAATGG-3′Quantitative real-time RT-PCR ID35′-GACTTCACCAAATCCCTTCC-3′5′-CCACTCCTTCCACACCTC-3′ CDKN1A5′-AGTGGACAGCGAGCAGCTGA-3′5′-CGAAGTTCCATCGCTCACGG-3′ CDKN1B5′-CGGTGGACCACGAAGAGTTAA-3′5′-GGCTCGCCTCTTCCATGTC-3′ CDKN2A5′-TGCCTTTTCACTGTGTTGGAGTT-3′5′-TCGCAAGAAATGCCCACAT-3′ CDKN2B5′-CCGCCCACAACGACTTTATT-3′5′-CAGCCTTCATCGAATTAGGTG-3′ CDC25A5′-GCCTGTCACCAACCTGAC-3′5′-CCAGGAGAATCTAGACAGAAACC-3′ MYC5′-CCACACATCAGCACAACTACGC-3′5′-CGGTTGTTGCTGATCTGTCTCA-3′ SMAD45′-GATACGTGGACCCTTCTGGA-3′5′-ACCTTTGCCTATGTGCAACC-3′ ACTB5′-TCACCCACACTGTGCCCATCTACGA-3′5′-CAGCGGAACCGCTCATTGCCAATGG-3′ Open table in a new tab Immunoblotting was performed as described previously.19Komuro A. Yashiro M. Iwata C. Morishita Y. Johansson E. Matsumoto Y. Watanabe A. Aburatani H. Miyoshi H. Kiyono K. Shirai Y.T. Suzuki H.I. Hirakawa K. Kano M.R. Miyazono K. Diffuse-type gastric carcinoma: progression, angiogenesis, and transforming growth factor beta signaling.J Natl Cancer Inst. 2009; 101: 592-604Crossref PubMed Scopus (60) Google Scholar Cultured cells were lysed in a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Nonidet P-40 surfactant, and 1% aprotinin (Calbiochem). We used antibodies to phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) (pSmad1/5/8; Cell Signaling Technology, Danvers, MA), phospho-Smad2 (Ser465/467) (pSmad2; Zymed Laboratories, South San Francisco, CA), Smad1 (Cell Signaling Technology), Smad2/3 (Cell Signaling Technology), Smad4 (Santa Cruz Biotechnology, Santa Cruz, CA), p21 (BD Biosciences, San Jose, CA), retinoblastoma protein (RB; BD Biosciences), poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology), GFP (MBL International, Woburn, MA), HA (3F10; Roche, Basel, Switzerland), FLAG (M2, Sigma-Aldrich), and α-tubulin (Sigma-Aldrich). RNA interference was performed as described previously.21Ehata S. Johansson E. Katayama R. Koike S. Watanabe A. Hoshino Y. Katsuno Y. Komuro A. Koinuma D. Kano M.R. Yashiro M. Hirakawa K. Aburatani H. Fujita N. Miyazono K. Transforming growth factor-beta decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.Oncogene. 2011; 30: 1693-1705Crossref PubMed Scopus (64) Google Scholar Stealth small interfering RNA (siRNA) duplex oligoribonucleotides against human SMAD4 (siRNA/SMAD4) or non-targeting control (siRNA/NTC) were synthesized by Invitrogen. OCUM-12 cells were transfected with each siRNA according to the manufacturer's protocols. Short hairpin RNA (shRNA) constructs against human p21 were designed using BLOCK-it RNAi Designer (Invitrogen), with the target sequence 5′-GCCTCTGGCATTAGAATTATT-3′. Before seeding cells, we used a poly-l-lysine solution (Sigma-Aldrich) to coat the chamber plates. Cells (2.5 × 104 cells) were seeded in eight-well chamber plates. On the next day, cells were treated with BMP-4. Cells were fixed in 3.7% formaldehyde and then permeabilized with PBS containing 0.1% Triton X-100 surfactant. A mouse monoclonal antibody against human Ki-67 (MIB-1; DakoCytomation, Carpinteria, CA) and Alexa Fluor 488-conjugated mouse secondary antibody (Invitrogen) were used to detect proliferating cells. The nuclei were counterstained with TOTO-3 fluorophore (Invitrogen). Fluorescence was examined using a Zeiss LSM 510 Meta confocal microscope and was measured with LSM Image Browser software version 3.5.0.359 (Carl Zeiss MicroImaging, Göttingen, Germany). Quantification was performed by counting Alexa Fluor 488-positive cells against TOTO3-positive cells in five fields. Cells were dissociated into single-cell populations and labeled with propidium iodide using a Cycletest Plus DNA Reagent Kit (BD Biosciences). Cell cycle distribution of cells was determined using an EPICS XL flow cytometer with EXPO32 ADC software (Beckman Coulter, Life Sciences, Indianapolis, IN). FlowJo software version 7.2.5 (Tree Star, Ashland, OR) was used to generate histograms. Cells (0.5 × 104 to 1.5 × 104 cells) were seeded in triplicate in 12-well plates. On the next day, cells were treated with BMP-4. Cells were counted with a hemocytometer. BALB/c nu/nu male mice (4 to 5 weeks of age) were obtained from the Oriental Yeast Company (Tokyo, Japan). A total of 5 × 106 cells in 100 μL of culture medium were injected into the right flank of each mouse, unless otherwise mentioned. Subcutaneous tumors were measured externally, and tumor volume was estimated as described previously.19Komuro A. Yashiro M. Iwata C. Morishita Y. Johansson E. Matsumoto Y. Watanabe A. Aburatani H. Miyoshi H. Kiyono K. Shirai Y.T. Suzuki H.I. Hirakawa K. Kano M.R. Miyazono K. Diffuse-type gastric carcinoma: progression, angiogenesis, and transforming growth factor beta signaling.J Natl Cancer Inst. 2009; 101: 592-604Crossref PubMed Scopus (60) Google Scholar All animal experiments were performed in accordance with the policies of the Animal Ethics Committee, University of Tokyo. Formalin-fixed, paraffin-embedded gastric tissues were obtained from patients with diffuse-type gastric carcinoma at the Osaka City University Hospital, Osaka, Japan, with informed consent. H&E staining of tissues was performed as described previously.19Komuro A. Yashiro M. Iwata C. Morishita Y. Johansson E. Matsumoto Y. Watanabe A. Aburatani H. Miyoshi H. Kiyono K. Shirai Y.T. Suzuki H.I. Hirakawa K. Kano M.R. Miyazono K. Diffuse-type gastric carcinoma: progression, angiogenesis, and transforming growth factor beta signaling.J Natl Cancer Inst. 2009; 101: 592-604Crossref PubMed Scopus (60) Google Scholar Antigen retrieval was performed with 10 mmol/L sodium citrate (pH 6.0) at 121°C for 10 minutes, and sections were immunostained with primary antibodies (pSmad1/5/8, p21, and MIB-1) and biotinylated secondary antibodies. Immunodetection was performed with a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine (DakoCytomation). Sections were weakly counterstained with hematoxylin. All studies were conducted using protocols approved by the Osaka City University Ethics Committee. The size of tumors was analyzed statistically by repeated-measures analysis of variance. Tukey-Kramer post hoc tests were used for examining differences between multiple groups. Two-tailed Student's t-tests were used to compare two groups. Results were considered to be statistically significant at P < 0.05. We first evaluated the expressions of BMP signal components in OCUM-12, HSC-39, and OCUM-2MLN cells using semi-quantitative RT-PCR (Figure 1A). In these cells, BMP type I receptor genes ACVR1 (encoding ALK-2), BMPR1A (encoding ALK-3), and BMPR1B (encoding ALK-6) were expressed; ACVRL1 (encoding ALK-1), which is mainly expressed in endothelial cells and transduces BMP-9 signaling, was not expressed. BMP type II receptor genes ACVR2A (encoding ACTR-IIA), ACVR2B (encoding ACTR-IIB), and BMPR2 were also expressed in these cells. We detected expression of SMAD4 transcripts in these cells. Among the three types of BMP-specific R-SMADs, SMAD1 and SMAD5 were expressed in all these cells, whereas SMAD8 was expressed only in HSC-39 cells. We also detected expression of BMP2 and/or BMP4 in all these cells. Expression levels of BMPR1B, SMAD1, and SMAD5 in OCUM-2MLN cells were lower than those in the other diffuse-type gastric carcinoma cells. Of the TGF-β signal components, TGFBR1 (TGF-β type I receptor, encoding ALK-5) and TGFBR2 (TGF-β type II receptor, encoding TGF-β receptor type 2, TβR-II) were expressed in these cells, as well as two TGF-β-specific R-SMADs, SMAD2 and SMAD3. We next examined phosphorylation of SMAD1/5/8 in OCUM-12, HSC-39, and OCUM-2MLN cells by immunoblotting (Figure 1B). In these cells, SMAD1/5/8 were phosphorylated by BMP-4 (in the BMP-2/4 group), which was suppressed by the small-molecule BMP inhibitor dorsomorphin.11Yu P.B. Hong C.C. Sachidanandan C. Babitt J.L. Deng D.Y. Hoyng S.A. Lin H.Y. Bloch K.D. Peterson R.T. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.Nat Chem Biol. 2008; 4: 33-41Crossref PubMed Scopus (822) Google Scholar In addition, phosphorylation of SMAD1/5/8 was induced by BMP-6 and BMP-9, whereas phosphorylation of SMAD2 was induced only by TGF-β1. Phosphorylation of SMAD1/5/8 was also induced by TGF-β1 in HSC-39 cells, as shown in certain other cells.28Liu I.M. Schilling S.H. Knouse K.A. Choy L. Derynck R. Wang X.F. TGFbeta-stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro-migratory TGFbeta switch.EMBO J. 2009; 28: 88-98Crossref PubMed Scopus (147) Google Scholar We also evaluated the expression of ID3 mRNA, one of the downstream targets of BMP-4, in these cells using quantitative real-time RT-PCR (Figure 1C). As expected, BMP-4 induced the expression of ID3 in these cells and dorsomorphin suppressed it, although the degrees of increased expression of ID3 by BMP-4 varied. To determine whether BMP signaling is responsible for the regulation of tumor growth of diffuse-type gastric carcinoma cells in vivo, we used diffuse-type gastric carcinoma cells stably expressing dnALK3 (OCUM-12-dnALK3 and HSC-39-dnALK3) (Figure 2A). BMP-4-induced expression of pSMAD1/5/8 in dnALK3-expressing cells was lower than that in the control GFP-expressing cells (OCUM-12-GFP and HSC-39-GFP). We also confirmed that the expression of dnALK3 had little effect on TGF-β signaling in diffuse-type gastric carcinoma cells (Figure 2B). Induction of ID3 mRNA by BMP-4 was suppressed in dnALK3-expressing cells (Figure 2C), indicating that BMP-2/4 signaling in these cells was successfully inhibited. We next xenografted GFP- and dnALK3-expressing cancer cells into BALB/c nude mice. We found that in vivo tumor growth of OCUM-12-dnALK3 or HSC-39-dnALK3 cells was significantly more accelerated than that of the corresponding control cells (OCUM-12-GFP or HSC-39-GFP; Figure 2D). TGF-β signaling was reported to regulate the vascular density and fibrosis in diffuse-type gastric carcinoma cells.19Komuro A. Yashiro M. Iwata C. Morishita Y. Johansson E. Matsumoto Y. Watanabe A. Aburatani H. Miyoshi H. Kiyono K. Shirai Y.T. Suzuki H.I. Hirakawa K. Kano M.R. Miyazono K. Diffuse-type gastric carcinoma: progression, angiogenesis, and transforming growth factor beta signaling.J Natl Cancer Inst. 2009; 101: 592-604Crossref PubMed Scopus (60) Google Scholar, 20Johansson E. Komuro A. Iwata C. Hagiwara A. Fuse Y. Watanabe A. Morishita Y. Aburatani H. Funa K. Kano M.R. Miyazono K. Exogenous introduction of tissue inhibitor of metalloproteinase 2 reduces accelerated growth of TGF-beta-disrupted diffuse-type gastric carcinoma.Cancer Sci. 2010; 101: 2398-2403Crossref PubMed Scopus (15) Google Scholar We therefore also examined the histology of the resultant tumor tissues by H&E staining. The appearance of the microenvironment in tumor tissues, including angiogenesis and fibrosis, was not obviously affected by the expression of dnALK3 (data not shown). We next evaluated the effects of BMP-4 on proliferation and apoptosis of diffuse-type gastric carcinoma cells in vitro. The in vitro proliferation of OCUM-12-GFP and HSC-39-GFP cells was inhibited by treatment with BMP-4, and growth inhibition by BMP-4 was abrogated in OCUM-12-dnALK3 and HSC-39-dnALK3 cells (Figure 3A). Because BMPs have been reported to induce apoptosis of certain types of cancer cells,29Kawamura C. Kizaki M. Yamato K. Uchida H. Fukuchi Y. Hattori Y. Koseki T. Nishihara T. Ikeda Y. Bone morphogenetic protein-2 induces apoptosis in human myeloma cells with modulation of STAT3.Blood. 2000; 96: 2005-2011Crossref PubMed Google Scholar, 30Fukuda N. Saitoh M. Kobayashi N. Miyazono K. Execution of BMP-4-induced apoptosis by p53-dependent ER dysfunction in myeloma and B-cell hybridoma cells.Oncogene. 2006; 25: 3509-3517Crossref PubMed Scopus (29) Google Scholar induction of apoptosis by BMP-4 in OCUM-12 and HSC-39 cells was examined. However, cleavage of PARP in these cells was not enhanced by BMP-4 (Figure 3B). Moreover, TUNEL staining revealed that BMP-4 did not induce DNA fragmentation in OCUM-12 cells (see Supplemental Figure S1 at http://ajp.amjpathol.org). Recent reports also suggest that BMPs negatively regulate cell cycle progression of cancer cells, including intestinal-type gastric carcinoma cells and prostate cancer cells.13Miyazaki H. Watabe T. Kitamura T. Mi