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Molecular and Cellular Pathobiology Autocrine BMP-4 Signaling Is a Therapeutic Target in Colorectal Cancer Yuichiro Yokoyama 1,2 , Toshiaki Watanabe 2 , Yusuke Tamura 1 , Yoshinobu Hashizume 3 , Kohei Miyazono 1 , and Shogo Ehata 1 Abstract Poor prognoses for colorectal cancer patients with meta- static lesions have driven demand for the development of novel targeted therapies. Here, we demonstrate that expres- sion of bone morphogenetic protein 4 (BMP-4) is universally upregulated in human colorectal cancer cells and tissues, resulting in activated BMP signaling. Inhibition of endoge- nous BMP signaling by the BMP type I receptor inhibitor LDN-193189 elevated expression of the phosphatase DUSP5 in colorectal cancer cells, inducing apoptosis via dephosphor- ylation of Erk MAPK. Administering LDN-193189 to mice diminished tumor formation of colorectal cancer cells. Our ndings suggest inhibition of autocrine BMP-4 as a candidate treatment strategy for colorectal cancer. Cancer Res; 77(15); 402638. Ó2017 AACR. Introduction Colorectal cancer is the third most common cancer and the fourth most common cause of cancer-related death worldwide (1). Although surgical resection can cure early-stage colorectal cancer, a combination of surgery and chemotherapeutic agents is recommended in advanced stages of colorectal cancer (1). In addition to conventional cytotoxic agents, new agents targeting VEGF signaling and EGFR signaling have been introduced during the last decade (2). Although colorectal cancer prognoses have steadily improved, the 5-year survival rate remains low, especially in patients with metastatic lesions (1). Thus, the development of new molecular targets for treatment of advanced colorectal cancer is critical for improving patient outcomes. Bone morphogenetic proteins (BMP) are members of the TGF-b family and are multifunctional cytokines (35). BMPs recognize two distinct receptors, termed type I and type II receptors, with serine/threonine and tyrosine kinase activities. Type I BMP receptors include activin receptor-like kinase (ALK)- 1, -2, -3, and -6, and type II receptors include BMP type II receptor (BMPR-II), activin type II receptor (ActR-II), and activin type IIB receptor (ActR-IIB). BMPs are classied into several subgroups, including the BMP-2/4 group, BMP-5/6/7/8 group, BMP-9/10 group, and growth and differentiation factor (GDF)-5/6/7 group, according to structural similarities and their ability to bind certain type I receptors. Upon binding to type I and type II receptors, BMPs form heterotetrameric com- plexes; the protein kinase of the type II receptor activates the protein kinase of the type I receptor, which in turn phosphor- ylates the BMP-specic receptor-regulated Smads (R-Smads), Smad1 and Smad5. Phosphorylated R-Smads induce a hetero- meric assembly with common-partner Smad (Co-Smad; Smad4) and translocate into the nucleus, regulating the tran- scription of target genes. BMPs can also activate non-Smad signaling pathways, including the MAPK pathway (3). Divergent roles of BMPs have been reported during cancer progression (46). BMPs inhibit proliferation of gastric cancer, breast cancer, and prostate cancer cells, induce differentiation of glioma-initiating cells, and inhibit glioblastoma tumor formation (5), indicating a tumor-suppressive role of BMPs. In contrast, BMPs have been reported to enhance the motility and invasive- ness of various types of cancer cells, such as breast cancer, prostate cancer, and malignant melanoma cells, suggesting that BMPs also function as tumor-promoting factors (4). In this study, the role of BMP-4 produced by colorectal cancer cells in cancer progression was investigated. We reveal for the rst time that inhibition of BMP-4 induces the apoptosis of colorectal cancer cells through the attenuation of MAPK activity in culture and that the small-molecule BMP inhibitor LDN-193189 diminishes colorectal cancer formation in vivo. Materials and Methods Cell culture and reagents Human colon adenocarcinoma cells HT29 and DLD-1 (Japa- nese Cancer Research Resource Bank) were cultured in RPMI containing 10% FBS, penicillin, and streptomycin. Human colon adenocarcinoma SW480 cells (ATCC) were cultured in DMEM containing 10% FBS, penicillin, and streptomycin. Routine Myco- plasma testing was performed by PCR regularly on these cells. The cells were bought in 2002 and have been stocked as cryopreserved aliquots in liquid N 2 . The cells were used within 8 passages after thawing and reauthenticated by short tandem repeat proling in 2017. LDN-193189 was obtained from Wako or RIKEN. 1 Department of Molecular Pathology, Graduate School of Medicine, The Univer- sity of Tokyo, Tokyo, Japan. 2 Department of Surgical Oncology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. 3 RIKEN Program for Drug Discovery and Medical Technology Platforms, Wako, Saitama, Japan. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Shogo Ehata, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 813-5841-3356; Fax: 813-5841-3354; E-mail: [email protected]; and Kohei Miyazono, [email protected] doi: 10.1158/0008-5472.CAN-17-0112 Ó2017 American Association for Cancer Research. Cancer Research Cancer Res; 77(15) August 1, 2017 4026 on October 5, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0112

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  • Molecular and Cellular Pathobiology

    Autocrine BMP-4 Signaling Is a TherapeuticTarget in Colorectal CancerYuichiro Yokoyama1,2, Toshiaki Watanabe2, Yusuke Tamura1,Yoshinobu Hashizume3, Kohei Miyazono1, and Shogo Ehata1

    Abstract

    Poor prognoses for colorectal cancer patients with meta-static lesions have driven demand for the development ofnovel targeted therapies. Here, we demonstrate that expres-sion of bone morphogenetic protein 4 (BMP-4) is universallyupregulated in human colorectal cancer cells and tissues,resulting in activated BMP signaling. Inhibition of endoge-nous BMP signaling by the BMP type I receptor inhibitor

    LDN-193189 elevated expression of the phosphatase DUSP5in colorectal cancer cells, inducing apoptosis via dephosphor-ylation of Erk MAPK. Administering LDN-193189 to micediminished tumor formation of colorectal cancer cells. Ourfindings suggest inhibition of autocrine BMP-4 as a candidatetreatment strategy for colorectal cancer. Cancer Res; 77(15);4026–38. �2017 AACR.

    IntroductionColorectal cancer is the third most common cancer and the

    fourth most common cause of cancer-related death worldwide(1). Although surgical resection can cure early-stage colorectalcancer, a combination of surgery and chemotherapeutic agents isrecommended in advanced stages of colorectal cancer (1). Inaddition to conventional cytotoxic agents, new agents targetingVEGF signaling and EGFR signaling have been introduced duringthe last decade (2). Although colorectal cancer prognoses havesteadily improved, the 5-year survival rate remains low, especiallyin patients with metastatic lesions (1). Thus, the development ofnewmolecular targets for treatment of advanced colorectal canceris critical for improving patient outcomes.

    Bone morphogenetic proteins (BMP) are members of theTGF-b family and are multifunctional cytokines (3–5). BMPsrecognize two distinct receptors, termed type I and type IIreceptors, with serine/threonine and tyrosine kinase activities.Type I BMP receptors include activin receptor-like kinase (ALK)-1, -2, -3, and -6, and type II receptors include BMP type IIreceptor (BMPR-II), activin type II receptor (ActR-II), andactivin type IIB receptor (ActR-IIB). BMPs are classified intoseveral subgroups, including the BMP-2/4 group, BMP-5/6/7/8group, BMP-9/10 group, and growth and differentiation factor(GDF)-5/6/7 group, according to structural similarities and

    their ability to bind certain type I receptors. Upon binding totype I and type II receptors, BMPs form heterotetrameric com-plexes; the protein kinase of the type II receptor activates theprotein kinase of the type I receptor, which in turn phosphor-ylates the BMP-specific receptor-regulated Smads (R-Smads),Smad1 and Smad5. Phosphorylated R-Smads induce a hetero-meric assembly with common-partner Smad (Co-Smad;Smad4) and translocate into the nucleus, regulating the tran-scription of target genes. BMPs can also activate non-Smadsignaling pathways, including the MAPK pathway (3).

    Divergent roles of BMPs have been reported during cancerprogression (4–6). BMPs inhibit proliferation of gastric cancer,breast cancer, and prostate cancer cells, induce differentiation ofglioma-initiating cells, and inhibit glioblastoma tumor formation(5), indicating a tumor-suppressive role of BMPs. In contrast,BMPs have been reported to enhance the motility and invasive-ness of various types of cancer cells, such as breast cancer, prostatecancer, andmalignant melanoma cells, suggesting that BMPs alsofunction as tumor-promoting factors (4).

    In this study, the role of BMP-4 produced by colorectal cancercells in cancer progression was investigated. We reveal for the firsttime that inhibition of BMP-4 induces the apoptosis of colorectalcancer cells through the attenuation of MAPK activity in cultureand that the small-molecule BMP inhibitor LDN-193189diminishes colorectal cancer formation in vivo.

    Materials and MethodsCell culture and reagents

    Human colon adenocarcinoma cells HT29 and DLD-1 (Japa-nese Cancer Research Resource Bank) were cultured in RPMIcontaining 10% FBS, penicillin, and streptomycin. Human colonadenocarcinoma SW480 cells (ATCC) were cultured in DMEMcontaining 10% FBS, penicillin, and streptomycin. RoutineMyco-plasma testing was performed by PCR regularly on these cells. Thecells were bought in 2002 and have been stocked as cryopreservedaliquots in liquid N2. The cells were used within 8 passages afterthawing and reauthenticated by short tandem repeat profiling in2017. LDN-193189 was obtained from Wako or RIKEN.

    1Department of Molecular Pathology, Graduate School of Medicine, The Univer-sity of Tokyo, Tokyo, Japan. 2Department of SurgicalOncology, TheUniversity ofTokyo, Bunkyo-ku, Tokyo, Japan. 3RIKEN Program for Drug Discovery andMedical Technology Platforms, Wako, Saitama, Japan.

    Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

    Corresponding Authors: Shogo Ehata, Graduate School of Medicine, TheUniversity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone:813-5841-3356; Fax: 813-5841-3354; E-mail: [email protected]; and KoheiMiyazono, [email protected]

    doi: 10.1158/0008-5472.CAN-17-0112

    �2017 American Association for Cancer Research.

    CancerResearch

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    Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0112

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  • Quantitative real-time RT-PCR analyses and chromatinimmunoprecipitation–qRT-PCR analyses

    Quantitative real-time RT-PCR (qRT-PCR) analysis and chro-matin immunoprecipitation (ChIP)–qRT-PCR analysis wereperformed as previously described (7, 8). Primer sequencesare described in Supplementary Table S1. Anti-TCF4 antibody(sc-8631) was purchased from Santa Cruz Biotechnology.

    ImmunoblottingImmunoblotting was performed as previously described (7).

    Antibodies are described in Supplementary Materials and Meth-ods. ImageJ (NIH) was used to quantify blot band intensities.

    Apoptosis assayTerminal deoxynucleotidyl transferase-mediated dUTP

    nick end labeling (TUNEL) assay was performed as previouslydescribed (7). Fluorescence was examined using a LeicaDMI6000 B.

    siRNAStealth RNAi Pre-Designed siRNAs targeting CTNNB1, BMP4

    and DUSP5 were synthesized by Thermo Fisher Scientific. Cellswere transfected in the presence of 30 nmol/L siRNA or controlsiRNA in a 500 mL volume with 3 mL RNAiMAX reagent (ThermoFisher Scientific) per well in a 6-well plate.

    Lentiviral production and infectionWe used a lentiviral vector system to induce specific gene

    introduction and knockdown as previously described (9, 10).The target sequences for shRNA are described in the Supplemen-tary Materials and Methods.

    RNA-sequence analysesRNA-sequence (RNA-seq) analysis was performed as described

    previously (11). Raw and processed data are available at GEO(GSE96914). Gene Ontology analysis was performed using CLCGenomics Workbench (Qiagen Bioinformatics).

    Subcutaneous xenograft modelBALB/c nu/nu female mice (4–5 weeks) were obtained from

    SankyoLaboServiceCorporation. A total of 5�106 cells in100mLof culture medium were subcutaneously inoculated. Tumor vol-ume was estimated as previously described (12). All animalexperiments were performed under the policies of the AnimalEthics Committee of The University of Tokyo (approval number:12312). The bioavailability of administered LDN-193189 wasexamined as described previously (13).

    IHCFormalin-fixed, paraffin-embedded human clinical samples

    were obtained from patients at The University of Tokyo Hospitalwith informed consent. The protocol was approved by theResearch Ethics Committee at The University of Tokyo, GraduateSchool of Medicine (approval number: 10475). IHC was per-formed as previously described (14). Antibodies are described inthe Supplementary Materials and Methods.

    ResultsColorectal cancer produces BMP-4 through aberrant activationof the Wnt/b-catenin pathway

    To investigate the expression of BMP mRNAs in colorectalcancer, data from several public databases were re-analyzed. The

    NCI-60 cell line panel indicated that, among various BMPs,expression of BMP4was commonly elevated in colon cancer cells(Fig. 1A).NCBIGEOdatabaseGSE14258 revealed that expressionof BMP4, but not of other BMPs, was significantly higher incolon cancer tissues than in normal colon tissues (Fig. 1B). Next,the correlation between BMP4 expression and colorectal cancerpatient prognosis was examined. GSE14333 showed that elevatedexpression of BMP4 was associated with poor prognosis inpatients with stage II colorectal cancer (Fig. 1C). Furthermore,multivariate analysis demonstrated that BMP4 expression was anindependent prognostic factor in stage II colorectal cancer (Sup-plementary Table S2). Expression of BMP-4 and activation ofSmad-dependent BMP signalingwas then examined using humancolorectal cancer tissues and cells. IHC analysis revealed thatexpression levels of BMP-4 and phosphorylated Smad1/5 wereupregulated in colorectal cancer tissues compared with those incorresponding normal tissues (Fig. 1D). ELISAs demonstratedthat these colorectal cancer cells produced BMP-4, whereas, withthe exception of SUIT-2 pancreatic cancer cells, other cancer cellsexamined did not (Fig. 1E). These findings suggest that colorectalcancer cells produce BMP-4, which may act in an autocrinemanner, and that BMP-4 expression may be related to colorectalcancer progression.

    Next, we sought to clarify the mechanism by which BMP4mRNA was elevated in colorectal cancer. Mutations in the APCgene occur in the early phase of colorectal cancer progression,which in turn increases the stability of b-catenin (15). BecauseBMP4 expression is reported to be regulated by theWnt/b-cateninpathway (16), the involvement ofWnt/b-catenin in the inductionofBMP4 expression in colorectal cancer cells was assessed. b-Cate-ninprotein levelswere elevated in colorectal cancer cells but not innon-colorectal cancer cells, such as pancreatic cancer cells (SUIT-2) and breast cancer cells (MDA-231-D; Fig. 2A). Knockdown ofthe CTNNB1 gene (encoding b-catenin) in colorectal cancer cellsby siRNAs suppressed BMP4 mRNA expression and BMP-4 pro-tein production, as well as expression of a direct downstreamtarget of the Wnt/b-catenin pathway, AXIN2 (Fig. 2B and C).Similar to colorectal cancer cells, introduction of siCTNNB1 toSUIT-2 cells also decreased BMP4 mRNA, indicating that regula-tion of BMP4 by the Wnt/b-catenin pathway was not restricted tocolorectal cancer cells (Fig. 2B). Although colorectal cancer cellswere stimulatedwithWnt-3a, increase ofAXIN2was not observed(Fig. 2D), suggesting that signaling activity of Wnt/b-cateninmight have already been saturated in colorectal cancer cells.Likewise, stimulation of pancreatic cancer cells with Wnt-3aincreased the expression of both AXIN2 and BMP4. Pretreatmentof BxPC-3 cells with cycloheximide did not suppress Wnt-3a–induced BMP4 expression (Supplementary Fig. S1). ChIP–qRT-PCR analysis revealed a binding of TCF4 to the previouslyreported enhancer region of BMP4 gene, which was enhanced byWnt-3a in SUIT-2 cells and attenuated by introduction ofsiCTNNB1 in HT29 cells (Fig. 2E; ref. 16). These findings suggestthat BMP-4 is directly regulated by the Wnt/b-catenin pathway.

    Colorectal cancer cells undergo apoptosis following theinhibition of autocrine BMP-4 signaling

    To examine the role of BMP-4 on colorectal cancer tumorgrowth, shRNA-targeting BMP4 was introduced into HT29 andSW480 cells. As a result, expression of inhibitor of DNA binding 1(ID1), a direct target gene of BMPs, and production of BMP-4protein in culture supernatants were significantly diminished

    BMP-4 as a New Therapeutic Target in Colorectal Cancer

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    Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0112

    http://cancerres.aacrjournals.org/

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    Figure 1.

    BMP-4 is produced in colorectal cancer cells and colorectal cancer tissues. A, Comprehensive gene-expression analysis from NCI-60 cell line panels showing geneexpression profiles of BMP mRNA in leukemia (n ¼ 6), breast cancer (n ¼ 5), central nervous system (CNS) tumor (n ¼ 6), colon cancer (n ¼ 7), renal cancer(n ¼ 8), lung cancer (n ¼ 9), ovarian cancer (n ¼ 7), prostate cancer (n ¼ 2), and melanoma (n ¼ 9) cells. Color indicates distance from log2 6. B, Gene expressionanalysis from the NCBI GEO database (GSE14258). Box plot reveals expression of BMP mRNA in normal colon epithelium (n ¼ 54) and colon cancer (n ¼ 186)tissues. C, Kaplan–Meier plot of disease-free survival of patients with stage II colorectal cancer (CRC; n ¼ 94) stratified by median BMP4 mRNA expression basedon data from NCBI GEO database (GSE14333). D, IHC of colorectal cancer tissues and corresponding normal colon tissues from two colorectal cancerpatients (Patients #1 and #2), stained with hematoxylin and eosin (H&E), anti–BMP-4 antibody, and antiphospho-Smad1/5 antibody (pSmad1/5); scale bars,50 mm. E, Concentrations of BMP-4 proteins in cancer cell culture supernatants (48 hours) determined by ELISA (n¼ 2). Figure data are shown as box whisker plots(B) or as means � SD (E). � , P < 0.05, ���, P < 0.001, as determined by Student t test (B) or by log-rank test (C).

    Yokoyama et al.

    Cancer Res; 77(15) August 1, 2017 Cancer Research4028

    on October 5, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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    http://cancerres.aacrjournals.org/

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    Elevated expression of BMP4 in colorectal cancer cells is due to aberrant activation of the Wnt/b-catenin pathway. A, Top, immunoblotting of cell lysates withindicated antibodies. Bottom, relative expression of b-catenin protein in indicated cells. B, qRT-PCR analysis of CTNNB1, AXIN2, and BMP4 expression incancer cells (n ¼ 2). Indicated cells were transfected with control siRNA (siNTC) or siRNA targeting CTNNB1 (siCTNNB1#1 and #2), cultured for 72 hours, andanalyzed by qRT-PCR.C,Concentrations of BMP-4 proteins in cells inB. Cell culture supernatants (48 hours)were examined by ELISA (n¼ 4).D, qRT-PCR analysis ofAXIN2 and BMP4 expression in indicated cancer cells after Wnt-3a stimulation (200 ng/mL) at the indicated time points (n¼ 2). E, ChIP-qRT-PCR analysis of TCF4-bound DNA using primers designed at the enhancer region of BMP4. SUIT-2 cells and HT29 cells were fixed and harvested 1.5 hours after Wnt-3a (200 ng/mL)stimulation or 48 hours after introduction of siCTNNB1, respectively. Sine oculis binding protein homolog (SOBP)was used as negative control. Figure data are shownas means � SD (B–E).

    BMP-4 as a New Therapeutic Target in Colorectal Cancer

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    (pg/

    mL)

    B

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    4sh

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    BMP-4 protein

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    HT29 DLD-1 SW480

    pSmad1/5

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    0

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    200

    300

    400

    500

    600

    0

    10

    20

    30

    40

    50

    Yokoyama et al.

    Cancer Res; 77(15) August 1, 2017 Cancer Research4030

    on October 5, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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    http://cancerres.aacrjournals.org/

  • (Fig. 3A and B). When these cells were subcutaneously inoculatedinto nudemice, knockdown of BMP-4 inhibited tumor formationof both HT29 and SW480 cells in vivo (Fig. 3C). To investigate therole of autocrine BMP-4onproliferation and survival of colorectalcancer cells, BMP-4 expression was silenced with siRNAs. Knock-down of BMP4 inhibited proliferation of colorectal cancer cellsunder serum-free conditions (Fig. 3D). TUNEL assay revealed thatthe number of TUNEL-positive cells increased following silencingof BMP4 (Fig. 3E). In addition, cleavage of PARP was enhanced inBMP4-silenced cells (Fig. 3F), suggesting that autocrine BMP-4prevents apoptosis of colorectal cancer cells, which in turn pro-motes tumor growth.

    We next examinedwhether LDN-193189, a BMP type I receptorinhibitor, induced apoptosis of colorectal cancer cells. Similar tothe extracellular BMP antagonist protein noggin, LDN-193189attenuated the phosphorylationof Smad1/5 and the expression ofID1 (Fig. 4A and B), demonstrating that LDN-193189 potentlyinhibits endogenous BMP signaling in colorectal cancer cells.LDN-193189 significantly suppressed proliferation of colorectalcancer cells under serum-free conditions (Fig. 4C). Apoptosis wasalso induced by LDN-193189 (Fig. 4D and E). Because LDN-193189 has been reported to exhibit off-target effects on variousprotein kinases, such as AMP-activated protein kinase (AMPK)and the tyrosine receptor kinases for platelet-derived growthfactor (PDGF) andVEGF (17, 18),we investigatedwhether noggincould reproduce the apoptosis-inducing activity of LDN-193189on colorectal cancer cells. As shown in Supplementary Fig. S2Aand S2B, apoptosis of colorectal cancer cells was induced bynoggin under serum-free conditions. Moreover, LDN-193189 didnot inhibit proliferation of BMP-4-negative cancer cells (Supple-mentary Fig. S2C). These results suggest that LDN-193189induces apoptosis of colorectal cancer cells through the inhibitionof BMP signaling.

    LDN-193189 induces apoptosis of colorectal cancer cellsthrough the induction of DUSP5

    To determine the underlying mechanism by which LDN-193189 induces apoptosis of colorectal cancer cells, downstreamtargets of BMP-4 were identified using RNA-seq analysis of HT29andDLD-1 cells. As a result, 62 geneswere commonly upregulatedand 24 were downregulated by LDN-193189 in HT29 cells andDLD-1 cells (Fig. 5A). Gene ontology (GO) analysis indicated thatLDN-193189 diminished MAPK activity in colorectal cancer cells(Fig. 5B), resulting in the elevation of genes encoding dualspecificity phosphatases (DUSP; Fig. 5C). RNA-seq analysis andqRT-PCR analysis revealed that, among the DUSP members incolorectal cancer cells, LDN-193189 had the greatest impact onthe expression of DUSP5 (Fig. 5D and E). Conversely, stimulation

    of cancer cells with BMP-4 attenuated DUSP5 mRNA expression,confirming that DUSP5 is regulated by BMP signaling (Fig. 5F).Because DUSP5 acts as an inducible nuclear MAPK phosphataseand specific dual phosphatase for extracellular signal-regulatedkinase (Erk) MAPK, the effects of LDN-193189 and noggin onMAPK signaling in colorectal cancer cells were examined.Although different effects on the phosphorylation of p38 MAPKand JNK were observed in a cell type-dependent manner, phos-phorylation of Erk MAPK was commonly attenuated in thecolorectal cancer cells examined (Fig. 5G; Supplementary Fig.S3A and S3B). Moreover, although siBMP4#2 exhibited a partialeffect probably because of inefficient knockdown of BMP-4,siBMP4#1 caused a similar result on DUSP5 expression toLDN-193189 (Supplementary Fig. S3C), suggesting that regula-tion of Erk MAPK by BMP signaling is mediated through DUSP5in colorectal cancer cells.

    To determine whether the prosurvival effect of BMP-4 ismediated by DUSP5, DUSP5 was knocked down in colorectalcancer cells with siRNAs (Fig. 6A). Knockdown of DUSP5attenuated LDN-193189-induced apoptosis (Fig. 6B and C).Furthermore, the enhancement of PARP cleavage and the atten-uation of Erk phosphorylation were diminished followingsilencing of DUSP5 in colorectal cancer cells (Fig. 6D). Becauseexpression levels of other DUSP members in colorectal cancercells were upregulated by the transfection of siDUSP5 (data notshown), siDUSP5 appeared to induce some off-target effects onother phosphatases, which might have influence on phosphor-ylation of Erk.

    To clarify the proapoptotic effect of DUSP5, DUSP5 wasintroduced into colorectal cancer cells using a lentiviral vector(Supplementary Fig. S4A). Under serum-free conditions, over-expression of DUSP5 resulted in cell-number reduction andelevated apoptosis of colorectal cancer cells (Supplementary Fig.S4B and S4C). PARP cleavage was also enhanced and Erk phos-phorylation was attenuated by overexpression of DUSP5 (Sup-plementary Fig. S4D).

    Finally, we examined whether attenuation of Erk MAPK resultsin apoptosis of colorectal cancer cells. Treatment ofU0126, aMEKinhibitor, abolished phosphorylation of Erk and enhanced cleav-age of PARP and apoptosis (Supplementary Fig. S5A and S5B).These results suggest that LDN-193189–induced apoptosis ofcolorectal cancer cells is regulated by DUSP5-mediated dephos-phorylation of Erk MAPK.

    LDN-193189 inhibits tumor formation in vivoOn the basis of the above findings, the efficacy of LDN-193189

    as a new therapeutic agent for colorectal cancer was evaluated.Colony formation of colorectal cancer cells in soft agar was

    Figure 3.Knockdown of BMP-4 inhibits tumor formation of colorectal cancer cells in vivo through induction of apoptosis. A, qRT-PCR analysis of ID1 and BMP4 expression incolorectal cancer cells (n¼ 2). Colorectal cancer cells were transducedwith control shRNA (shNTC) and shRNA targeting BMP4 (shBMP4) using lentiviral vector andanalyzed by qRT-PCR. B, Concentrations of BMP-4 proteins in cells in A. Cell culture supernatants (48 hours) were examined by ELISA (n ¼ 4). C, Tumor-formingability of BMP-4-silenced colorectal cancer cells. BALB/c nu/nu female mice received subcutaneous transplants of HT29-shNTC (n ¼ 5) and HT29-shBMP4 (n ¼ 5)cells or SW480-shNTC (n¼ 5) and SW480-shBMP4 (n¼ 5) cells. Left, representative photographs 19 days (HT29) and 15 days (SW480) after injection. Right, tumorvolumes at the indicated time points.D, Proliferation of BMP-4-silenced colorectal cancer cells. Colorectal cancer cells were transfected with control siRNA (siNTC)–or siRNA-targeting BMP4 (siBMP4 #1 and #2). On the following day, cells were deprived of serum and cultured for 3 days. Cell numbers are indicated (n¼ 2 for HT29and DLD-1 cells and n ¼ 4 for SW480 cells). E, TUNEL staining of cells in D. Top, red, TUNEL; blue, SYTOX green. Bottom, the percentage of TUNEL-positive cellsamong SYTOX green-positive cells. Data represent the mean of six microscopic fields. F, Top, immunoblotting of lysates from cells in D with indicated antibodies.Bottom, cleavageof PARPprotein in indicated cells. Data represent a fold increase comparedwith negative control (n¼ 2 for HT29 and n¼ 3 for DLD-1 cells). Data areshown as means � SD (A, B, and D–F) or as means � SEM (C). �, P < 0.05; �� , P < 0.01; ��� , P < 0.001; n.s., nonsignificant, as determined byStudent t test (D–F) or by two-way ANOVA (C).

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  • 0

    5

    10

    15

    20

    25

    30

    pSmad1/5

    Smad1

    NogginLDN

    SW480

    - - +- + -

    ADLD-1

    - - +- + -

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    LDN

    **

    Cel

    l num

    ber (

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    *

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    ID1

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    0.2

    0.4

    0.6

    0.8

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    1.2

    1.4ID1

    (-)

    LDN

    Nog

    gin

    DLD-1

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    0.2

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    gin

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    Apo

    ptot

    ic c

    ells

    (%)

    (-) LDN (-) LDN (-) LDN0

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    0

    10

    20

    30

    40

    50 ****** ***

    HT29 DLD-1 SW480

    SYTOX Green TUNEL

    pSmad1/5

    α-Tubulin

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    3

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    0

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    avag

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    Figure 4.

    LDN-193189 induces apoptosis of colorectal cancer cells. A, Immunoblot analysis of colorectal cancer cells treated with BMP inhibitors. Colorectal cancer cells werecultured with 0.2–0.3 mmol/L LDN-193189 (LDN) or 50 ng/mL noggin for 2 hours. Immunoblotting of cell lysates was conducted with indicated antibodies. B,qRT-PCR analysis of ID1 expression in colorectal cancer cells in A (n ¼ 2). C, Effects of LDN-193189 on proliferation of colorectal cancer cells. Cells were seeded in6-well plates. On the following day, cells were deprived of serum and cultured with DMSO or 0.2–0.3 mmol/L LDN-193189 for 3 days. Cell numbers are indicated(n ¼ 2). D, TUNEL staining of cells in C. Left, red, TUNEL; blue, SYTOX green. Right, the percentage of TUNEL-positive cells among SYTOX green-positivecells. Data represent the mean of six microscopic fields. E, Left, immunoblotting of lysates from cells in C with indicated antibodies. Right, cleavage of PARPprotein in indicated cells. Data represent a fold increase compared with untreated control (n ¼ 2). Data are shown as means � SD (C–E). � , P < 0.05;�� , P < 0.01; ��� , P < 0.001, as determined by Student t test (C–E).

    Cancer Res; 77(15) August 1, 2017 Cancer Research4032

    Yokoyama et al.

    on October 5, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

    Published OnlineFirst June 13, 2017; DOI: 10.1158/0008-5472.CAN-17-0112

    http://cancerres.aacrjournals.org/

  • PGene ontology1.24E-11Ion transport

    Positive regulation of transcription from RNA polymerase II promoter 1.52E-102.35E-08Amino acid transport

    Negative regulation of transcription from RNA polymerase II promoter 5.59E-089.37E-07Negative regulation of transcription, DNA-dependent3.38E-06Chromatin silencing4.4E-06Amino acid transmembrane transport4.61E-06Response to cAMP2.92E-05Inflammatory response5.15E-05Transcription, DNA-dependent6.27E-05Skeletal muscle cell differentiation

    transcription factor activityPositive regulation of NF-kappaB 0.0001640.000196Cellular response to hormone stimulus0.000258Negative regulation of fat cell differentiation0.000303Mammary gland branching involved in thelarche0.000363Cellular response to insulin stimulus0.000405Inactivation of MAPK activity0.000405Response to progesterone stimulus0.000457Liver development0.000542Placenta blood vessel development

    PGene ontology9.44E-05Digestion0.000132Endoderm formation

    Carbohydrate metabolic process 0.0001560.000167Transmembrane transport0.000373Heterotypic cell-cell adhesion0.000373Notch signaling involved in heart development0.000498Skeletal muscle cell differentiation

    Negative regulation of cell proliferation 0.0009150.001274Inactivation of MAPK activity

    Positive regulation of leukocyte chemotaxis 0.001462Proteasomal protein catabolic process 0.001462

    Negative regulation of protein autophosphorylation 0.001462G-Protein coupled receptor signaling pathway 0.001979

    0.002047Gluconeogenesis0.002047Response to calcium ion

    Positive regulation of cell migration 0.002959Glycosaminoglycan metabolic process 0.00328

    0.003457Lung-associated mesenchyme development0.003457Cellular response to zinc ion0.003457Glycerol metabolic process

    DLD-1

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    Genes upregulated by LDN-193189

    687255 24

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    Genes downregulated by LDN-193189

    A

    D

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    DUSP9

    DUSP10

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    DLD-1HT29

    0

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    30

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    10

    20

    30

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    50

    60

    70

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    Figure 5.

    LDN-193189 inactivates MAPK through induction of DUSP5 in colorectal cancer cells. A, Identification of genes regulated by LDN-193189 in colorectal cancer cellsusing RNA-seq analysis. Cells were seeded in 6-well plates. On the following day, cells were deprived of serum and cultured with DMSO or 0.2–0.3 mmol/LLDN-193189 for 3 days. All genes whose RPKM values were >3 were included in analysis. Left Venn diagrams, number of genes upregulated >1.5-fold by LDN-193189.Right Venn diagrams, number of genes downregulated >1.5-fold by LDN-193189. B, Gene ontology analysis of genes upregulated by LDN-193189 in indicatedcells. C, Genes upregulated by LDN-193189 (>1.5-fold) that belong to the gene ontology "inactivation of MAPK activity." Red genes belong to DUSP family.D, Expression of DUSP family genes in colorectal cancer cells. Data were extracted from RNA-seq analysis. E, qRT-PCR analysis of DUSP5 expression in colorectalcancer cells (n ¼ 2). Cells were seeded in 6-well plates. On the following day, cells were deprived of serum and cultured with DMSO or 0.2–0.3 mmol/LLDN-193189 (LDN) for 3 days. F, qRT-PCR analysis of DUSP5 expression in cancer cells (n ¼ 2). Cells were seeded in 6-well plates. On the following day, cells weredeprived of serum and cultured with BMP-4 (30 ng/mL) for 3 days (MDA-231-D cells) or 24 hours (A549 cells). G, Left, immunoblotting of lysates from cellsin Ewith indicated antibodies. Right, expression of pErk MAPK protein in indicated cells. Data represent as a fold decrease compared with untreated control (n¼ 2).Data are shown as means � SD (E–G). � , P < 0.05; �� , P < 0.01; ��� , P < 0.001, as determined by Student t test (G).

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  • pErk

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    siN

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    siN

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    siN

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    USP

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    ptot

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    Figure 6.

    Silencing of DUSP5 in colorectal cancer cells abolishes proapoptotic effect of LDN-193189.A, qRT-PCR analysis ofDUSP5 expression in colorectal cancer cells (n¼ 2).Colorectal cancer cells were transfected with control siRNA (siNTC)– or siRNA-targeting DUSP5 (siDUSP5 #1 and #2). On the following day, cells were deprived ofserum and cultured with DMSO or 0.2–0.3 mmol/L LDN-193189 for 3 days, followed by qRT-PCR analysis. B, Number of cells in A (n ¼ 2). C, TUNEL staining ofcells in A. Left, red, TUNEL; blue, SYTOX green. Right, the percentage of TUNEL-positive cells among SYTOX green-positive cells. Data represent the meanof sixmicroscopic fields.D, Top, immunoblotting of lysates from cells inAwith indicated antibodies. Bottom, cleavage of PARP and expression of pErk MAPK proteinin indicated cells. Data represent a fold change compared with negative control. Data are shown as means � SD (A–C). �, P < 0.05; �� , P < 0.01; ��� , P < 0.001; n.s.,nonsignificant, as determined by Student t test (B and C).

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    on October 5, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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    http://cancerres.aacrjournals.org/

  • D pErkpSmad1/5

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    Type Ireceptors

    PP

    DUSP5

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    Survival

    ErkP

    APCMutation

    BMP-4

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    BMPR-II

    CRC Cells

    Type Ireceptors

    PP

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    Erk

    Survival

    ErkP

    APCMutation

    LDN-193189

    BMP-4

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    Figure 7.

    (Continued on the following page.)

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  • significantly suppressed by LDN-193189 (Fig. 7A). To confirm thebioavailability of administered LDN-193189, an ex vivo bioassaywas performed using DLD-1 cells. qRT-PCR analysis revealed thatserum from LDN-193189–treated mice successfully suppressedID1 expression (Fig. 7B). Body-weight loss was not observed inLDN-193189–treated mice (data not shown), suggesting thatLDN-193189 acted as a potent BMP inhibitor in vivo withoutsevere toxicity. Finally, the effect of LDN-193189 on the tumorformation of colorectal cancer cells was investigated in vivo.Notably, LDN-193189 inhibited tumorigenesis in mice bearingcolorectal cancer cells (Fig. 7C). Furthermore, LDN-193189 atten-uated the phosphorylation of Smad1/5 and ErkMAPK in vivo (Fig.7D). These results indicate that the inhibition of endogenous BMPsignaling by LDN-193189 may represent a potential strategy fortreatment of colorectal cancer.

    DiscussionIn this study, we demonstrated the protumorigenic role of

    BMP-4 in colorectal cancer (Fig. 7E). Aberrant activation of theWnt/b-catenin pathway increases BMP4 expression in colorectalcancer cells. Autocrine BMP-4 signaling protects cells from apo-ptosis through suppression of DUSP5-mediated dephosphoryla-tion of Erk MAPK. When autocrine BMP-4 signaling is inhibitedby LDN-193189, ErkMAPK is dephosphorylated via induction ofDUSP5, resulting in colorectal cancer cell apoptosis. These resultssuggest that autocrine BMP-4 represents a potential target forcolorectal cancer treatment.

    We demonstrated that endogenous BMP signaling was activat-ed by autocrine BMP-4 in colorectal cancer cells and tissues.Elevated BMP4 expression is unique to colorectal cancer, as otherBMPs are not elevated in colorectal cancer cells, andBMP4 appearsto be elevated only in colorectal cancer. Elevated expression ofBMP ligands in colorectal cancer was shown previously and wasreported to correlate with poor prognosis (4, 5, 19); however,activation of endogenous signaling by autocrine BMPs in colo-rectal cancer remains controversial. Kodach and colleagues (20)reported that Smad1/5 phosphorylation was not observed inmost colorectal cancer cases due to mutations in Smad4 orBMPR-II. In contrast, Beck and colleagues (21) showed thatSmad1 phosphorylation was detected in colorectal cancer tissuesand observed in colorectal cancer cells with BMPR2mutations. Inthis study, BMP-4 expression and Smad1/5 phosphorylationwereobserved in colorectal cancer tissues and correlated with eachother. Furthermore, Smad1/5 phosphorylation was detected inDLD-1 cells, which are reported to carry mutations in BMPR-II(21), suggesting that BMP signaling is transduced in these cells

    through ActR-II and ActR-IIB. On the basis of these findings, weconcluded that BMP signaling was activated in colorectal cancercells and tissues.

    The role of BMP signaling during colorectal cancer progressionhas not been fully elucidated. Some studies have revealed thatBMPs promote invasiveness and tumor formation of colorectalcancer cells (4, 5), whereas other reports have demonstratedgrowth-suppressive roles of BMPs in colorectal cancer (22, 23).Smad4 and p53 are reported to be key molecules affecting thefunctional roles of BMPs in colorectal cancer progression. Voor-neveld and colleagues reported that loss of Smad4 switchesBMPs from antitumorigenic to protumorigenic, whereas p53mutations suppress the enhancement of chemosensitivity by BMPsignaling in colorectal cancer cells with wild-type Smad4 (24, 25).Because p53 was mutated in all colorectal cancer cells used in thecurrent study (26), the association between p53 status and theprotumorigenic role of BMP signaling in colorectal cancer couldnot be assessed in our study. However, we demonstrated thatinhibition of endogenous BMP signaling induced apoptosis ofnot only Smad4-null cells (HT29 and SW480) but also cells withwild-type Smad4 (DLD-1). Although factors other than Smad4and p53 may modulate the role of BMP signaling in colorectalcancer progression, our findings suggest that autocrine BMP-4exerts a prosurvival effect on colorectal cancer cells regardless ofSmad4 status.

    Erk enhances the proliferation, survival, and metastasis ofcancer cells and acts as an oncogenic signaling pathway (27).Although BMPs have been known to phosphorylate Erk MAPKand enhance its activity, the underlying molecular mechanism isnot fully understood (28–30). Using RNA-seq, we identifiedDUSP5 as a BMP target gene and determined that DUSP5 wasimportant for the Erk-mediated prosurvival effect of BMP-4 oncolorectal cancer cells. DUSP5 is a member of the four induciblenuclear MAPK phosphatases and dephosphorylates Erk1/2 (31,32). DUSP5 has been implicated as a tumor suppressor in varioustypes of cancer, including skin, gastric, prostate, colon, and lungcancer (33–36). In this study, we also showed that enhancementof Erk MAPK phosphorylation by BMP-4 is mediated by a reduc-tion in DUSP5. Although we did not examine whether theregulation of DUSP family genes by BMPs occurs via a Smad-dependent pathway, our findings may provide insight into themechanism of regulation of MAPK signaling by BMP signaling.

    Because BMPs provide a potential target for cancer treatment,various BMP inhibitors have been developed. Calpe and col-leagues (37) showed that neutralizing antibodies for BMP-4increase the chemosensitivity of HT29 cells. Dorsomorphin wasidentified as a potent BMP inhibitor, and its analogue, DMH-1,

    (Continued.) LDN-193189 attenuates tumor formation of colorectal cancer cells in vivo. A, Colony formation assay of colorectal cancer cells with or without LDN-193189. Top, representative photographs. Bottom, box plot reveals tumor areas of eightmicroscopic fields (n¼ 2).B, Ex vivo bioassay to confirm the bioavailability ofadministered LDN-193189. Separatedmouse serumwas diluted 2-fold with RPMI containing 10% FBS. DLD-1 cells were treatedwith 200 mL of serum in 12-well plates.After 2 hours, ID1 expression was examined by qRT-PCR analysis (n ¼ 2). C, Tumor formation of colorectal cancer cells with or without LDN-193189. BALB/cnu/nu female mice received subcutaneous transplants of SW480 (n ¼ 7) cells. Vehicle (2.5% DMSO) or 6 mg/kg LDN-193189 in 2.5% DMSO was injectedintraperitoneally twice a day, starting 2 days before inoculation of SW480 cells. Top, tumor volumes at the indicated time points. Bottom, representativephotographs and tumor weights 21 days after injection. D, Tumor tissues in C stained with hematoxylin and eosin (H&E), antiphospho-Erk antibody (pErk), andantiphospho-Smad1/5 antibody (pSmad1/5). Representative photographs are shown. E, Scheme of autocrine BMP-4 signaling in colorectal cancer cells with orwithout BMP inhibitors. Left, in colorectal cancer cells, aberrant activation of theWnt/b-catenin pathway induces expression of BMP4mRNA, which in turn activatesendogenous BMP signaling. This endogenous BMP signaling promotes phosphorylation of Erk through downregulation of DUSP5, which results in survival ofcolorectal cancer cells. Right, BMP inhibitors, including LDN-193189, inhibit endogenous BMP signaling. This results in dephosphorylation of Erk through elevation ofDUSP5, which in turn induces apoptosis of colorectal cancer cells. Data are shown as means � SD (B) or as means � SEM (C, top). � , P < 0.05; ��, P < 0.01;��� , P < 0.001, as determined by Student t test (A and C, bottom) or two-way ANOVA (C, top).

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  • disturbs lung cancer growth and breast cancer metastasis(38, 39). LDN-193189 was reported to inhibit growth of breastand prostate cancers in vivo and prolong survival of micebearing ovarian cancer cells (40–42). In colorectal cancer,Voorneveld and colleagues demonstrated that LDN-193189reduced the viability and enhanced the chemosensitivity ofSmad4-silenced colorectal cancer cells in vitro (25). On thebasis of these reports, we attempted to determine whether LDN-193189 inhibits colorectal cancer tumor formation in vivo.Tumor formation in mice bearing colorectal cancer cells wassignificantly diminished by LDN-193189, suggesting that thistherapeutic strategy may potentially be of use in colorectalcancer treatment. However, the risk of intestinal carcinogenesismust be noted when LDN-193189 is used in vivo. Becauseinhibition of BMP signaling in mice by transgenic expressionof noggin under control of the villin promoter or by condi-tional knockout of Bmpr1a leads to intestinal polyposis (43,44), inhibition of BMP signaling in colon epithelial cells mayincrease the risk of intestinal carcinogenesis. Whissell andcolleagues (16) demonstrated that orally administered LDN-193189 increased intestinal tumor formation in conditionalApc knockout mice; however, we did not detect intestinal tumorformation in LDN-193189–treated mice (data not shown).One possible explanation for this discrepancy is a differencein the route of LDN-193189 administration. Intermittent dos-ing of inhibitors may be required to avoid the risk of tumor-igenesis. Another possible explanation is the use of differentstrains of mice in the experiments. The balance between BMPsignaling and Wnt/b-catenin pathways is important for main-tenance of homeostasis of intestinal epithelial regeneration(44). Because Apc knockout mice exhibit activation of theWnt/b-catenin pathway in the intestine, Apc knockout miceare more sensitive to BMP inhibitors than wild-type mice.

    In this study, we also demonstrated that LDN-193189 atten-uatedphosphorylation of not only Smad1/5but also ErkMAPK incolorectal cancer tissues in mice. Because the Ras–Raf–MEK–Erksignaling cascade is activated by mutations in the signalingcomponents of cancer cells, this signaling pathway is consideredan important target for cancer treatment (27). Indeed, Ras muta-tions are detected in 50% of colorectal cancer cases (45). Anti-EGFR antibodies targeting this signaling, such as cetuximab andpanitumumab, are effective against certain types of colorectalcancer; however, their usefulness is limited to colorectal cancercases without KRASmutations (1). We showed that LDN-193189

    was also effective against SW480 cells harboring KRASmutationsin vivo (46). Together, these results suggest that BMP inhibitors,especially small-molecule kinase inhibitors such as LDN-193189,may represent attractive new therapeutic strategies for colorectalcancer treatment.

    Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

    Authors' ContributionsConception and design: Y. Yokoyama, K. Miyazono, S. EhataDevelopment of methodology: Y. Yokoyama, S. EhataAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Yokoyama, Y. Tamura, S. EhataAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Yokoyama, Y. Tamura, S. EhataWriting, review, and/or revision of the manuscript: Y. Yokoyama,Y. Hashizume, K. Miyazono, S. EhataAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Yokoyama, T. Watanabe, S. EhataStudy supervision: T. Watanabe, K. Miyazono, S. EhataOther [preparation of chemical tool and evaluation of the properties;designed the administration method (solvent, administration route, anddosage) based on the above data]: Y. Hashizume

    AcknowledgmentsWe thank Yasuyuki Morishita (The University of Tokyo) for technical

    assistance and Hiroyuki Miyoshi (Keio University) for providing lentiviralvectors.

    Grant supportThis work was supported by a KAKENHI Grant-in-Aid for Scientific Research

    on Innovative Areas, Integrative Research on Cancer Microenvironment Net-work (22112002) from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT; to K. Miyazono); a KAKENHI Grant-in-Aid forScientific Research (C) (15K08393) from the Japan Society for the Promotion ofScience (JSPS; to S. Ehata); a grant for Leading Advanced Projects for MedicalInnovation (LEAP; 16am0001003h0003) from the Japan Agency for MedicalResearch and Development (AMED; to K. Miyazono); and Specific ResearchGrants fromTheCell Science Research Foundation (to S. Ehata), and the YasudaMedical Foundation (to K. Miyazono).

    The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

    Received January 12, 2017; revised April 5, 2017; accepted June 5, 2017;published OnlineFirst June 13, 2017.

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  • 2017;77:4026-4038. Published OnlineFirst June 13, 2017.Cancer Res Yuichiro Yokoyama, Toshiaki Watanabe, Yusuke Tamura, et al. CancerAutocrine BMP-4 Signaling Is a Therapeutic Target in Colorectal

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