Myeloid Differentiation Factor 88 Signaling in Bone Marrow …cancerpreventionresearch.aacrjournals.org/content/... · pathway and Wnt signaling. Notably, ... induces chronic gastritis,

Research Article

Myeloid Differentiation Factor 88 Signaling inBone Marrow–Derived Cells Promotes GastricTumorigenesis by Generation of InflammatoryMicroenvironmentYusuke Maeda1,2,3, Kanae Echizen1,4, Hiroko Oshima1, Liang Yu5, Natthiya Sakulsak6,Osamu Hirose7, Yoichi Yamada7, Tadatsugu Taniguchi8, Brendan J. Jenkins5,Hideyuki Saya2, and Masanobu Oshima1,4

Abstract

It has been established that COX-2 and downstream signal-ing by prostaglandin E2 (PGE2) play a key role in tumorigenesisthrough generation of inflammatory microenvironment. Toll-like receptor (TLR) signaling through myeloid differentiationfactor 88 (MyD88) also regulates inflammatory responses intumors. However, the relationship between these distinct path-ways in tumorigenesis is not yet fully understood. We hereininvestigated the role of MyD88 in gastric tumorigenesis usingGan mice, which develop inflammation-associated gastrictumors due to the simultaneous activation of the COX-2/PGE2pathway and Wnt signaling. Notably, the disruption of Myd88in Gan mice resulted in the significant suppression of gastrictumorigenesis with the inhibition of inflammatory responses,even though COX-2/PGE2 pathway is constitutively activated.Moreover, Myd88 disruption in bone marrow–derived cells

(BMDCs) in Gan mice also suppressed inflammation andtumorigenesis, indicating that MyD88 signaling in BMDCsregulates the inflammatory microenvironment. We also foundthat expression of Tlr2 and its coreceptor Cd14 was inducedin tumor epithelial cells in Gan mice, which was suppressedby the disruption of Myd88. It has already been shown thatTLR2/CD14 signaling is important for stemness of intestinalepithelial cells. These results indicate that MyD88 in BMDCs,together with COX-2/PGE2 pathway, plays an essential role inthe generation of the inflammatory microenvironment, whichmay promote tumorigenesis through induction of TLR2/CD14pathway in tumor epithelial cells. These results suggest thatinhibition of TLR/MyD88 signaling together with COX-2/PGE2pathway will be an effective preventive strategy for gastric cancer.Cancer Prev Res; 9(3); 253–63. �2016 AACR.

IntroductionGastric cancer is the second leading cause of death from

malignancy worldwide (1).Helicobacter pylori (H. pylori) infectioninduces chronic gastritis, which is closely associated with gastriccancer (2, 3). It has been shown that COX-2 expression is induced

by H. pylori infection in the stomach (4), and that the activationof downstream signaling by prostaglandin E2 (PGE2) plays a keyrole in tumorigenesis in a variety of organs (5). We previouslyshowed that induction of COX-2/PGE2 pathway in the gastricmucosa ofK19-C2mEmice by expression of Ptgs2 and Ptges causesinflammatory responses in the glandular stomach (6). Moreover,Wnt signaling activation in the gastric mucosa of K19-Wnt1miceby expression of Wnt1 results in the development of limiteddysplastic lesions; however, the simultaneous activation of Wntsignaling and COX-2/PGE2 pathway in Gan mice leads to devel-opment of inflammation-associated gastric tumors (7, 8). Theseresults indicate the role of COX-2/PGE2 pathway-induced inflam-mation in gastric tumorigenesis. In the gastric tumor stroma,proinflammatory cytokines including TNFa, IL1b, and IL11 areexpressed and promote tumorigenesis through enhancement ofundifferentiated status, recruiting of myeloid-derived suppressorcells and increasing tumor cell survival (9–11).

On the other hand, it has been shown that innate immuneresponses through Toll-like receptors (TLRs) promote tumordevelopment through the regulation of inflammatory andimmune responses in tumor tissues (12, 13). Mouse geneticsstudies have indicated that signaling through myeloid differen-tiation factor 88 (MyD88), which is an effector molecule of theTLRs, is required for the development of sporadic intestinaltumors (14–16), colitis-associated colon cancer (17, 18), gastriccancer (19), liver cancer (20), skin tumors, and sarcomas (21).

1Division of Genetics, Cancer Research Institute, Kanazawa University,Kanazawa, Japan. 2Division of Gene Regulation, Institute forAdvancedMedical Research, Keio University,Tokyo, Japan. 3ResearchFellow of Japan Society for the Promotion of Science (JSPS), Tokyo,Japan. 4AMED-CREST, AMED, Japan Agency for Medical Researchand Development, Tokyo, Japan. 5Centre for Innate Immunity andInfectious Diseases, Hudson Institute of Medical Research, MonashUniversity, Clayton, Australia. 6Department of Anatomy, Faculty ofMedical Science, Naresuan University, Phitsanulok, Thailand. 7Facultyof Electrical and Computer Engineering, Institute of Science andEngineering, Kanazawa University, Kanazawa, Japan. 8Department ofMolecular Immunology, Institute of Industrial Science, The Universityof Tokyo, Tokyo, Japan.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Masanobu Oshima, Division of Genetics, CancerResearch Institute, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan.Phone: 81-76-264-6760; Fax: 81-76-234-4519; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-15-0315

�2016 American Association for Cancer Research.

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MyD88 has also been shown to promote tumor metastasis (22).Moreover, MyD88 signaling induces the expression of COX-2 andinflammatory cytokines in tumor tissues, which contribute to theinflammatory microenvironment generation (14, 17, 18, 20).However, relationship between the COX-2/PGE2 pathway andTLR/MyD88 signaling in the inductionof inflammatory responsesand the promotion of tumorigenesis is not yet fully understood.

Here, we examined the role of MyD88 in gastric tumorigenesisusing Gan mice, in which COX-2/PGE2 pathway is constitutivelyactivated. Importantly, we show that MyD88 signaling in BMDCsis required for the generation of the inflammatory microenviron-ment in tumors even though the COX-2/PGE2 pathway is acti-vated. Moreover, inflammatory responses induce the expressionof TLR2 and CD14 in tumor epithelial cells, which have beenshown to play tumor-promoting roles through enhancement ofstem cell properties (16, 23, 24). These results indicate that boththe MyD88 signaling and the COX-2/PGE2 pathway are requiredfor generation of the inflammatory microenvironment, whichmay promote tumorigenesis through the induction of TLR2/CD14 signaling in tumor cells. These results suggest that targetingboth TLR/MyD88 signaling and the COX-2/PGE2 pathwaywill bean effective preventive strategy for gastric cancer.

Materials and MethodsAnimal model experiments

The construction ofGanmice, the gastric tumormodel that wasused in the current study, is described in Supplementary Infor-mation. Myd88-mutant mice were purchased (Oriental Bio-Service), and Irf5-mutant mice were constructed as describedpreviously (25). For the gastric tumor phenotype analyses, eachgenotype mice were euthanized and examined at 40 to 50 weeksof age. For the preneoplastic lesion analyses, K19-Wnt1mice wereexamined at 45 to 50 weeks of age. To induce gastric mucosalregeneration, tamoxifen (150 mg/kg) was administered to 10-week-old wild-type mice by oral gavage for 5 continuous days.This resulted in a loss of parietal cells, leading to mucosalregeneration (26). All of the mice used in this study were bornand raised in the same room of the Kanazawa University SPFfacility (Kanazawa, Japan). The protocols of all animal experi-ments were approved by the Committee on Animal Experimen-tation of Kanazawa University (Kanazawa, Japan).

The measurement of tumor volume and scoring ofpreneoplastic lesions

Ganmouse tumors are not polypotic, thus the numbers cannotbe counted. Thus, the tumor heightwasmeasuredusing histologicsections, and the relative tumor height was compared with thecontrol. The number of preneoplastic lesions of K19-Wnt1mousestomach was counted using six independent histologic sectionsfromeachmouse, and themeannumber of lesions per sectionwascalculated.

Histology and IHCTissues were fixed in 4% paraformaldehyde, paraffin-embed-

ded, and sectioned at 4-mm thickness. The sections were stainedwith haematoxylin and eosin (H&E) or processed for IHC. Theantibodies and visualization methods are described in Supple-mentary Information. The mean index for Ki-67 or F4/80 wascalculated by counting the labeled cells per microscopic field at200� in five fields. The immunostaining-positive area in the field

was measured using ImageJ 1.48 software program (NIH,Bethesda, MD). Apoptotic cells were detected using an ApopTagPeroxidase In Situ Apoptosis Detection Kit (Millipore).

PCR array analysesTotal RNAwas extracted from themouse gastric tumors and the

normal wild-typemouse stomach (n¼ 3 for each genotype) usingISOGEN (Nippon Gene), and purified using an RNeasy Mini Kit(Qiagen). Inflammation-related genes were analyzed using theRT2 Profiler PCR Array Mouse Innate & Adaptive ImmuneResponses (Qiagen). Thedatawere analyzedusing theRT2ProfilerPCR Array Data Analysis version 3.5 software (Qiagen) and werecompared with the wild-type mice.

Western blotting analysesTissues were homogenized in lysis buffer and the supernatant

protein sample was separated in SDS-polyacrylamide gel. Theantibodies are described in Supplementary Information. The ECLDetection System (GE Healthcare) was used to detect the signals.

Bone marrow transplantation and X-ray CTBonemarrow cells were prepared from the femurs and tibias of

donormice. Recipientmicewere irradiated (9Gy), followed by anintravenous injection of 2� 106 bone marrow cells. The x-ray CTimages of the gastric tumors were examined using a LaTheta LCT-100 instrument (Aloka) at every 4weeks until 20weeks after bonemarrow transplantation. The tumor areas of the slice images weremeasured using the ImageJ 1.48 software program (NIH) asdescribed previously (9).

Real-time RT-PCRTotal RNAs were extracted from cells or tumor tissues using

ISOGEN (Nippon Gene), reverse-transcribed using the Prime-Script RT reagent kit (Takara), and PCR-amplified by a StratageneMx3000P instrument (Agilent Technologies) using SYBR PremixExTaqII (Takara). The primers used for the real-time RT-PCRexcept for Cd14 were purchased from Takara, and the primer IDsand sequences for Cd14 are shown in Supplementary Table S1.

Cell culture experimentsThe human gastric cancer cell lines, AGS (ATCC), MKN74

(RIKEN BioResource Center, Japan), SNU484 [Korean Cell LineBank (KCLB)], and the mouse macrophage cell line, RAW264(RIKEN BioResource Center, Japan), were cultured in RPMI1640medium supplemented with 10% FBS. Cell lines were authenti-cated by isoenzyme analysis or short tandem repeat by the ATCC,RIKEN, and KCLB. All cell lines were initially expanded andcryopreserved within 1 month of receipt. Cells were used for 3months after thawing frozen vials. Construction of knockdowncell lines and the preparation of the conditioned medium (CM)are described in Supplementary Information.

Next-generation sequencing analysesTotal RNAwas extracted from themouse gastric tumors and the

normal wild-typemouse stomach (n¼ 3 for each genotype) usingISOGEN (Nippon Gene) and purified using an RNeasy Mini Kit(Qiagen). A sequencing analysis and upstream pathway analysiswere performed according to the methods described in Supple-mentary Information. The sequence results were deposited in theGene Expression Omnibus, as accession GSE70135.

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Statistical analysisThe data were analyzed using the unpaired t test and are

presented as the mean � SE or SD. A value of P < 0.05 wasconsidered to be statistically significant.

ResultsThe suppression of gastric tumor developmentin Gan Myd88�/� mice

Innate immune signaling throughTLR/MyD88has been shownto regulate immune responses in tumors (12, 13). IFN regulatoryfactor 5 (IRF5) has also been shown to play a role in the TLR/MyD88 pathway through the induction of proinflammatorycytokines, including TNFa (25). Because TNFa plays a tumor-promoting role in Gan mice (9), we examined the roles of bothMyD88 and IRF5 in the current study. The expression of bothMYD88 and IRF5was significantly increased in human intestinal-type gastric cancer, and significant upregulation ofMyd88was alsofound in Ganmouse gastric tumors (Supplementary Fig. S1). Wethus crossedGanmicewithMyd88 and Irf5 gene knockoutmice togenerate GanMyd88�/� and Gan Irf5�/�mice, in whichMyd88 orIrf5 was disrupted in both epithelial and stromal cells, respec-tively. Although the control Gan mice developed large gastrictumors by 40 to 50 weeks of age, gastric tumorigenesis wassignificantly suppressed in the Gan Myd88�/� mice, but not inthe Gan Irf5�/� mice (Fig. 1A). The mean tumor sizes in the GanMyd88�/�micewere 49%of the controlGanmice (Fig. 1B). Theseresults indicate that MyD88, but not IRF5, plays a role in gastrictumor development.

Apoptotic cells were found on the tumor surface in both thecontrol Gan and the Gan Irf5�/� mice, and similar apoptotic celllocalization was found in theGanMyd88�/�mice, indicating thatMyD88 pathway does not affect apoptosis (Fig. 1C). In contrast,Ki-67–labeled cells were found throughout the tumor tissues ofGan andGan Irf5�/�mice,whereas theyweremostly limited to theproliferating zone at the gland neck in the Gan Myd88�/� mice.Consistently, the Ki-67–labeling index was reduced, to a signif-icant extent, in the Gan Myd88�/� mouse tumors, suggesting thatMyD88 signaling promotes tumor cell proliferation (Fig. 1D).

The suppression of the inflammatory responses inGan Myd88�/� tumors

In Gan mouse gastric tumors, macrophages infiltrated thetumor stroma and generated an inflammatorymicroenvironment(Fig. 2A). In the Gan Myd88�/� mouse tumors, the number ofmacrophages was significantly decreased in comparison to Ganand Gan Irf5�/� mouse tumors (Fig. 2A and B).

We thus performed PCR array analyses to examine the expres-sion of inflammation-related genes in the respective genotypemouse tumors. In the control Gan mouse tumors, expressionlevels of proinflammatory cytokines Il17a, Tnf, Il1a, Il1b, Il6, andIl23a, chemokine receptors Ccr4 and Ccr6, and TLRs, Tlr1, Tlr2,and Tlr5 were significantly increased (Fig. 2C and D). Althoughthe upregulation of Il1b, Il6,Ccr4, andCcr6was suppressed inGanIrf5�/� mouse tumors, most other inflammation-related geneswere upregulated, suggesting that IRF5 signaling may be lessinvolved in the inflammatory responses in gastric tumors. Impor-tantly, in the Gan Myd88�/� mouse tumors, induction of inflam-mation-related genes was significantly suppressed, indicating thatMyD88 signaling is essential for inflammatorymicroenvironmentgeneration (Fig. 2C and D).

We also found substantially decreased levels of phosphorylatedIkB and NF-kB in Gan Myd88�/� mouse tumors (Fig. 2E). More-over,we confirmedby IHC thatNF-kB is activated inmacrophagesof Gan mouse tumors, which was suppressed in Gan Myd88�/�

mice (Fig. 2F). InGanmouse tumors, the COX-2/PGE2 pathway isconstitutively activated by transgenic expression of Ptgs2 and Ptgesencoding COX-2 and mPGES-1, respectively; we confirmedthe expression of COX-2 and mPGES-1 in Gan Myd88�/� mousetumors. In two Gan Myd88�/� mice, mPGES-1 levels weredecreased in comparison to control, possibly due to the decreasein endogenous mPGES-1 levels by suppression of inflammation(Fig. 2G). We previously showed that Il1r1 disruption did notaffect the inflammation phenotype in K19-C2mEmice, indicatingthat the IL1R/MyD88 pathway is not required for COX-2/PGE2pathway-induced inflammation (27). Accordingly, these resultsindicate that TLR/MyD88 signaling is required for the generationof the COX-2/PGE2 pathway-induced inflammatory microenvi-ronment through the activation of NF-kB. Interestingly, the levelof active b-catenin was also decreased by Myd88 disruption (Fig.2E), suggesting that MyD88 has a role in activation of Wntsignaling in tumor cells (see below).

We next examined the expression of MyD88-dependent genesin another inflammation-associated gastric tumor model,gp130F/Fmice, in which hyperactivation of Stat3 promotes gastrictumorigenesis via the TLR2/MyD88 cascade (19, 28, 29). Theexpression levels of Tnf, Cd14, Casp1, Il1a, and Tlr2 were signif-icantly increased in gp130F/F tumors; however, this upregulationwas not suppressed in the gp130F/F Myd88�/� mouse tumors, inwhich Myd88 was disrupted in both epithelial and stromalcells (Supplementary Fig. S2). It is therefore possible that thehyperactivation of Stat3 (in gp130F/F mice) can mimic the role ofCOX-2/PGE2 and MyD88 (in Gan mice) in the generation of theinflammatory microenvironment; however, this possibilityremains to be investigated.

The suppression of gastric tumorigenesis by Myd88 disruptionin BMDCs

To examine the role of MyD88 in BMDCs in gastric tumor-igenesis, we next performed bone marrow transplantation fromMyd88�/�, Irf5�/�, or GFP transgenic mice into Gan mice togenerate the respective bone marrow chimeric mice. In thesemodels, Myd88 or Irf5 was disrupted in the BMDCs but not inthe epithelial cells of the tumor tissues. Chronologic X-ray CTanalyses showed a significant increase in the gastric tumorvolume of the bone marrow–transplanted (BMT)-Gan micethat received bone marrow from GFP or Irf5�/� mice duringthe 20-week observation period (Fig. 3A and B). In contrast,gastric tumor growth was significantly suppressed in the BMT-Gan mice that received Myd88�/� mouse bone marrow. Mac-rophage infiltration into the tumor tissues was significantlydecreased by the disruption of Myd88 in BMDCs (Fig. 3C andD). We further examined the gene expression in gastric tumorsof BMT-Gan mice by next-generation sequencing. We foundthat the levels of inflammatory cytokines and chemokines weredecreased in the BMT-Gan from Myd88�/� mice in comparisonwith the control Gan mice (Fig. 3E). Consistently, NF-kBactivation was suppressed in macrophages by the disruptionof Myd88 in BMDCs (Fig. 2F). Taken together, these resultsindicate that MyD88 signaling in BMDCs is important for theinflammatory microenvironment generation, which may play atumor-promoting role.

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The induction of TLR2 and CD14 expression in tumor cells byactivated macrophages

Among the MyD88-dependent upregulated genes in Ganmouse tumors, we focused on TLR2 and its coreceptor CD14because we had previously demonstrated that epithelial TLR2expression is important for gastric tumorigenesis in gp130F/Fmice(28). We found that the expression levels TLR2 and CD14 wereincreased significantly in human gastric cancer and Gan mousetumor tissues which were amixture of epithelial and stromal cells(Supplementary Fig. S3). We thus performed laser microdissec-tion–based RT-PCR, and found that the expression of Tlr2 andCd14 in Gan mouse tumors was predominantly detected in E-cadherin (Cdh1)-expressing tumor epithelial cells rather thanstromal cells (Fig. 4A). These results suggest that the inflammatorymicroenvironment induces the expression of Tlr2 and Cd14 intumor epithelial cells.

To further confirm the inflammation-dependent induction ofTLR2 and CD14 in tumor cells, we treated gastric cancer cells withCM from activated RAW264 macrophages treated with LPS.Importantly, we found that the expression levels of TLR2 andCD14 were significantly increased in AGS, MKN74, and SNU484cells after treatment with LPS-stimulated RAW264 CM but notwith control CM (Fig. 4C). Moreover, the induction of TLR2 andCD14 in gastric cancer cells was significantly suppressed whenMyd88 expression was knocked down by CRISPR/Cas9 system(KD-Myd88) in RAW264 cells (Fig. 4B and C). These resultsindicate that MyD88-dependent macrophage-derived factorsupregulate TLR2 and CD14 in cancer cells. Moreover, the expres-sion of stem cell marker CD44 was significantly increased in AGScells when the cells were treated with LPS CM, whereas it was

suppressed by knockdown of Myd88 expression in RAW264macrophages (Fig. 4D). These results indicate thatMyD88-depen-dent macrophage-derived factor(s) induces the TLR2/CD14 sig-naling pathway in cancer cells, whichmaypromote tumorigenesisby enhancing the undifferentiated status of the tumor cells.

TheMyD88 pathway helpsmaintain the undifferentiated statusof tumor cells

Tamoxifen treatment induces loss of parietal cells in mousestomach, which leads to expansion of undifferentiated cell pop-ulation and regeneration of the gastric mucosa (SupplementaryFig. S4) as described previously (26). Notably, the expressionlevels of Tlr2 and Cd14 were significantly increased in theseregenerating gastric mucosa, suggesting a role for TLR2/CD14signaling in undifferentiated epithelial cells in the stomach.

We next examined the differentiation status of the gastrictumors of Gan Myd88�/� mice, in which Myd88 was disruptedin both epithelial and stromal cells. Importantly, the expressionlevels of intestinal stem cell markers, Cd44, Ephb3, Cd133, andSox9 (30), were significantly decreased in the Gan Myd88�/�

mouse tumors but not in Gan Irf5�/� mice compared with thecontrol Ganmice (Fig. 5A). CD44 expression was not detected inthe normal glandular stomach, however, CD44-expressing cellswere found throughout the tumor tissues ofGanmice (Fig. 5B). Incontrast, there were substantial decreases in the numbers ofCD44-positive cells in Gan Myd88�/� mice and BMT-Gan fromMyd88�/� mice (Fig. 5B). We also found that the expression ofMUC5AC and HþKþ/ATPase (differentiation markers) was notexpressed in control Gan mouse tumors, but induced in thetumors of Gan Myd88�/� and BMT-Gan from Myd88�/� mice

Figure 1.The suppression of gastric tumordevelopment by Myd88 disruption.A, representative macroscopicphotographs (top) and histologicphotographs of whole views (bottom)of the indicated genotype mousegastric tumors. Arrow on theGan Myd88�/� tumor indicates asuppressed tumor lesion. Bars, 5 mm.B, the gastric tumor size of the GanMyd88�/� and Gan Irf5�/� micerelative to the mean level of controlGan mouse tumors is shown (mean%� SE). �, P < 0.05. C, representativephotographs of ApopTag (top) andanti-Ki-67 immunostaining (bottom)of the indicated genotype mousetumors. The insets are enlargedimages. Asterisk indicates aproliferating zone in a Gan Myd88�/�

mouse tumor. Bars, 200 mm. D, themean Ki-67 labeling indices of GanMyd88�/� and Gan Irf5�/� tumorsrelative to that of Gan mouse tumorsare shown (mean% � SE). � , P < 0.05.

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Figure 2.Suppression of the inflammatory microenvironment generation by Myd88 disruption. A, representative photographs of F4/80 immunostaining of the indicatedgenotype mouse gastric tumors. Bars, 100 mm. B, the mean numbers of F4/80-positive cells per field in the respective genotype mouse tumors are shown (mean�SE). � , P < 0.05. C, The fold changes (Log2) in gene expression in Gan (top), Gan Myd88�/� (middle), and Gan Irf5�/� (bottom) mouse gastric tumors in comparisonwith wild-type mouse stomach analyzed by PCR array are shown. The gene symbols for each bar are indicated at the bottom. D, the expression profilesof the top 21 upregulated genes in the Gan mouse tumors in (C) are shown with the heatmap for the indicated genotype mouse tumors. E, immunoblotting of theindicated genotype mouse gastric tumors as well as wild-type mouse stomach. GAPDH was used as an internal control. F, representative photographs of thefluorescence immunohistochemical analysis of F4/80 and phosphorylated NF-kB in the indicated genotype mouse gastric tumors. Arrowheads indicateddouble positive cells. Bars, 200 mm. G, immunoblotting of the COX-2 and mPGES-1 in the indicated genotype mouse gastric tumors as well as wild-type mousestomach. b-Actin was used as an internal control.






Figure 3.Suppression of gastric tumor development by Myd88 disruption in BMDCs. A, representative x-ray CT images of bone marrow–transplanted (BMT)-Gan fromGFP transgenic (left), Myd88�/� (center), or Irf5�/� mice (right) at 0 week (top) and 20 weeks (middle) after BMT. The stomach areas are indicated withyellow dashed lines. Representative macroscopic photographs of gastric tumors at 20 weeks after BMT are shown (bottom). Bars, 5 mm. B, thecalculated mean tumor areas from the X-ray CT images of the respective BMT-Ganmice relative to those at 0 week are indicated as line graphs (mean� SE). � ,P < 0.05 to control BMT-Gan from GFP mice. C, representative photographs of the F4/80 immunostaining of the indicated mouse tumors. Bars, 100 mm. D, themean numbers of F4/80-positive cells per field are shown (mean � SE). � , P < 0.05. E, the expression levels of cytokines and chemokines in thegastric tumors of Gan mice and BMT-Gan from Myd88�/� mice relative to the levels in wild-type mouse stomach as determined by next-generationsequencing (n ¼ 3 for each genotype).

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(Fig. 5B). Notably, there are chimeric glands consisting of bothCD44-positive cells and HþKþ/ATPase-positive cells in BMT-Ganfrom Myd88�/� mice, suggesting that tumor epithelial cells aredifferentiated by Myd88 disruption in BMDCs (Fig. 5B, whitearrowheads).

Activation of Wnt signaling by MyD88 pathway ingastric tumors

We performed next-generation sequencing using the tumortissues ofGanMyd88�/�mice and BMT-Gan fromMyd88�/�miceto examine the gene expression profiles of the respective mousetumors. In the Gan Myd88�/� mouse tumors, Myd88 was dis-rupted in both the epithelial and stromal cells, whereas Myd88was disrupted only in the BMDCs in the BMT-Gan fromMyd88�/�

mice. We found that the numbers of genes that were down-regulated below 0.5-fold or upregulated above 2.0-fold in tumorsin both Gan Myd88�/� mice and BMT-Gan from Myd88�/� micecompared with control Gan mouse levels were 908 and 318,respectively (Fig. 6A). Using the sequencing results, we performedan Ingenuity Pathway Analysis (IPA) to examine the pathwaysthat were inhibited by disruption ofMyd88 in BMDCs. We foundthat NF-kB pathway was themost inhibited byMyd88 disruption,and inflammatory cytokine pathways were also significantlyinhibited (Fig. 6B and Supplementary Fig. S5). Notably, the IPAanalysis showed that the Wnt/b-catenin (CTNNB1) signaling wasalso significantly suppressed by Myd88 disruption, which wasconsistent with the immunoblotting results (Fig. 2E). These

results suggest that MyD88 signaling in BMDCs promotes tumor-igenesis through activation of Wnt signaling in tumor cells.

We previously showed that macrophages were infiltrated in thestroma of preneoplastic lesions of the K19-Wnt1mouse stomach,and thatWnt signaling activity was promoted in epithelial cells ofthese lesions, suggesting that macrophage-derived factors activateWnt signaling (31).Notably, thenumbers of preneoplastic lesionsin K19-Wnt1 Myd88�/� mice were significantly decreased incomparison with control K19-Wnt1 mice (Fig. 6C and D). It istherefore possible that the MyD88 signaling in BMDCs contri-butes to promotion of Wnt signaling activity in the tumor epi-thelial cells, which may contribute to the acceleration of theundifferentiated status and tumorigenesis.

Taken together, the current study strongly suggests that MyD88signaling inBMDCs togetherwithCOX-2/PGE2pathwaypromotetumorigenesis through the induction of the TLR2/CD14 pathwayas well as activation of Wnt signaling in tumor cells.

DiscussionIt has been established that chronic inflammation plays an

important role in cancer development through a variety ofmechanisms (32, 33), and that the COX-2/PGE2 pathway playsa key role in the promotion of tumorigenesis through the gener-ation of the inflammatory microenvironment (5, 34–36). Nota-bly, the induction of COX-2 expression in intestinal tumors wassuppressed inApcMinMyd88�/�mice (14), suggesting thatMyD88

Figure 4.The induction of TLR2 and CD14 intumor epithelial cells by activatedmacrophage-derived factors. A, themean expression levels of Cdh1, Tlr2,and Cd14 in tumor epithelial cells (GanEpi) and tumor stromal cells (Gan St)from Gan mice relative to the meanlevel of wild-type mouse epithelialcells (WTEpi) are shown (mean� SE).� , P < 0.05. B, immunoblotting ofMyD88 in the CRISPR/Cas9-mediatedMyd88-knockdown RAW264 cells.b-Actin was used as an internalcontrol. C, the mean expression levelsof CD14 and TLR2 in the indicated celllines treated with CM of parental orMyd88-knockdown (KD-Myd88)RAW264 cells with or without LPSstimulation relative to those ofuntreated control (NT) cells (shown asthe mean � SD). �, P < 0.05. D, theexpression levels of CD44 in AGS cellstreated with each RAW264 CMrelative to that treated with thecontrol CM are shown. Real-timeRT-PCR (mean � SD; left) and flowcytometry analysis (right). � , P < 0.05.CD44high population ratios areindicated in the flow cytometry graph.






signaling induces COX-2 expression in tumor tissues possiblythrough NF-kB activation, which induces inflammatoryresponses. However, in the current study, we have shown thatthe inflammatory responses are significantly suppressedbyMyd88disruption in BMDCs, even though theCOX-2/PGE2 pathwaywasconstitutively activated. It is therefore possible that the simulta-neous activation of both MyD88 signaling in BMDCs and theCOX-2/PGE2 pathway is required to generate the inflammatorymicroenvironment in tumor tissues (Fig. 6E). We previouslyshowed that IL1R signaling is not required for the maintenanceof COX-2/PGE2 pathway-induced inflammatory responses (27),thus TLR/MyD88 rather than IL1R/MyD88pathway plays a role inthe generation of inflammatory microenvironment in gastrictumors.

It has consistently been shown that the disruption ofMyd88 orTlr2, 4, 9 in the BMDCs of Apc-mutant mice caused a reduction incytokine expression and the suppression of intestinal tumorigen-esis (37). Moreover, it has been demonstrated that the IKKb-induced NF-kB activation in myeloid cells is important for

inflammatory responses and proliferation in the stomach afterHelicobacter infection (38). Taken together, these results indicatethat the TLR/MyD88/NF-kB signaling in BMDCs plays a key rolein generating the inflammatory microenvironment in tumortissues.

It is intriguing to determine the specific TLRs and ligands thatare required for the activation of the MyD88/NF-kB pathway inBMDCs in tumor tissues. TLR2 and TLR4 have been demonstratedto be important for the homeostasis of intestinal epithelial cells(39), and polymorphisms of TLR2 and TLR4 have been shown tobe associated with an increased risk of gastric cancer (40, 41).Accordingly, TLR2 or TLR4 are important candidates for respon-sible TLRs in gastric tumorigenesis. TLRs recognize not onlypathogen-associated molecular patterns (PAMP) but also endog-enous ligands such as damage-associated molecular patterns(DAMP; refs. 13, 42). It has been reported that TLR2/4 recognizehigh-mobility group box 1 and extracellular matrix proteoglycanversican that are produced by cancer cells (24, 43, 44). Accord-ingly, cancer cell–derived ligands as well as infectious agents may

Figure 5.The differentiation of gastric tumor epithelialcells by Myd88 disruption. A, the meanexpression levels of stem cell markers in gastrictumors fromGanMyd88�/� andGan Irf5�/�micerelative to those fromGanmice are shown (mean� SE). �, P < 0.05 and P values to Gan mouselevel. B, representative photographs of theimmunostaining of CD44 (left), MUC5AC(center), and HþKþ/ATPase (right) in the gastrictumors from the respective genotypemice and inthe normal stomach of wild-type mice areshown. Ratios (%) of positively stained cell areain the field are indicated. Bars, 500mm.Asterisks,CD44-positive tumor cells (top left). Arrowsindicate CD44-, MUC5AC-, or HþKþ/ATPase-expressing cells. The white arrowheads on theBMT-Gan (Myd88-/-) tumor indicate a chimericgland consisting of CD44-positive cells andHþKþ/ATPase-positive cells.

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activate TLR2 and/or TLR4 in tumor tissues, thereby generatingthe inflammatory microenvironment.

We also showed that inflammatory responses induced theexpression of TLR2 and CD14 in tumor epithelial cells (Fig.6E). We previously showed that TLR2/MyD88 signaling in tumorcells promotes gastric tumorigenesis in gp130F/F mice (19, 28).Consistent with these findings, it has been shown that TLR2signaling in cancer cells plays a tumor-promoting role throughthe enhancement of stemness (23, 24). Moreover, the epithelialcell–specific deletion ofMyd88was observed to suppress intestinal

tumorigenesis in ApcMin mice (16). These results suggest that theTLR2/CD14 signaling in "epithelial cells" promotes tumor devel-opment through the maintenance of the undifferentiated status oftumor cells. Notably, it has been reported that H. pylori infectionactivates TLR2 but not TLR4 (45). Accordingly, it is possible thatH. pylori infection can promote gastric tumorigenesis through theactivationof TLR2on tumorepithelial cells.On theotherhand, theNF-kBpathway is activated inbothdifferentiated (tubular typeandpapillary type) and undifferentiated (mucinous type) humangastric cancers, which suggests that the MyD88/NF-kB pathway is

Figure 6.The suppression of Wnt signaling byMyd88 disruption. A, a Venn diagram of the genes downregulated below <0.5-fold (top) or upregulated >2.0-fold (bottom) inbothGanMyd88�/� and BMT-Gan fromMyd88�/�mice are shown. B, the list of upstream regulators inhibited in the gastric tumors in bothGanMyd88�/� and BMT-Gan from Myd88�/� mice as determined by the IPA (z-score < �2.9). The orange-colored lines indicate cytokine pathways. The red-colored lines indicatetheNF-kB,MyD88, andWnt/b-catenin (CTNNB1) pathways. C, representative photographs of H&E (top) and immunostaining for Ki-67 (bottom) using serial sectionsof the gastricmucosa of the indicated genotypemice. Arrows indicate preneoplastic lesions ofK19-Wnt1mice. Bars, 200 mm. D, the numbers of preneoplastic lesionsin control K19-Wnt1 and K19-Wnt1 Myd88�/� mice are shown (mean � SE). Asterisks indicate P < 0.05. E, a schematic drawing showing the role of MyD88 ingastric tumorigenesis. TLR/MyD88 signaling and COX-2/PGE2 pathways in BMDCs cooperatively generate inflammatory microenvironment, which inducesexpression of TLR2 and CD14 in tumor epithelial cells. The promotion of Wnt signaling through activation of NF-kB is a possible mechanism underlying TLR2/CD14-dependent tumorigenesis in tumor epithelial cells.






less involved in the regulation of the histologic subtypes ofestablished gastric cancer (Supplementary Table S2).

The expression of Tlr2 and Cd14 in intestinal and mammaryepithelial cells is associated with the activation of Wnt signal-ing, which may explain the increased stemness and tumorige-nicity (16). We herein showed that Wnt/b-catenin signaling issignificantly suppressed and that differentiation is induced ingastric tumors by Myd88 disruption, supporting the idea thatTLR2/CD14 signaling enhances the stemness of tumor cellsthrough activation of Wnt signaling. It has been reported thatNF-kB activation promotes Wnt signaling in the intestinalepithelial cells, which causes dedifferentiation and the acqui-sition of stem cell properties (46). It is therefore possible thatthe activation of NF-kB by TLR2/CD14/MyD88 signalingincreases the Wnt signaling activity in tumor epithelial cells,thereby contributing to the maintenance of undifferentiatedstatus of tumor cells (Fig. 6E).

In the current study, we showed that TLR/MyD88 signaling inBMDCs is required for the generation of the inflammatory micro-environment in cooperation with the activation of the COX-2/PGE2pathway.MyD88-dependentmacrophage-derived factors inthe inflammatory microenvironment induce the expression ofTLR2 and CD14 in tumor cells. Wnt signaling activation is apossible mechanism underlying the TLR2/CD14/MyD88 signal-ing–induced tumor promotion. On the basis of these findings, wepropose that the targeting of TLR/MyD88 signaling together withthe COX-2/PGE2 pathway will be an effective preventive strategyagainst gastric cancer.

Disclosure of Potential Conflicts of InterestH. Saya reports receiving commercial research grants from Daiichi Sankyo

Co., Ltd. and Eisai Co., Ltd. No potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. OshimaDevelopment of methodology: Y. MaedaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Maeda, K. Echizen, H. Oshima, L. Yu, N. Sakulsak,B.J. JenkinsAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Maeda, K. Echizen, H. Oshima, O. Hirose,Y. Yamada, T. Taniguchi, B.J. JenkinsWriting, review, and/or revision of the manuscript: B.J. Jenkins, M. OshimaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Taniguchi, H. SayaStudy supervision: H. Saya, M. Oshima

AcknowledgmentsThe authors thank Manami Watanabe and Ayako Tsuda for technical

assistance. The computations were partially performed on the NIG supercom-puter at the ROIS National Institute of Genetics, Japan.

Grant SupportThisworkwas supported byAMED-CREST, AMED, JapanAgency forMedical

Research and Development, Japan (to M. Oshima), and Grants-in-Aid forScientific Research on Innovative Areas from theMinistry of Education, Culture,Sports, Science and Technology of Japan (#22114005 and #15H02362; toM. Oshima). This work was also supported in part by the National Health andMedical Research Council of Australia (to B. Jenkins) as well as the OperationalInfrastructure Support Program by the Victorian Government of Australia (to B.Jenkins).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 14, 2015; revised November 19, 2015; accepted December11, 2015; published OnlineFirst February 17, 2016.

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2016;9:253-263. Published OnlineFirst February 17, 2016.Cancer Prev Res Yusuke Maeda, Kanae Echizen, Hiroko Oshima, et al. Inflammatory MicroenvironmentDerived Cells Promotes Gastric Tumorigenesis by Generation of

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