Blockade of B-Catenin–Induced CCL28 Suppresses Gastric Cancer

Published OnlineFirst March 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3074

CANCER RESEARCH | TUMOR BIOLOGY AND IMMUNOLOGY

Blockade of b-Catenin–Induced CCL28 Suppresses Gastric Cancer Progression via Inhibition of Treg Cell Inﬁltration Lu Ji1,2, Wei Qian3, Liming Gui1,2, Zhongzhong Ji1,2, Pan Yin1,2, Guan Ning Lin3, You Wang4, Bin Ma1,2, and Wei-Qiang Gao1,2

ABSTRACT ◥ Dysregulation of Wnt/b-catenin signaling is frequently observed the stomach. Moreover, an anti-CCL28 antibody attenuated Treg cell in human gastric cancer. Elucidation of the tumor immune micro- infiltration and tumor progression in H. felis/MNU mouse models. environment is essential for understanding tumorigenesis and for the Diphtheria toxin–induced Treg cell ablation restrained gastric cancer development of immunotherapeutic strategies. However, it remains progression in H. felis/MNU-treated DEREG (Foxp3-DTR) mice, unclear how b-catenin signaling regulates the tumor immune clarifying the tumor-promoting role of Treg cells. Thus, the b-cate- microenvironment in the stomach. Here, we identify CCL28 as a nin–CCL28–Treg cell axis may serve as an important mechanism for direct transcriptional target gene of b-catenin/T-cell factor (TCF). immunosuppression of the stomach tumor microenvironment. Our Protein levels of b-catenin and CCL28 positively correlated in human findings reveal an immunoregulatory role of b-catenin signaling in gastric adenocarcinoma. b-Catenin–activated CCL28 recruited reg- stomach tumors and highlight the therapeutic potential of CCL28 ulatory T (Treg) cells in a transwell migration assay. In a clinically blockade for the treatment of gastric cancer. relevant mouse gastric cancer model established by Helicobacter (H.) felis infection and N-methyl-N-nitrosourea (MNU) treatment, inhi- Significance: These findings demonstrate an immunosuppressive bition of b-catenin/TCF activity by a pharmacologic inhibitor role of tumor-intrinsic b-catenin signaling and the therapeutic iCRT14 suppressed CCL28 expression and Treg cell infiltration in potential of CCL28 blockade in gastric cancer.

Introduction Wnt/b-catenin signaling has been identified in more than 70% patients with gastric cancer (6). In addition to genetic mutations, alterations in Gastric cancer is the fifth most common cancers and the third many Wnt pathway components may occur through various mechan- leading cause of cancer-related deaths worldwide (1). The initiation isms either to upregulate the expression of the positive regulators or and progression of gastric cancer is attributable to complex genetic and downregulate the expression of the negative regulators, eventually environmental interactions. Majority of stomach tumors are adeno- leading to an aberrant activation of the canonical Wnt pathway (7). It carcinomas that are traditionally divided into two main histologic has also been shown that H. pylori infection promotes Wnt/b-catenin subtypes: intestinal and diffuse types. Among all the environmental activation in gastric epithelial cells (8, 9). Together, these findings factors, Helicobacter pylori (H. pylori) infection is overwhelmingly the pinpoint a crucial role of Wnt/b-catenin signaling in the pathogenesis greatest risk factor for gastric cancer and is associated with approx- of gastric cancer. imately 90% of cases (2, 3). Recent comprehensive molecular profiling Immune evasion has been recognized as an emerging hallmark of has provided new classifications and highlighted the molecular com- cancer (10). Understanding the tumor immune microenvironment is plexity of gastric adenocarcinoma (4, 5). Canonical Wnt pathway- essential for the discovery of new therapeutic targets as well as prediction related genes including APC and CTNNB1 are among the most and guidance of immunotherapeutic responsiveness. Data from murine significantly mutated genes (4, 5). Importantly, dysregulation of models or patient samples have suggested the involvement of myeloid- derived suppressor cells (MDSC) and M2 macrophages in the immu- 1State Key Laboratory of Oncogenes and Related Genes, Renji-Med-X Clinical nosuppression of gastric cancer (11, 12). Regulatory T (Treg) cells are Stem Cell Research Center, Renji Hospital, School of Medicine and School of another group of immunosuppressive cells that accelerate tumor pro- Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China. 2Med- gression in a broad range of cancer types (13, 14). However, the X Research Institute, Shanghai Jiao Tong University, Shanghai, China. 3School of prognostic role of Treg cells in gastric cancer is still controversial based Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China. on previous clinical studies (15–20) and the tumor-promoting or 4 Department of Obstetrics and Gynecology, Renji Hospital, School of Medicine, tumor-inhibiting function of Treg cells in gastric adenocarcinoma is Shanghai Jiao Tong University, Shanghai, China. still not verified. Moreover, tumor immune microenvironment or Note: Supplementary data for this article are available at Cancer Research immunotherapy in gastric cancer is much less well explored than many Online (http://cancerres.aacrjournals.org/). other cancer types such as melanoma and lung cancer (21–23). Corresponding Authors: Wei-Qiang Gao, Shanghai Jiao Tong University, 160 Recent studies have underlined a strong impact of oncogenic Pujian Road, Shanghai 200017, China. Phone: 86-21-62932049; Fax: 86-21- pathways on evasion of antitumor responses (24). For example, in 68383916; E-mail: [email protected]; and Bin Ma, Med-X Research mouse models of live carcinoma, p53 maintains the expression of Institute, 1954 Huashan Road, Shanghai 200030, China. Phone: 86-21- – – 62933631; E-mail: [email protected] natural killer (NK) cell recruiting chemokines and NK cell mediated antitumor responses (25, 26); melanoma-intrinsic b-catenin signaling Cancer Res 2020;80:1–13 reduces dendritic and T-cell infiltration via downregulation of doi: 10.1158/0008-5472.CAN-19-3074 CCL4 (27). However, it remains to be elucidated how oncogenic 2020 American Association for Cancer Research. pathways such as Wnt/b-catenin and tumor-derived chemokines

AACRJournals.org | OF1

Ji et al.

determine the composition of gastric cancer immune microenviron- After washing with PBS, slides were incubated with anti-mouse or anti- ment. The present study was set forth to answer this important rabbit HRP–conjugated secondary antibodies (1:1,000 dilution) and question. Our experiments revealed that activation of b-catenin in visualized using a DAB Peroxidase Substrate Kit (Gene Tech). Staining gastric cancer caused an upregulation of CCL28 expression and was visualized by Olympus BX53 System Microscope. Staining of subsequent Treg cell recruitment. Blockade of CCL28 suppressed Treg b-catenin and CCL28 on human gastric cancer tissue sections was cell infiltration and gastric cancer progression, thus shedding new light analyzed by ImageJ. To quantify the histopathologic score of mouse on the role of b-catenin signaling in shaping the gastric cancer immune stomach, tissue sections were stained with hematoxylin-eosin and was microenvironment. evaluated as described previously (28). Immunofluorescence of gastric cancer cells or mouse stomach tissue Materials and Methods sections was performed using the rabbit anti-b-catenin (clone E247, Abcam, 1:300 dilution) or rabbit polyclonal anti-GFP antibody Cell lines and plasmid transfection (Abcam, 1:300 dilution). Secondary antibody was Alexa Fluor 488– Human gastric cancer cell lines SGC7901, AGS, and BGC823 conjugated polyclonal goat anti-rabbit IgG (Abcam, 1:1,000 dilution). were obtained from the Cell Bank of Chinese Academy of Sciences Immunofluorescence images were acquired using Zeiss LSM 710 (Shanghai, China). MKN28 and MKN45 cell lines were obtained from Confocal Microscope. the Japanese Collection of Research Bioresources (JCRB) Cell Bank. Human normal gastric epithelial cell line GES-1 was a kind gift from Chromatin immunoprecipitation assay Dr. Helen H. Zhu (Renji Hospital, Shanghai Jiao Tong University Chromatin immunoprecipitation (ChIP) assay was performed as School of Medicine, Shanghai, China). Authentication of these cell described previously (29). Relative enrichment was calculated as lines was performed by Shanghai Biowing Applied Biotechnology Co. relative binding of anti-b-catenin over control IgG on the potential Ltd. using the short tandem repeat genetic analysis. All cell lines were DNA binding sites. maintained in RPMI1640 supplemented with 10% FBS and 1% penicillin–streptomycin (Thermo Fisher Scientific), cultured for no Luciferase reporter assay more than 2–3 weeks after thawing, and routinely checked for Myco- Wnt/b-catenin pathway reporter plasmid M50 Super 8X TOP- plasma infection using PlasmoTest Kit (InvivoGen). Transfection of Flash (Addgene plasmid, catalog no. 12456; ref. 30) was a kind gift þ plasmids into gastric cancer cells was performed using jetPRIME from Randall Moon. A 2.8-kb promoter ( E2576/ 205 relative to CCL28 fi fl transfection reagent (Polybus Transfection) following the manufac- transcription start site) of human gene was cloned into a re y turers’ protocols. luciferase reporter construct pGL4. Mutations on potential T-cell factor/lymphoid enhancer factor (TCF/LEF) biding sites on CCL28 RNA purification and qPCR promoter were introduced using Hieff Mut Site-Directed Mutagenesis Total RNA was extracted from the gastric cancer cells or murine Kit (Yeasen) to generate mutant CCL28 promoter reporter constructs stomach tissues using RNeasy Mini Kit (Qiagen). The cDNA synthesis (pGL4-CCL28.mut). Plasmid transfection efficiency was normalized was performed using PrimeScript RT Reagent Kit (Takara). qPCR by cotransfection with a pRL-CMV reporter containing Renilla lucif- experiments were conducted using SYBR Green PCR Master Mix Kit erase gene. Firefly and Renilla luciferase activities were measured (Takara) and ABI 7900HT Fast Real-Time PCR System (Applied using the Dual-Glo Luciferase Assay System (Promega). Biosystems). mRNA expressions of target genes were normalized to GAPDH C Computational analysis of human stomach TCGA/Oncomine and calculated by the DD t method. database Western blotting To study the correlation of CCL28 expression with Wnt/b-catenin Proteins of gastric cancer cells or mouse stomach tissues were pathway genes, the raw dataset of The Cancer Genome Atlas (TCGA) collected with RIPA lysis buffer (Thermo Fisher Scientific) supplemen- stomach adenocarcinoma (STAD) gene expression from 417 patient ted with protease and phosphatase inhibitors (Roche). Protein concen- samples was accessed from UCSC Xena (http://xena.ucsc.edu/). tration was quantified by BCA Protein Assay Reagent (Thermo Fisher Positive/negative Wnt/b-catenin score was calculated as the geometric Scientific). Equal amounts of proteins (10–20 mg) were separated by mean of a set of genes (31): TCF7L2 (encoding TCF4), CDH17 for SDS-PAGE using criterion precast gels (EpiZyme), transferred to poly- positive and NKD1, SFRP1, SFRP2, SFRP4, SOX10, SULF1 for negative. vinylidene difluoride membranes (Millipore). Images were developed We transformed expression unit from RPKM to TPM for better using a chemiluminescent horseradish peroxidase (HRP) substrate comparison between samples, and offset 0.01 for friendly plotting (Millipore). Primary antibodies used were anti-b-catenin (Abcam), view. Spearman rank correlation (R) was used with corresponding anti-CCL28 (R&D Systems), antiactive b-catenin (Merck), anti- P value. The comparison of expression levels of CCL28 gene in the ERK1/2 (Cell Signaling Technology), anti-phospho-ERK1/2 (Cell Sig- gastric cancer and normal stomach tissues was performed using naling Technology), anti-GAPDH (Abcam), and anti-b-tubulin Oncomine database. (Abcam). Densitometric analysis of blots was performed using ImageJ. Mouse models and treatments Histology, IHC, and immunofluorescence Mouse model of Helicobacter felis (H. felis) and N-methyl-N- IHC was performed on paraffin-embedded tissue arrays of human nitrosourea (MNU)-induced gastric cancer was established as gastric cancer (Alenabio) or formalin-fixed, paraffin-embedded mouse described previously with slight modifications (32). Briefly, mice were stomach tissue sections. Tissues were deparaffinized and rehydrated infected by oral gavage with H. felis (ATCC 49179) three times in a firstly, then blocked with 5% BSA in PBS at room temperature for week on every other day. Two weeks after the initiation of H. felis 1 hour. Afterward, slides were incubated overnight at 4 C with rabbit infection, mice were given drinking water containing 240 ppm MNU anti-b-catenin (clone E247, Abcam, 1:300 dilution), rabbit polyclonal on alternative weeks for a total of 12 weeks (total exposure of 6 weeks). þ þ anti-CCL28 (Abcam, 1:300 dilution), and mouse anti-H K ATPase b iCRT14 (BOC Sciences) was dissolved in DMSO to make 25 mg/mL (ATP4B; 2G11; Abcam, 1:100 dilution) antibodies in blocking buffer. stock solution, then diluted in PEG300, Tween 80 (Sigma-Aldrich),

OF2 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

b-Catenin Induces CCL28 Expression and Immunosuppression

and saline to prepare 5 mg/mL working solution freshly before Cell proliferation assay and scratch wound-healing assay injection. Intraperitoneal injection of iCRT14 was given at a dose of GES-1 or SGC7901 cells were plated in 96-well cell culture plate. Cell 50 mg/kg twice per week for 6 weeks before sacrifice of the mice at the Counting Kit-8 (CCK-8; Dojindo) was used to evaluate the cell growth end of week 32. For antibody treatment, mice were administered rates at 0, 1, 2, or 3 days. Absorbance values were determined on the intraperitoneally with 50 mg/kg CCL28 mAb (R&D) to block CCL28 microplate reader SpectraMax i3 (Molecular Devices) at 450 nm. or isotype IgG once per week starting from week 27. For Treg cell To determine the cell migratory ability, GES-1 or SGC7901 cells ablation, diphtheria toxin (DT; Sigma) was administered intraperito- were grown to 95% confluence after transfected with CCL28 or control neally at a dose of 6.25 mg/kg of body weight once weekly to DEREG plasmids. A straight scratch was made using a pipette tip. The cells mice (MMRRC Stock No: 32050-JAX; ref. 33) starting from week 27. were washed with PBS and cultured for 24 to 30 hours. The gap width For evaluation of tumor formation and histopathology, antibody- or of scratch repopulation was measured and compared with the initial DT-treated mice were sacrificed at the end of week 36. All animal gap width at 0 hour. experiments were performed in accordance with institutional guidelines. ELISA CCL28 in lysates of mouse stomach was measured by CCL28 ELISA FACS analysis Kit according to the manufacturer's instructions (R&D Systems). For the detection of immune cells in the spleen, blood, and stomach, Protein concentration of tissue lysates was quantified by BCA Protein single-cell suspensions of spleens and stomach tissue were prepared Assay Reagent (Thermo Scientific Scientific). using gentleMACS Octo dissociator (Miltenyi Biotec). 1 mg/mL Col- lagenase type IV (Thermo Fisher Scientific) and 50 mg/mL DNase I Statistical analysis (Sigma-Aldrich) were used to dissociate stomach tissue. Red blood Data were expressed as mean SEM. Statistical significance cells in tissues and whole blood were removed using RBC lysis buffer between groups was calculated by two-tailed, unpaired Student t test (BioLegend). For detection of intracellular cytokine, separated cells (GraphPad Prism). One-way ANOVA is used to compare the means of were stimulated for 6 hours with cell activation cocktail (BioLe- three or more groups to determine the statistical significance (Graph- gend). Intracellular cytokine staining was performed using Cyto- Pad Prism). Pearson correlation analysis was used to study the Fast Fix/Perm Buffer Set (BioLegend). Nuclear proteins were correlation between b-catenin and CCL28 expression in human gastric stained using the Foxp3/Transcription Factor Staining Buffer Set cancer samples. P < 0.05 was considered statistically significant. (eBioscience). Fluorescence data were acquired on a FACS Aria II cytometer (BD Biosciences) and analyzed using FlowJo software. Study approval Anti-mouse antibodies for FACS analysis used were as follows: All animal procedures were approved by the Institutional Animal CD3e (145-2C11), CD11c (N418), CD25 (PC61), CD11b (M1/70), Care and Use Committee of Shanghai Jiao Tong University, Shanghai, CD206 (C068C2), CD279 (PD-1; 29F.1A12), F4/80 (BM8), Foxp3 China. Collection and study of human gastric adenocarcinoma sam- (MF14), IFNg (XMG1.2), and MHC-II (I-A/I-E; M5/114.15.2) from ples was approved by the Renji Hospital Ethics Committee and BioLegend; CD4 (GK1.5), and CD8a (53-6.7) from BD Biosciences; conducted with the patients’ written informed consent in accordance CD45 (30-F11) and Ly-6G/Ly-6C (Gr1; RB6-8C5) from with the Declaration of Helsinki guidelines. eBioscience.

Gene expression profiling of mouse stomach tissues Results RNA samples were isolated from stomachs of control or H. felis/ b-Catenin signaling elevates CCL28 expression in gastric cancer MNU-treated mice. Transcriptome sequencing and data analysis cells were performed by Novogene. Gene Ontology (GO) or Kyoto To establish a possible immunomodulatory role of the gastric Encyclopedia of Genes and Genomes (KEGG) gene set enrichment oncogenic b-catenin signaling, we sought out to explore the effects analysis (GSEA) of differentially expressed genes was implemented of b-catenin signaling on chemokine expression in gastric cancer cells. by the cluster Profiler R package, in which gene length bias was We first evaluated the expression levels of b-catenin in several gastric corrected. GO terms with corrected P value less than 0.05 were cancer cell lines, among which, SGC7901 and MKN45 showed the considered significantly enriched by differential expressed genes. lowest and highest expression levels, respectively (Supplementary Fig. S1A). We next overexpressed a constitutively active mutant PBMC isolation and transwell migration assay S33Y.b-catenin, in which the presumptive GSK3b phosphorylation The human periphery blood mononuclear cells (PBMC) were site serine was replaced by tyrosine, in SGC7901 cells with the lowest isolated via a Ficoll-Paque Plus (GE Healthcare) gradient. PBMCs b-catenin level. As shown in Fig. 1A, overexpression of S33Y.b- were washed and resuspended in PBS to create a single-cell suspension. catenin led to the nuclear accumulation of b-catenin. We further The PBMCs were counted with a hemocytometer. analyzed expression of all chemokines in SGC7901 cells overexpres- Supernatants from SGC7901 gastric cancer cell culture were sing S33Y.b-catenin or transfected with empty vectors by qRT-PCR. plated onto the lower compartment of 5-mm-pore transwell cham- Among the detectable genes, CCL28 was the most highly upregulated bers (Corning). Two million fresh human PBMCs were seeded in chemokine by SGC7901 S33Y.b-catenin overexpression (Fig. 1B). PBS containing 1% FBS in the upper compartment of transwell In both SGC7901 and AGS cell lines that exhibited relatively low chambers.After6-hourincubationat37C, PBMC cells migrated b-catenin levels, CCL28 protein expression was elevated by over- to the lower chambers, the migrated cells were collected and used expression of wild-type (WT) or S33Y.b-catenin (Fig. 1C). Of note, þ þ for FACS analysis of Treg cells, CD4 , and CD8 T cells, using S33Y.b-catenin overexpression did not induce higher CCL28 expres- antibodies against human CD3 (SK7; eBioscience), CD4 (RPA-T4; sion as compared with WT.b-catenin. This was possibly due to that the eBioscience), CD8a (OKT8; eBioscience), CD25 (BC96; BioLegend), overexpression of the WT.b-catenin had reached the maximum and FOXP3 (206D; BioLegend). upregulating effect in these cells, and/or that other genes/pathways

AACRJournals.org Cancer Res; 80(10) May 15, 2020 OF3

Ji et al.

Figure 1. b-Catenin elevates CCL28 expression in gastric cancer cells. A, Representative immunoﬂuorescence images of SGC7901 gastric cancer cells transfected with empty vectors or S33Y.b-catenin plasmids. Cells were stained for b-catenin (green) and nuclei by DAPI (blue). Scale bar, 50 mm. B, qPCR analysis of chemokine expression in SGC7901 cells transfected with empty vectors or S33Y.b-catenin plasmids (normalized to GAPDH; n ¼ 3 biological replicates). Representative data from two independent experiments. C, Immunoblotting analysis for b-catenin and CCL28 in SGC7901 and AGS gastric cancer cells transfected with empty vectors, wild-type (WT), or S33Y.b-catenin plasmids. Representative data from three independent experiments. D, Immunoblotting analysis for b-catenin and CCL28 in MKN45 and BGC823 cells transfected with plasmids expressing scrambled shRNA (shScr) or shRNA against b-catenin (shCat). E, Representative images of IHC staining of b-catenin and CCL28 in human stomach tumors. Scale bar, 100 mm. F, Correlation between b-catenin and CCL28 protein expression in human stomach tumors, analyzed by Pearson correlation (n ¼ 89). G, Analysis of gene expression correlation between Wnt positive genes (TCF7L2 [TCF4]andCDH17)andCCL28 (left), or between Wnt negative genes (NKD1, SFRP1, SFRP2, SFRP4, SOX10, and SULF1)andCCL28 (right) by Spearman rank correlation (n ¼ 417).

were also regulating CCL28 expression downstream b-catenin. Con- CCL28 expression levels (Supplementary Fig. S1A). SGC7901 cells versely, in MKN45 and BGC823 cell lines that displayed relatively high expressed lowest amounts of both b-catenin and CCL28 while MKN45 b-catenin levels, knockdown of b-catenin led to a decrease in CCL28 cells exhibited highest expression levels for both proteins. protein expression (Fig. 1D). In agreement with S33Y.b-catenin IHC analysis further showed a signiﬁcant positive correlation overexpression, the GSK3 inhibitor LiCl rapidly resulted in a nuclear between b-catenin and CCL28 protein expression in human gastric accumulation of b-catenin and upregulation of CCL28 expression in cancer samples (Fig. 1E and F). Bioinformatic analysis of TCGA SGC7901 cells (Supplementary Fig. S1B–S1D). Highest level of active database also revealed that CCL28 mRNA expression was positively b-catenin was correlated with strongest CCL28 expression at 8 hours correlated with the mRNA expression of Wnt pathway positive poststimulation (Supplementary Fig. S1D). In the human gastric regulators while negatively correlated with the expression of Wnt cancer cell lines, b-catenin expression levels generally correlated with pathway negative regulators (Fig. 1G). A signiﬁcant although less

OF4 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

b-Catenin Induces CCL28 Expression and Immunosuppression

strong correlation between CTNNB1 (b-catenin) and CCL28 mRNA but not the promoters with mutations in either of the two potential expression was also observed (Supplementary Fig. S1E). binding sites (site 1 and 3; Fig. 2C). Among the TCF transcription factor family members, TCF1 and TCF4, but not LEF1, were found to b-Catenin/TCF binds to and activates CCL28 promoter in gastric mediate CCL28 promoter activity (Fig. 2D and E). The b-catenin/TCF cancer cells inhibitor iCRT14 (35), also abrogated S33Y.b-catenin–induced CCL28 The molecular mechanism for the regulation of CCL28 expression promoter activity (Supplementary Fig. S1F). These results demon- by b-catenin pathway was further investigated. By searching a 4-kb strate that CCL28 is a direct transcriptional target of b-catenin/TCF in human CCL28 promoter region (3,000 bp before and 1,000 bp down- gastric cancer cells. stream the transcription start site), we found three potential b-catenin/ In contrast, overexpression of WT or S33Y.b-catenin did not change TCF binding sites matching the core consensus TCF binding CCL28 expression or the reporter activity in normal gastric epithelial sequences (34) (Fig. 2A). ChIP analysis showed that site 1 and 3, but cell line GES-1 (Supplementary Fig. S2A and S2B), suggesting that the not site 2, were actual binding sites for b-catenin (Fig. 2B). By regulatory effect of b-catenin was speciﬁc in gastric cancer cells. It was generating a CCL28 promoter activity reporter system, we demon- reported that CCL28 might regulate b-catenin and ERK signaling in strated that S33Y.b-catenin elevated the activity of the 2.8-kb promoter other tumor types (36, 37), thus we further examined this issue in

Figure 2. b-Catenin/TCF binds to and activates the CCL28 promoter in gastric cancer cells. A, Scheme of CCL28 promoter luciferase reporter constructs illustrating the wild-type or mutated sequences of potential b-catenin/TCF binding sites. B, ChIP analysis of the binding of b-catenin to the CCL28 promoter in SGC7901 gastric cancer cells (n ¼ 3 biological replicates). C, Luciferase reporter activities of promoterless control (pGL4), CCL28 promoter (pGL4-CCL28), or promoters with mutated binding sites (pGL4-CCL28.mut-1/-3) in SGC7901 cells transfected with vector or S33Y.b-catenin plasmids (n ¼ 3 biological replicates). D, CCL28 reporter activities in SGC7901 cells transfected with plasmids overexpressing shRNA against TCF1 (shTCF1), TCF4 (shTCF4), LEF1 (shLEF1) or scrambled shRNA (shScr), in combination with S33Y.b-catenin plasmid (n ¼ 3 biological replicates). E, CCL28 reporter activities in SGC7901 cells transfected with TCF1-, TCF4-, LEF1-overexpressing or empty vector plasmids (n ¼ 3 biological replicates). Representative data from two to three independent experiments. , P < 0.05; , P < 0.01; , P < 0.001; N.S., not signiﬁcant.

AACRJournals.org Cancer Res; 80(10) May 15, 2020 OF5

Ji et al.

gastric cancer cells. In AGS and BGC823 cancer cell lines, CCL28 simultaneously upregulated in H. felis/MNU-induced gastric tumors, overexpression induced active and normal b-catenin expression but and that CCL28 expression is enriched in gastric epithelial cells with not ERK phosphorylation or expression (Supplementary Fig. S3A). upregulated b-catenin levels. Upregulation of b-catenin signaling activity by CCL28 was also To verify whether the elevated CCL28 expression was mediated by observed in normal gastric epithelial cell line GES-1 (Supplementary the canonical Wnt/b-catenin pathway, H. felis/MNU-treated mice Fig. S3B). However, overexpression of b-catenin or CCL28 did not were given a b-catenin/TCF inhibitor iCRT14 (39) to block the influence the proliferation or migration of GES-1 (Supplementary transcriptional activity. Western blotting and ELISA results both Fig. S4). Therefore, the mutual promoting effects between b-catenin showed that CCL28 protein levels were downregulated by iCRT14 and CCL28 may serve as a positive feedback loop in gastric cancer cells. treatment (Fig. 3F and G). In contrast, treatment of healthy control mice with iCRT14 did not change CCL28 expression (Supplementary CCL28 expression is associated with progression of clinical Fig. S9A), suggesting that CCL28 might be regulated by Wnt/b-catenin gastric cancer pathway in transformed cells where b-catenin had been activated, but To better understand the pathogenic relevance of CCL28 in clinical not in normal healthy gastric cells. In the H. felis/MNU mice, long- gastric cancer, we performed bioinformatic analysis using Oncomine term iCRT14 treatment was found to be associated with signs of database and found that CCL28 mRNA expression was higher in both toxicities such as fatigue and abdominal swelling, which precluded us intestinal-type and diffuse-type gastric tumors than normal tissues from analyzing tumor growth and pathology at week 36. Overall, in (Supplementary Fig. S5A). Analysis based on IHC of patient tumor agreement with the in vitro findings in gastric cancer cells (Fig. 1C samples revealed that higher CCL28 protein levels were also associated and D; Supplementary Fig. S1F), these results indicate that activation with higher pathologic grades (Supplementary Fig. S5B). Taken of b-catenin signaling in the stomach leads to an increase in CCL28 together, these data suggest a potentially pathogenic role of CCL28 expression in H. felis/MNU-induced gastric cancer in vivo. in gastric cancer. CCL28 blockade inhibits H. felis/MNU-induced gastric cancer CCL28 is upregulated by b-catenin in H. felis/MNU-induced progression gastric cancer We further explored whether CCL28 was indeed a pathogenic factor To determine whether b-catenin regulates CCL28 in vivo,we and contributed to gastric cancer progression in vivo. Mice were given established the mouse model of H. felis/MNU-induced gastric cancer anti-CCL28 mAb to block CCL28 activity once per week starting from (Supplementary Fig. S6A; ref. 32), which mimicked the proposed week 27 after the start of MNU treatment. After a 10-week treatment pathogenesis of human gastric carcinogenesis to a great extent. with antibodies, the anti-CCL28 antibody significantly reduced tumor Tumors formed 36 weeks after the start of MNU treatment (Supple- size in the stomachs of H. felis/MNU mouse models as compared with mentary Fig. S6B). Alcian blue staining indicated the feature of isotype control (Fig. 4A). Histologic analysis showed an apparent intestinal-type transformation. Pathologic scores for inflammation, alleviation in dysplastic grade and intestinal-type transformation by epithelial defects, metaplastic, and dysplastic grades were drastically anti-CCL28 therapy (Fig. 4B). Loss of parietal cells was reversed as þ þ enhanced in H. felis/MNU-treated mice (Supplementary Fig. S6C; shown by immunochemical staining of H K ATPase (Fig. 4B). The ref. 28). We further performed FACS analysis to characterize the pathologic degrees of epithelial defects, metaplasia, and dysplasia were changes in immune cells in this model at the end of week 36 all significantly reduced by anti-CCL28 therapy especially in the gastric (Supplementary Fig. S7 and S8). Treg cell numbers were found to be antrum, although not in the corpus (Fig. 4C). These data demonstrate increased in the stomachs and spleens of H. felis/MNU-treated mice that CCL28 blockade suppresses H. felis/MNU-induced gastric cancer þ þ (Supplementary Fig. S8). There was a reduction in IFNg CD8 T cells progression and that CCL28 is a pathogenic factor in gastric cancer. þ þ but an elevation in PD-1 CD8 T cells in the stomachs. MDSCs were increased in the spleens and blood. These findings pinpoint an b-Catenin–upregulated CCL28 recruits Treg cells in vitro and enhanced immunosuppressive phenotype of H. felis/MNU-induced in vivo gastric cancer. We next sought out to elucidate the mechanisms for the antitumor Interestingly, GSEA analysis of mRNA expression unraveled the effects of CCL28 blockade. It has been shown that CCL28 mediates significant enrichment of a set of genes involved in negative regulation Treg cell migration (40, 41). We examined whether b-catenin– of the canonical Wnt signaling pathway (GO:0090090) in control activated tumor cells recruit human Treg cells in vitro through CCL28. stomachs, but no significant change in the gene set for positive Notably, CCL28 secreted into the supernatants of SGC7901 cells was regulators (GO:0090263; Fig. 3A), suggesting an activation of Wnt/ raised by S33Y.b-catenin and decreased by CCL28 shRNA as analyzed b-catenin signaling in the H. felis/MNU-treated stomachs at least by ELISA (Supplementary Fig. S9B and S9C). Supernatants from partly through downregulation of the negative regulators. Many SGC7901 cells transfected with S33Y.b-catenin, CCL28 shRNA, or chemokine pathway genes were enriched in H. felis/MNU-treated control plasmids were collected to recruit immune cells in an in vitro stomachs (Fig. 3A), and, in particular, Ccl28 was among the top cell migration assay. Western blotting confirmed a reduction in upregulated chemokine genes (Fig. 3B). Consistently, Western blot- S33Y.b-catenin–induced CCL28 protein expression by shRNA ting and ELISA analyses demonstrated that both b-catenin and CCL28 knockdown (Fig. 5A). In the transwell migration assay, PBMCs that proteins of the stomach tissues were also upregulated in the stomachs migrated to the supernatants from tumor cell culture were analyzed by þ of H. felis/MNU-treated mice as compared with control mice (Fig. 3C flow cytometry (Fig. 5B). The ratio of Treg cells in total CD4 T cells and D). In line with previous findings that CCL28 was mainly and the absolute migrated Treg cell number were significantly expressed in epithelial cells of various mucosal tissues (38), CCL28 increased under S33Y.b-catenin overexpression condition and expression was also observed in gastric epithelium of H. felis/MNU- decreased following CCL28 knockdown (Fig. 5C and D), indicating treated mice (Fig. 3E). Moreover, IHC of sequential sections revealed a that b-catenin–activated gastric cancer cells recruit Treg cells through coexpression of b-catenin and CCL28 in the same areas in both corpus CCL28 in vitro. Conditioned medium did not show any difference in þ þ and antrum. Thus, these data suggest that b-catenin and CCL28 are the recruitment of total CD4 or CD8 T cells, suggesting a specific

OF6 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

b-Catenin Induces CCL28 Expression and Immunosuppression

Figure 3. b-Catenin upregulates CCL28 expression in H. felis/MNU-induced gastric cancer. A, GSEAs for negative regulation of the canonical Wnt signaling pathway (GO:0090090) and chemokine signaling pathway (KEGG:MMU04062) gene sets in H. felis/MNU-induced stomach tumors compared with control stomach tissues collected 36 weeks after initiation of MNU treatment. B, Heatmap for mRNA expression of top 10 upregulated chemokines in H. felis/MNU-induced stomach tumors compared with control stomach tissues (n ¼ 3–4/group). C, Levels of active b-catenin (ABC), total b-catenin, and CCL28 in control and H. felis/MNU-treated (HþM) stomach tissues were analyzed by Western blotting and densitometry (n ¼ 3/group). D, ELISA for CCL28 expression in control and H. felis/MNU-treated stomach tissues and tumors (n ¼ 3–5/group). E, IHC for b-catenin and CCL28 in the stomachs of H. felis/MNU-treated mice. Scale bar, 100 mm. F and G, Detection of CCL28 expression by Western blotting (F) and ELISA (G) in stomach tissues of H. felis/MNU mice treated with iCRT14 or vehicle at the end of week 32 (n ¼ 3–4/ group). , P < 0.05. effect of the b-catenin/CCL28 axis on Treg cells. Furthermore, over- itor iCRT14 (Supplementary Fig. S11). Interestingly, such splenic Treg expression of CCL28, in contrast, did not change the proliferation or cell ratio was also signiﬁcantly decreased, implying that there might migratory ability of gastric cancer cells (Supplementary Fig. S10), also be a Wnt/b-catenin–dependent mechanism in the spleens. Treg suggesting that the tumor-promoting effect of CCL28 might rely on its cell numbers in the blood were not inﬂuenced by iCRT14 treatment, immunoregulatory function. suggesting that the reduction in Treg cell numbers in the stomach and We also examined the effects of b-catenin/TCF inhibitor iCRT14 on spleens was likely due to a change in recruitment but not differenti- the recruitment of Treg cells in vivo in H. felis/MNU-treated mice at ation, proliferation, or viability of the cells. the end of week 32, and found that the ratio of stomach Treg cells To ascertain whether CCL28 controls Treg cell migration in vivo,we þ versus total CD45 cells was downregulated by b-catenin/TCF inhib- treated H. felis/MNU mice with anti-CCL28 or isotype antibodies from

AACRJournals.org Cancer Res; 80(10) May 15, 2020 OF7

Ji et al.

Figure 4. CCL28 blockade inhibits progression of H. felis/MNU- induced gastric cancer. Mice were treated with anti- CCL28 (aCCL28) or isotype antibodies for 10 weeks and euthanized at the end of week 36. A, Represen- tative macroscopic images of stomachs of H. felis/ MNU mice treated with aCCL28 or isotype antibodies (left) and quantiﬁcation of tumor areas (right; n ¼ 3/ group). Scale bar, 50 mm. B, Hematoxylin and eosin (H&E), Alcain blue (AB), and Hþ/Kþ ATPase staining of the stomach sections of H. felis/MNU mice treated with aCCL28 or isotype. Black arrowheads, invasive tumor cells in the gastric mucosa. Scale bars, 100 mm. C, Histopathologic scores for mouse stomachs (n ¼ 3–4/group). , P < 0.05; , P < 0.01; , P < 0.001.

the beginning of week 27 as described above. FACS analysis was In addition to its effect on stomachs, the influence of anti-CCL28 performed at the end of week 32 and confirmed a significant reduction antibody in the spleens may also contribute to the overall therapeutic of Treg cells in the stomachs of anti-CCL28 antibody-treated mice effects of the treatment because effective cancer immunotherapy (Fig. 6A). Interestingly, anti-CCL28 treatment also decreased the ratio requires immune activation in the periphery and coordinated systemic of Treg cells in the spleens but not in the blood, in line with previous immunity (35). Together, these data suggest that anti-CCL28 findings in iCRT14-treated mice, suggesting that there was a therapy effectively inhibits the recruitment of Treg cells without b-catenin–CCL28-dependent mechanism for Treg cell recruitment influencing the function of stomach Treg cells to counteract the þ þ in the spleens as well as the stomachs. IFNg CD4 T cells did not show immunosuppressive state, which might account for the mechanism significant changes by the treatment (Fig. 6B), while there was a underlying its antitumor effects in H. felis/MNU-induced gastric þ þ significant increase in IFNg CD8 T-cell ratio in the spleens of cancer. anti-CCL28–treated mice (Fig. 6C). The expression of the inhibitory immune checkpoint molecule CTLA4 on Treg cells in the stomach or Ablation of Treg cells restrains H. felis/MNU-induced gastric blood was not affected by anti-CCL28 treatment, but was significantly cancer progression decreased on Treg cells from the spleens (Supplementary Fig. S12). Although Treg cells have been shown to be tumor-promoting in Interestingly, the number and immunosuppressive function of the many types of cancers, an explicit role of Treg cells in gastric cancer has splenic Treg cells were both attenuated by the anti-CCL28 treatment. not been established and remains debatable based on clinical

OF8 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

b-Catenin Induces CCL28 Expression and Immunosuppression

Figure 5. b-Catenin–upregulated CCL28 recruits Treg cells in vitro. A, Immunoblotting analysis of b-catenin and CCL28 in SGC7901 gastric cancer cells transfected with plasmids as indicated. B, Immune cells that migrated to conditioned media from SGC7901 cells were analyzed by flow cytometry. C and D, The percentages of CD25þFOXP3þ Treg cells in CD4þ T cells and CD8þ T cells in total live PBMCs (C) and the absolute numbers of migrated immune cells (D) are shown (n ¼ 3 biological replicates). Representative data from two independent experiments. , P < 0.05; , P < 0.01. studies (15–20). Our above experiments showed that the anti-CCL28 was injected for 10 weeks before DEREG mice were sacrificed at the treatment effectively blocked the infiltration of Treg cells in stomach end of week 36. Ablation of Treg cells by DT indeed resulted in tumors, but whether the therapeutic effects of CCL28 blockade depend significantly reduced tumor areas (Fig. 7A). Histologically, DT-treated on modulation of Treg cells and what biological function of the Treg mice regained relatively normal phenotype of stomach (Fig. 7B) and cells exert in the progression of H. felis/MNU-induced gastric cancer exhibited much lower pathologic degrees of epithelial defects, meta- are unknown. To address these questions, we next examined the role of plasia, and dysplasia (Fig. 7C). IHC staining of GFP demonstrated a Treg cells by utilizing the DEREG (Foxp3-DTR/GFP) mice in which decrease in Treg cell infiltration in both gastric corpus and antrum by Treg cells could be depleted by DT treatment (34). In these mice, GFP DT treatment (Fig. 7D). FACS analysis revealed that Treg cell expression correlated with Foxp3 expression as shown by FACS numbers were decreased in the spleens and blood of DT-treated mice analysis (Supplementary Fig. S13A–S13C) and GFP staining could (Fig. 7E), confirming a successful ablation of Treg cells by DT þ þ identify Treg cells in the stomach (Supplementary Fig. S13D). As treatment. In contrast, CD4 and CD8 T-cell numbers were þ expected, DT treatment for a period of 4 weeks significantly decreased increased in the spleens and blood, implying that ablation of Foxp3 Treg ratios in the spleens, blood, and stomachs of DEREG mice cells might influence the proliferation, viability, and/or differentiation (Supplementary Fig. S14). of the effector T cells. In contrast, DT treatment did not affect CCL28 We then treated DEREG mice with H. felis and MNU to induce expression in the stomachs of normal DEREG mice (Supplementary gastric cancer as described above (Supplementary Fig. S5A). DT or PBS Fig. S15A) or H. felis/MNU-treated DEREG mice (Supplementary

AACRJournals.org Cancer Res; 80(10) May 15, 2020 OF9

Ji et al.

Figure 6. CCL28 blockade alleviates Treg cell inﬁltration in H. felis/MNU-induced gastric cancer. Mice were treated with anti-CCL28 (aCCL28) or isotype antibodies for 6 weeks and FACS analysis was performed at the end of week 32. A–C, Gating strategy (top) and quantiﬁcation (bottom) for Treg cells (A), IFNgþCD4þ T cells (B), and þ þ IFNg CD8 T cells (C) in the spleens, blood, and stomachs of H. felis/MNU mice treated with aCCL28 or isotype antibodies (n ¼ 4/group). , P < 0.05; , P < 0.001.

Fig. S15B), suggesting DT specifically depleted Treg cells by inducing create a favorable immune microenvironment for their growth. direct cell death without influencing the CCL28 chemotaxis. Together, Because a direct effect of CCL28 on proliferation or migration of these results indicate that Treg cells contribute to the progression of gastric cancer cells was not observed, we proposed and proved that the H. felis/MNU-induced gastric cancer and that anti-CCL28 therapy at tumor-promoting function of CCL28 largely depended on the recruit- least partly depends on the blockade of Treg cell infiltration. ment of immunosuppressive Treg cells. We demonstrated that, in addition to those cell growth–related genes such as MYC (c-myc) and CCND1 (cyclin D 1), CCL28 was a novel transcriptional target gene of Discussion b-catenin/TCF. In agreement with a previous report showing that In the present study, we uncover that b-catenin signaling in gastric CCL28 was increased in H. pylori–induced gastric mucosa (46), cancer upregulates CCL28 expression and increases Treg cell infiltra- H. felis/MNU treatment also upregulated CCL28 expression in the tion subsequently, which shapes an immunosuppressive microenvi- mouse stomach at least partly through the b-catenin/TCF pathway. ronment. Our study not only extends previous understandings of the This notion is supported by the fact that Helicobacter infection induced oncogenic effects of the Wnt/b-catenin pathway mainly via its control b-catenin activation in both human and mouse stomachs and further on cell proliferation, survival, and differentiation in gastric can- proven by the evidence that b-catenin/TCF inhibitor iCRT14 blocked cer (7, 42, 43), but also indicates that the immunoregulatory function CCL28 expression in Helicobacter-treated mouse stomach. These of b-catenin signaling also plays a pivotal role in tumor progression. findings pinpoint an important role of b-catenin–CCL28 axis in Heli- More importantly, the present work demonstrates that CCL28 block- cobacter-involvedgastriccancer.RecentanalysisofallTCGAtumorsby ade exhibits a striking antitumor effect by suppressing Treg cell others revealed a correlation of b-catenin signaling with non-T-cell– infiltration and may serve as a therapeutic strategy for gastric cancer. inflamedphenotype,althoughthemechanismswerenotelucidated(47). It is important to point out that regarding the immunoregulatory In melanoma, activation of the Wnt/b-catenin pathway results in mechanism by which the Wnt/b-catenin pathway contributes to lymphocyte exclusion via downregulation of CCL4 expression. How- gastric cancer progression, our study identifies CCL28 as a key linker ever, we did not observe a decrease in CCL4 expression upon b-catenin between the oncogenic b-catenin signaling and the stomach tumor activation in gastric cancer. The discrepancy in the two findings may be microenvironment. Chemokines have been shown to be critical for due to intrinsic differences in gastric tumor cells versus melanoma tumor development and progression by influencing tumor cell pro- cells(27).Whilemost ofpreviousresearches onthe connectionbetween liferation or metastasis or by shaping the tumor immune microenvi- oncogenic pathways and the tumor microenvironment focused on the ronment (44, 45). In particular, tumor cells tend to downregulate the immune effector cells (24), the conceptual advancement of our current expression of chemokines that recruit antitumor effector immune cells study is the regulation of immunosuppressive Treg cells by tumor and upregulate chemokines that attract immunosuppressive cells to b-catenin signaling. The control of Treg cell infiltration by b-catenin

OF10 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

b-Catenin Induces CCL28 Expression and Immunosuppression

Figure 7. Ablation of Treg cells restrains gastric cancer progression. A, Representative macroscopic images of the stomachs of H. felis/MNU-treated DEREG mice further injected with DT or PBS (left) and quantiﬁcation of tumor areas (right; n ¼ 3/group). Scale bar, 50 mm. B, Hematoxylin and eosin (H&E) staining of the stomachs in H. felis/MNU-treated DEREG mice. Scale bar, 100 mm. C, Histopathologic scores for DEREG mouse stomachs (n ¼ 3–4/group). D, IHC for GFP (Foxp3) in DEREG þ þ mouse stomachs. Scale bar, 100 mm. E, FACS analysis of Treg, CD4 T, and CD8 T cells in the spleens and blood of H. felis/MNU-treated DEREG mice given DT or PBS. FACS analysis was performed at the end of week 36 (n ¼ 3/group). , P < 0.05; , P < 0.01; , P < 0.001; N.S., not signiﬁcant.

signaling in H. felis/MNU-induced stomach tumor was clearly dem- The most signiﬁcant clinical implication of our work is that onstrated by the effect of iCTR14 treatment in the present experiments, blockade of CCL28 activity effectively suppresses gastric cancer pro- further supporting such conceptual advancement. gression. Although Wnt/b-catenin pathway has been an attractive

AACRJournals.org Cancer Res; 80(10) May 15, 2020 OF11

Ji et al.

target for cancer treatment, application of its inhibitors may be risky strategy to target Treg cells without affecting the viability of Treg because it is involved in a multitude of developmental processes and cells. Alternatively, the CCL28 chemotaxis can be also interrupted the maintenance of adult tissue homeostasis (48–50). Up to now, no by targeting the CCL28 receptor CCR10 on Treg cells (41), which Wnt pathway inhibitors have become successful drugs for cancer needs to be investigated in the future. Given the fact that the treatment (48, 51). Targeting the pathogenic factors downstream b-catenin pathway is frequently dysregulated in gastric cancer (7), Wnt/b-catenin pathway may provide an alternative strategy. In this the b-catenin–CCL28-Treg regulatory axis should be widely regard, the present work showing that targeting the b-catenin target involved. The anti-CCL28 therapy may apply to a broad range of gene CCL28 by a neutralizing antibody leads to decreased Treg patients with gastric cancer and represent a novel and promising infiltration in the stomach and represses tumor growth is of particular treatment in the future. interest. Consistent with our work, it has been shown that hypoxia induces CCL28 expression and blockade of its receptor CCR10 Disclosure of Potential Conflicts of Interest attenuates tumor growth in ovarian cancer (41). It will be interesting No potential conflicts of interest were disclosed. to investigate whether CCL28 expression is regulated by hypoxia and whether CCR10 blockade exerts an antitumor effect in gastric cancer. Authors’ Contributions Despite the possibility that CCL28 is also regulated by other pathways Conception and design: L. Ji, B. Ma, W.-Q. Gao besides b-catenin signaling, we propose that CCL28 could be a Development of methodology: L. Ji, B. Ma Acquisition of data (provided animals, acquired and managed patients, provided promising therapeutic target for gastric cancer. facilities, etc.): L. Ji, W. Qian, L. Gui, Z. Ji, P. Yin, G.N. Lin, Y. Wang, B. Ma Because Treg cells are regulated by CCL28-mediated chemotaxis Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and appear to be tumor-promoting in many cancers, the antitumor computational analysis): L. Ji, Z. Ji, B. Ma, W.-Q. Gao function of CCL28 blockade is presumed to depend on the inhibition Writing, review, and/or revision of the manuscript: L. Ji, B. Ma, W.-Q. Gao of the chemotaxis for Treg cell infiltration. However, the exact Administrative, technical, or material support (i.e., reporting or organizing data, biological function of Treg cell in gastric cancer development and constructing databases): L. Ji, B. Ma Study supervision: B. Ma, W.-Q. Gao progression has not been verified yet, and the prognostic value of tumor-infiltrating Treg cells in patients with gastric cancer remains Acknowledgments controversial (15–20). Here, by using the DT/DTR-system–mediated fi fi þ This work was supported by funds from Ministry of Science and Technology of the speci c and ef cient depletion of Foxp3 cells in DEREG mice, we People's Republic of China (2017YFA0102900 to W.-Q. Gao), National Natural H. felis found that ablation of Treg cells drastically attenuated /MNU- Science Foundation of China (81602484 to B. Ma; 81872406 and 81630073 to W.-Q. induced gastric cancer, in accordance with their tumor-promoting role Gao), Science and Technology Commission of Shanghai Municipality (16JC1405700 in many other cancer types (14, 52). Thus, Treg cells are also an to W.-Q. Gao), Shanghai Jiao Tong University Scientific and Technological Inno- attractive therapeutic target in gastric cancer. Toward this direction, vation Funds (2019TPB07 to W.-Q. Gao), KC Wong Foundation (to W.-Q. Gao), anti-CD25 antibody is one of the most widely used strategy to deplete and the Shanghai Young Eastern Scholar Funds (QD2016005 to B. Ma). Finally, we thank Shengguo Jia for his assistance in FACS analysis. Treg cells (14, 53), but CD25 is also expressed on other subpopulations þ of CD4 T cells besides Treg cells and may not be a perfect target. On The costs of publication of this article were defrayed in part by the payment of page the other hand, a recent study reveals that apoptotic Treg cells become charges. This article must therefore be hereby marked advertisement in accordance even more immunosuppressive than live Treg cells (54), implying that with 18 U.S.C. Section 1734 solely to indicate this fact. treatments-induced Treg apoptosis may have compromised therapeutic efficacy. To avoid these complications, by blocking CCL28- Received October 2, 2019; revised January 7, 2020; accepted March 5, 2020; mediated Treg cell infiltration, we provide an additional therapeutic published first March 10, 2020.

References 1. Global Burden of Disease Cancer Collaboration, Fitzmaurice C, Allen C, Barber 9. Murata-Kamiya N, Kurashima Y, Teishikata Y, Yamahashi Y, Saito Y, Higashi RM, Barregard L, Bhutta ZA, et al. Global, regional, and national cancer H, et al. Helicobacter pylori CagA interacts with E-cadherin and deregulates the incidence, mortality, years of life lost, years lived with disability, and disabil- beta-catenin signal that promotes intestinal transdifferentiation in gastric epi- ity-adjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis thelial cells. Oncogene 2007;26:4617–26. for the global burden of disease study. JAMA Oncol 2017;3:524–48. 10. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 2. Plummer M, Franceschi S, Vignat J, Forman D, de Martel C. Global burden of 144:646–74. gastric cancer attributable to helicobacter pylori. Int J Cancer 2015;136:487–90. 11. Tu S, Bhagat G, Cui G, Takaishi S, Kurt-Jones EA, Rickman B, et al. Over- 3. Amieva M, Peek RM Jr. Pathobiology of helicobacter pylori-induced gastric expression of interleukin-1beta induces gastric inflammation and cancer and cancer. Gastroenterology 2016;150:64–78. mobilizes myeloid-derived suppressor cells in mice. Cancer Cell 2008;14:408–19. 4. Cancer Genome Atlas Research Network. Comprehensive molecular character- 12. Lin C, He H, Liu H, Li R, Chen Y, Qi Y, et al. Tumour-associated macrophages- ization of gastric adenocarcinoma. Nature 2014;513:202–9. derived CXCL8 determines immune evasion through autonomous PD-L1 5. Cristescu R, Lee J, Nebozhyn M, Kim KM, Ting JC, Wong SS, et al. Molecular expression in gastric cancer. Gut 2019;68:1764–73. analysis of gastric cancer identifies subtypes associated with distinct clinical 13. Crunkhorn S. Cancer immunotherapy: targeting regulatory T cells. Nat Rev Drug outcomes. Nat Med 2015;21:449–56. Discovery 2017;16:754. 6. Ooi CH, Ivanova T, Wu J, Lee M, Tan IB, Tao J, et al. Oncogenic pathway 14. Tanaka A, Sakaguchi S. Regulatory T cells in cancer immunotherapy. Cell Res combinations predict clinical prognosis in gastric cancer. PLoS Genet 2009;5: 2017;27:109–18. e1000676. 15. Shen Z, Zhou S, Wang Y, Li RL, Zhong C, Liang C, et al. Higher intratumoral 7. Chiurillo MA. Role of the Wnt/beta-catenin pathway in gastric cancer: an in- infiltrated Foxp3þ Treg numbers and Foxp3þ/CD8þ ratio are associated with depth literature review. World J Exp Med 2015;5:84–102. adverse prognosis in resectable gastric cancer. J Cancer Res Clin Oncol 2010;136: 8. Franco AT, Israel DA, Washington MK, Krishna U, Fox JG, Rogers AB, et al. 1585–95. Activation of beta-catenin by carcinogenic helicobacter pylori. Proc Natl Acad 16. Kim HI, Kim H, Cho HW, Kim SY, Song KJ, Hyung WJ, et al. The ratio of intra- Sci U S A 2005;102:10646–51. tumoral regulatory T cells (Foxp3þ)/helper T cells (CD4þ) is a prognostic factor

OF12 Cancer Res; 80(10) May 15, 2020 CANCER RESEARCH

b-Catenin Induces CCL28 Expression and Immunosuppression

and associated with recurrence pattern in gastric cardia cancer. J Surg Oncol 36. Park J, Zhang X, Lee SK, Song NY, Son SH, Kim KR, et al. CCL28-induced 2011;104:728–33. RARbeta expression inhibits oral squamous cell carcinoma bone invasion. J Clin 17. Huang Y, Liao H, Zhang Y, Yuan R, Wang F, Gao Y, et al. Prognostic value of Invest 2019;129:5381–99. tumor-infiltrating FoxP3þ T cells in gastrointestinal cancers: a meta analysis. 37. Yang XL, Liu KY, Lin FJ, Shi HM, Ou ZL. CCL28 promotes breast cancer growth PLoS One 2014;9:e94376. and metastasis through MAPK-mediated cellular anti-apoptosis and pro-metas- 18. Liu K, Yang K, Wu B, Chen H, Chen X, Chen X, et al. Tumor-infiltrating immune tasis. Oncol Rep 2017;38:1393–401. cells are associated with prognosis of gastric cancer. Medicine 2015;94:e1631. 38. Xiong N, Fu Y, Hu S, Xia M, Yang J. CCR10 and its ligands in regulation of 19. Kim KJ, Lee KS, Cho HJ, Kim YH, Yang HK, Kim WH, et al. Prognostic epithelial immunity and diseases. Protein Cell 2012;3:571–80. implications of tumor-infiltrating FoxP3þ regulatory T cells and CD8þ 39. Gonsalves FC, Klein K, Carson BB, Katz S, Ekas LA, Evans S, et al. An RNAi- cytotoxic T cells in microsatellite-unstable gastric cancers. Hum Pathol 2014; based chemical genetic screen identifies three small-molecule inhibitors of the 45:285–93. Wnt/wingless signaling pathway. Proc Natl Acad Sci U S A 2011;108:5954–63. 20. Choi HS, Ha SY, Kim HM, Ahn SM, Kang MS, Kim KM, et al. The prognostic 40. Eksteen B, Miles A, Curbishley SM, Tselepis C, Grant AJ, Walker LS, et al. effects of tumor infiltrating regulatory T cells and myeloid derived suppressor Epithelial inflammation is associated with CCL28 production and the recruit- cells assessed by multicolor flow cytometry in gastric cancer patients. Oncotarget ment of regulatory T cells expressing CCR10. J Immunol 2006;177:593–603. 2016;7:7940–51. 41. Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang LP, et al. 21. Chung HW, Lim JB. Role of the tumor microenvironment in the pathogenesis of Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T(reg) gastric carcinoma. World J Gastroenterol 2014;20:1667–80. cells. Nature 2011;475:226–30. 22. Niccolai E, Taddei A, Prisco D, Amedei A. Gastric cancer and the epoch of 42. Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, et al. immunotherapy approaches. World J Gastroenterol 2015;21:5778–93. Lgr5(þve) stem cells drive self-renewal in the stomach and build long-lived 23. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. gastric units in vitro. Cell stem cell 2010;6:25–36. Science 2018;359:1350–5. 43. Radulescu S, Ridgway RA, Cordero J, Athineos D, Salgueiro P, Poulsom R, et al. 24. Spranger S, Gajewski TF. Impact of oncogenic pathways on evasion of anti- Acute WNT signalling activation perturbs differentiation within the adult tumour immune responses. Nat Rev Cancer 2018;18:139–47. stomach and rapidly leads to tumour formation. Oncogene 2013;32:2048–57. 25. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al. 44. Viola A, Sarukhan A, Bronte V, Molon B. The pros and cons of chemokines in Senescence and tumour clearance is triggered by p53 restoration in murine liver tumor immunology. Trends Immunol 2012;33:496–504. carcinomas. Nature 2007;445:656–60. 45. Nagarsheth N, Wicha MS, Zou W. Chemokines in the cancer microenvironment 26. Iannello A, Thompson TW, Ardolino M, Lowe SW, Raulet DH. p53-dependent and their relevance in cancer immunotherapy. Nat Rev Immunol 2017;17: chemokine production by senescent tumor cells supports NKG2D-dependent 559–72. tumor elimination by natural killer cells. J Exp Med 2013;210:2057–69. 46. Hansson M, Hermansson M, Svensson H, Elfvin A, Hansson LE, Johnsson E, 27. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic beta-catenin signalling et al. CCL28 is increased in human helicobacter pylori-induced gastritis and prevents anti-tumour immunity. Nature 2015;523:231–5. mediates recruitment of gastric immunoglobulin a-secreting cells. Infect Immun 28. Rogers AB, Taylor NS, Whary MT, Stefanich ED, Wang TC, Fox JG. Helico- 2008;76:3304–11. bacter pylori but not high salt induces gastric intraepithelial neoplasia in B6129 47. Luke JJ, Bao R, Sweis RF, Spranger S, Gajewski TF. WNT/beta-catenin pathway mice. Cancer Res 2005;65:10709–15. activation correlates with immune exclusion across human cancers. Clin Cancer 29. Ma B, Fey M, Hottiger MO. WNT/beta-catenin signaling inhibits CBP-mediated Res 2019;25:3074–83. RelA acetylation and expression of proinflammatory NF-kappaB target genes. 48. Kahn M. Can we safely target the WNT pathway? Nat Rev Drug Discovery 2014; J Cell Sci 2015;128:2430–6. 13:513–32. 30. Veeman MT, Slusarski DC, Kaykas A, Louie SH, Moon RT. Zebrafish prickle, a 49. Tai D, Wells K, Arcaroli J, Vanderbilt C, Aisner DL, Messersmith WA, et al. modulator of noncanonical Wnt/Fz signaling, regulates gastrulation move- Targeting the WNT signaling pathway in cancer therapeutics. Oncologist 2015; ments. Curr Biol 2003;13:680–5. 20:1189–98. 31. Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and genetic 50. Wang B, Tian T, Kalland KH, Ke X, Qu Y. Targeting Wnt/beta-catenin signaling properties of tumors associated with local immune cytolytic activity. Cell 2015; for cancer immunotherapy. Trends Pharmacol Sci 2018;39:648–58. 160:48–61. 51. Duchartre Y, Kim YM, Kahn M. The Wnt signaling pathway in cancer. Crit Rev 32. Tomita H, Takaishi S, Menheniott TR, Yang X, Shibata W, Jin G, et al. Inhibition Oncol Hematol 2016;99:141–9. of gastric carcinogenesis by the hormone gastrin is mediated by suppression of 52. Pastille E, Bardini K, Fleissner D, Adamczyk A, Frede A, Wadwa M, et al. TFF1 epigenetic silencing. Gastroenterology 2011;140:879–91. Transient ablation of regulatory T cells improves antitumor immunity in colitis- 33. Klingenberg R, Gerdes N, Badeau RM, Gistera A, Strodthoff D, Ketelhuth DF, associated colon cancer. Cancer Res 2014;74:4258–69. et al. Depletion of FOXP3þ regulatory T cells promotes hypercholesterolemia 53. Arce Vargas F, Furness AJS, Solomon I, Joshi K, Mekkaoui L, Lesko MH, et al. Fc- and atherosclerosis. J Clin Invest 2013;123:1323–34. optimized anti-CD25 depletes tumor-infiltrating regulatory T cells and syner- 34. Cadigan KM, Waterman ML. TCF/LEFs and Wnt signaling in the nucleus. gizes with PD-1 blockade to eradicate established tumors. Immunity 2017;46: Cold Spring Harb Perspect Biol 2012;4. DOI: 10.1101/cshperspect.a007906. 577–86. 35. Spitzer MH, Carmi Y, Reticker-Flynn NE, Kwek SS, Madhireddy D, Martins 54. Maj T, Wang W, Crespo J, Zhang H, Wang W, Wei S, et al. Oxidative stress MM, et al. Systemic immunity is required for effective cancer immunotherapy. controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade Cell 2017;168:487–502 e15. resistance in tumor. Nat Immunol 2017;18:1332–41.

AACRJournals.org Cancer Res; 80(10) May 15, 2020 OF13

Blockade of β-Catenin−Induced CCL28 Suppresses Gastric Cancer Progression via Inhibition of Treg Cell Infiltration

Lu Ji, Wei Qian, Liming Gui, et al.

Cancer Res Published OnlineFirst March 10, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-3074

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/03/10/0008-5472.CAN-19-3074.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2020/04/29/0008-5472.CAN-19-3074. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.