Forkhead Box F2 Suppresses Gastric Cancer Through a Novel FOXF2

Published OnlineFirst January 26, 2018; DOI: 10.1158/0008-5472.CAN-17-2403

Cancer Molecular Cell Biology Research

Forkhead Box F2 Suppresses Gastric Cancer through a Novel FOXF2–IRF2BPL–b-Catenin Signaling Axis Akira Higashimori1,2, Yujuan Dong1,3, Yanquan Zhang1, Wei Kang4, Geicho Nakatsu1, Simon S.M. Ng3, Tetsuo Arakawa2, Joseph J.Y. Sung1, Francis K.L. Chan1, and Jun Yu1

Abstract

DNA methylation has been identified as a hallmark of gastric inducing b-catenin protein ubiquitination and degradation inde- cancer (GC). Identifying genes that are repressed by DNA promoter pendently of GSK-3b. FOXF2 directly bound the promoter of E3 methylation is essential in providing insights into the molecular ligase interferon regulatory factor 2-binding protein-like (IRF2BPL) pathogenesis of gastric cancer. Using genome-wide methylation and induced its transcriptional expression. IRF2BPL in turn inter- studies, we identified that transcription factor forkhead box F2 acted with b-catenin, increasing its ubiquitination and degradation. (FOXF2) was preferentially methylated in gastric cancer. We then Multivariate Cox regression analysis identified FOXF2 hypermethy- investigated the functional significance and clinical implication of lation as an independent prognostic factor of poor survival in early- FOXF2 in gastric cancer. FOXF2 was silenced in gastric cancer cell stage gastric cancer patients. In conclusion, FOXF2 is a critical tumor lines and cancer tissues by promoter methylation, which was suppressor in gastric carcinogenesis whose methylation status serves negatively associated with mRNA expression. Ectopic expression as an independent prognostic factor for gastric cancer patients. of FOXF2 inhibited proliferation, colony formation, G1–S cell-cycle Significance: FOXF2-mediated upregulation of the E3 ligase transition, induced apoptosis of gastric cancer cell lines, and sup- IRF2BPL drives ubiquitylation and degradation of b-catenin in pressed growth of xenograft tumors in nude mice; knockdown of gastric cancer, blunting Wnt signaling and suppressing carcino- FOXF2 elicited opposing effects. FOXF2 inhibited Wnt signaling by genesis. Cancer Res; 78(7); 1643–56. 2018 AACR.

Introduction and treatment of this disease (4). Using the promoter methylation array for a genome-wide screen of hypermethylated candidates in Gastric cancer (GC) is one of the most common cancers all over gastric cancer, we identified that the promoter of Forkhead box F2 the world, especially in Asia (1). The prognosis of gastric cancer (FOXF2) gene was preferentially methylated in gastric cancer cell patients is still poor, in spite of the improved surgical and lines. FOXF2 was first identified to be frequently rmethylated in adjuvant treatment approaches. Five-year overall survival of gas- childhood acute lymphoblastic leukemia, colorectal cancer, and tric cancer is generally 25%–30% (2). There are accumulating kidney cancer (5). However FOXF2 methylation status in gastric evidences that aberrant DNA methylation is a hallmark of gastric cancer has not been studied yet. cancer (3). Identification of novel tumor suppressor genes FOXF2, located on chromosome 6p25 and encoded a 444 repressed by DNA promoter methylation will provide new amino-acid protein, belongs to the forkhead family of transcrip- insights into the molecular pathogenesis of gastric cancer, and tional regulators (6, 7). It has been described as an essential will be useful in discovering potential biomarkers for diagnosis signaling molecule for embryogenesis and tissue development (8–11). Recently, decreased FOXF2 was shown in certain human

1 cancers of prostate, breast, liver, and esophageal squamous cell Institute of Digestive Disease and Department of Medicine and Therapeutics, – State Key Laboratory of Digestive Disease and Li Ka Shing Institute of Health carcinoma (12 15). However, the role of FOXF2 in human gastric Sciences, Shenzhen Research Institute, The Chinese University of Hong Kong, cancer is still unknown. In this study, we investigated functional Shatin, Hong Kong. 2Department of Gastroenterology, Osaka City University signiﬁcance, molecular mechanisms, and clinical impact of Graduate School of Medicine, Abeno-ku, Osaka, Japan. 3Department of Surgery, FOXF2 in gastric cancer. The Chinese University of Hong Kong, Shatin, Hong Kong. 4Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin, Hong Kong. Materials and Methods Note: Supplementary data for this article are available at Cancer Research Gastric tissue samples Online (http://cancerres.aacrjournals.org/). Forty paired primary gastric cancer and adjacent nontumor A. Higashimori and Y. Dong contributed equally to this article. samples were obtained during operation of gastric cancer Corresponding Author: Jun Yu, Department of Medicine and Therapeutics, patients diagnosed in the Prince of Wales Hospital of the Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Chinese University of Hong Kong from 2015 to 2017. In Hong Kong. Phone: 852-3763-6099; Fax: 852-2144-5330; E-mail: addition, 103 primary gastric tumors DNA were obtained from [email protected] Zhejiang University, China. We used the same inclusion and doi: 10.1158/0008-5472.CAN-17-2403 exclusion criteria for both cohorts. Gastric adenocarcinoma 2018 American Association for Cancer Research. patients with age >18 were enrolled in this study. Pregnant or

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nursing patients were excluded. Written informed consents were Primer sequences are listed in Supplementary Table S1. Stable obtained from subjects or their authorized representatives. The transfections were selected for 1 week with G418 antibiotic. study protocol was approved by the Ethics Committee of the Chinese University of Hong Kong and the Ethics Committee of FOXF2 knockdown Zhejiang University. This study was carried out in accordance Cells were transfected with FOXF2 siRNA (RiboBio) or con- with the Declaration of Helsinki of the World Medical trol siRNA using Lipofectamine 2000 (Thermo Fisher Scientif- Association. ic). The sequences of the siRNA used are listed in Supplemen- tary Table S1. Cell lines Gastric cancer cell lines (AGS, HGC27, MKN1, MKN28, Semiquantitative PCR and quantitative real-time PCR analyses MKN45, MKN74, NCI-N87, SNU719), L cell, L Wnt-3a cell were Semiquantitative PCR was performed by AmpliTaq Gold DNA purchased from the ATCC and MGC803 was purchased from polymerase (Applied Biosystems; Thermo Fisher Scientific). Chinese Academy of Sciences Cell Bank (CAS Cell Bank, Beijing, Quantitative real-time PCR was performed by SYBR Green PCR China). 293ft cell line was purchased from Invitrogen (Thermo Master Mix (Applied Biosystems; Thermo Fisher Scientific) on fi Fisher Scienti c). Cell lines were maintained according to the 7500HT Fast Real-Time PCR System (Applied Biosystems; protocols from ATCC. All cell lines were obtained between 2013 Thermo Fisher Scientific). Primer sequences are listed in Supple- fi and 2015 and cell identities were con rmed by short tandem mentary Table S1. repeat profiling. Routine Mycoplasma testing was performed by PCR. Cells were grown for no more than 25 passages in total for Western blotting any experiment. Proteins were separated on 12% SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare). Blots were fi fi Genome-wide pro ling by Illumina In nium Human- immunostained with primary antibody and secondary antibody. Methylation450 arrays Independent experiments were performed at least twice. The Genomic DNA samples isolated from three gastric cancer cell antibodies used and their dilutions are listed in Supplementary lines (AGS, MKN45, MGC803), normal gastric cell line GES1, and Table S2. two normal stomach tissues were employed for promoter methylation analysis by the Infinium HumanMethylation450 Bead- Chip array (Illumina). Cell viability and colony formation After 24 hours of transfection, cells were seeded for 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- Analysis of FOXF2 single-gene signatures in TCGA cohort fophenyl)-2H-tetrazolium (MTS; Promega) assay to measure the To externally validate the epigenetic regulation of FOXF2 cell viability. Cells were seeded on 6-well plates. After five days, gene expression in gastric cancer, we analyzed publicly avail- cells were fixed with 70% ethanol for 10 minutes and stained able datasets of methylome profiling on the Infinium DNA with 0.5% crystal violet solution for 10 minutes. Colony with Methylation Array [Stomach Adenocarcinoma (STAD) Methy- more than 50 cells per colony was counted. The experiment was lation450k and Methylation27k datasets] and transcriptome conducted in three independent triplicates. sequencing on the Illumina HiSeq 2000 platform provided by TheCancerGenomeAtlas(TCGA)multicenterproject.IDAT files and raw mRNA sequence counts were obtained from the Cell-cycle assay and apoptosis assay fi TCGA Data Portal (https://gdc.cancer.gov/). Transfected cells were xed in 70% ethanol and stained with PI/RNase Staining Buffer (BD Biosciences). The cells were sorted Bisulfite genomic sequencing by FACSCalibur system (BD Biosciences) and analyzed by Flowjo DNA BGS analysis was performed as described previously (16). 7.6 software (BD Biosciences).Transfected cells were stained with Ten CpG sites spanning from 1037 to 836 bp relative to the Annexin V-APC (BD Biosciences) and 7-aminoactinomycin transcription start site (TSS) were evaluated. Primer sequences for (7-AAD). The cells were sorted by FACSCalibur system and – BGS are listed in Supplementary Table S1. Annexin V positive cells were counted as apoptotic cells.

Treatment with 5-aza-20-deoxycytidine, Trichostatin A, MG132, Immunocytochemistry and chloroquine Cells were transfected with the indicated plasmids and stained Cell lines were treated with demethylation reagent 5-aza-20- with primary antibody and secondary antibody. Images were fl deoxycytidine (5-Aza; 2 mmol/L, Sigma-Aldrich) for 5 days. The captured by uorescent microscopy. The antibodies used and 5-Aza was replenished every day. Some cell lines were further their dilutions are listed in Supplementary Table S2. treated with histone deacetylase inhibitor Trichostatin A (TSA, 300 nmol/L) for additional 24 hours. Cells were treated with Immunoprecipitation assay cell-permeable proteasome inhibitor MG132 (Millipore) and Cells were chemically cross-linked by formaldehyde and son- chloroquine (Sigma-Aldrich) for 12 hours before harvesting for icated at 4 C. Cell was then cleared by centrifugation and super- Western blot or immunoprecipitation assays. natant was incubated overnight at 4 C with 100 mL of Protein G magnetic beads that had been preincubated with 10 mg of the Construct wild-type and deletion of FOXF2 (DFOXF2) appropriate antibody for at least 3 hours. Bound complexes were Human FOXF2 (NM_001452) ORF Clone (RC212020) eluted from the beads by heating at 65C and cross-linking was was purchased from Origene. The deletion of FOXF2 (DFOXF2) reversed by overnight incubation at 65C. Immunoprecipitated (N-terminal deletion of residues 1–200) was amplified by PCR. DNA was purified and used for gene-specific PCR.

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Dual-luciferase reporter assay The bisulfite sequencing result was positively correlated with two Cells in a 24-well plate were cotransfected with luciferase CpG probe values (Spearman r ¼ 0.66, P < 0.05, Supplementary reporter plasmid, and pRL-TK control vector by Lipofectamine Fig. S2B; Supplementary Table S3). In addition, the promoter 2000. Plasmids of FOXF2, DFOXF2, b-catenin wild-type (WT), methylation level was significantly higher in gastric cancer cell b-catenin (S33Y), WNT1, or empty vector were cotransfected as lines compared with normal gastric tissues (P < 0.05; Fig. 1B). In indicated per well. After 24 hours of transfection, luciferase addition, treatment with demethylation agent 5-Aza and histone activities were analyzed by Dual-Luciferase Reporter Assay System deacetylase inhibitor TSA restored FOXF2 expression in all gastric (Promega) and normalized to the control Renilla. cancer cell lines (Fig. 1C). We next analyzed FOXF2 expression in paired gastric cancer In vivo subcutaneous xenograft models and adjacent normal tissues. FOXF2 mRNA expression was MKN45 cells (1 107 cells in 0.1-mL PBS) stably transfected significantly downregulated in 40 gastric cancer tumors as with FOXF2 expression vector or empty vector were injected compared with their adjacent normal tissues by real-time PCR subcutaneously into the dorsal right flank of 4-week-old female (Fig. 1D, left). The downregulation of FOXF2 was validated Balb/c nude mice (n ¼ 5 per group). Tumor diameter was independently in TCGA cohort (Fig. 1D, right). Moreover, measured every 3 days for 3 weeks. Tumor volume (mm3) was FOXF2 expression was negatively correlated with promoter estimated by measuring the longest and shortest diameters of the methylation of gastric cancer tissues in Hong Kong cohort (N tumor and calculating as described previously (16). The excised ¼ 20, Spearman r ¼0.57, P ¼ 0.0068) and in TCGA Methy- tissues were either fixed in 10% neutral-buffered formalin or snap lation450k dataset (N ¼ 348, average value of probe frozen in liquid nitrogen. Tumor sections from paraffin-embed- cg06005891 and cg03848675, Spearman r ¼0.28, P ¼ 1.7 ded blocks were used for histologic examination. All experimental 10 7; Fig. 1E). Starburst plot integrating alterations in DNA procedures were approved by the Animal Ethics Committee of the methylation and gene expression showed that FOXF2 was Chinese University of Hong Kong. hypermethylated and downregulated in gastric cancer tissues from TCGA datasets (cg03848675, Methylation450k and Statistical analysis Methylation27k datasets, Supplementary Fig. S3). Consistently, Values are expressed as mean SD. The independent Student FOXF2 protein was reduced in gastric cancer tissues compared t test was used to compare the difference between two groups. with adjacent normal tissues as determined by Western blot One-way ANOVA was used to compare the difference between analysis (P < 0.001). Proliferating cell nuclear antigen (PCNA) multiple groups. Correlation analysis (Spearman r)wasusedto protein was served as a marker of tumor tissues in the Western measure the strength of association between FOXF2 methyla- blot analysis (Fig. 1F). We further evaluated FOXF2 in 10 pairs tion and expression. The x2 test was used to compare the of gastric cancer clinical samples by IHC. FOXF2 was readily clinicopathologic characteristics of gastric cancer patients and expressed in normal gastric tissues but was reduced in cancer FOXF2 methylation. Univariate and multivariate Cox regres- counterpart (P < 0.01; Fig. 1G). sion analysis was performed to assess the prognostic value of FOXF2 methylation. Overall survival in relation to methylation Ectopic expression of FOXF2 inhibits cell growth and status was evaluated by Kaplan–Meier survival curve and log- suppresses migration and invasion of gastric cancer cells rank test. Differences with P < 0.05 were considered to be The frequent silencing of FOXF2 in gastric cancer cell lines and statistically significant. tissues suggests that FOXF2 may have a tumor-suppressive function. To test this hypothesis, we examined the effect of FOXF2 overexpression on the cell growth in AGS and MKN45, which Results showed complete silencing of FOXF2. Reexpression of FOXF2 was FOXF2 is downregulated by promoter hypermethylation in confirmed by semiquantitative PCR and Western blot analysis gastric cancer (Fig. 2A). FOXF2 markedly reduced cell viability and colony We first examined the FOXF2 expression in normal human formation ability in AGS (P < 0.01) and MKN45 (P < 0.01) as tissues and found that FOXF2 was predominantly expressed in compared with empty vector (Fig. 2A). We further investigated the gastrointestinal tract including stomach (Supplementary Fig. S1). effect of FOXF2 knockdown by two FOXF2 siRNAs in HCG27 and Compared with its readily expression in normal gastric tissues, MGC803, which have relatively high FOXF2 expression. Silencing FOXF2 was silenced in 6 of 9 gastric cancer cell lines (Fig. 1A). To of FOXF2 at mRNA and protein level in HCG27 and MGC803 was confirm whether FOXF2 expression was repressed by promoter confirmed by semiquantitative PCR and Western blot analysis methylation, methylation status of FOXF2 was evaluated by (Fig. 2B). Knockdown of FOXF2 significantly enhanced cell via- bisulfite genome sequencing (BGS) analysis in the CpG island bility (P < 0.01) and increased colony numbers in both cell lines of the promoter region covering 10 CpG sites from 1037 to (P < 0.01; Fig. 2B). 836 of the FOXF2 gene (Fig. 1B). Full or partial methylation was We next investigated the effect of FOXF2 on migration and detected in six gastric cancer cell lines (AGS, MKN28, MKN45, invasion abilities of gastric cancer cells using in vitro transwell MKN74, NCI-N87, and SNU719), which showed silencing of with or without Matrigel matrix layer. Ectopic expression of FOXF2, whereas relatively less methylation was detected in FOXF2 in AGS and MKN45 significantly suppressed cell migra- HCG27, MGC803, and MKN1, which showed FOXF2 expression tion and invasive capabilities in both cell lines (Fig. 2C). In (Fig. 1B). The BGS product covers two CpG probes of 450K array: keeping with this, Western blot analyses showed that FOXF2 cg06005891 and cg03848675. These two probes locate at the regulated the epithelial–mesenchymal transition (EMT) through promoter region of FOXF2 within 1500 bp upstream of tran- upregulation of epithelial markers (E-cadherin) and downregu- scription start site (Supplementary Fig. S2A). We also evaluated lation of mesenchymal markers (N-cadherin and Vimentin; the correlation between 450K array and bisulfite sequencing data. Fig. 2D). AGS cells are negative for E-cadherin due to the presence

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Figure 1. FOXF2 is inactivated by promoter hypermethylation in gastric cancer (GC). A, Expression of FOXF2 mRNA in gastric cancer cell lines and normal gastric epithelial cell lines was determined by semiquantitative PCR. B, BGS analysis of FOXF2 CpG island region. Dense methylation was observed in gastric cancer cell lines, but not in normal stomach tissue. C, Treatment of demethylating agent 5-aza and histone deacetylase inhibitor TSA restored FOXF2 expression in gastric cancer cell lines. D, Expression levels of FOXF2 mRNA in 40 paired primary gastric cancer tissues and TCGA gastric cancer dataset. E, Correlation analysis of the association between FOXF2 methylation and expression in 20 gastric cancer tissues and in TCGA gastric cancer datasets. F1, Expression levels of FOXF2 in 14 paired primary GC tissues. F2, Quantitative analysis of relative FOXF2 expression in adjacent normal and gastric cancer tissues is shown. G, Representative image of FOXF2 staining in clinical gastric cancer specimens by IHC. Nuclear staining of FOXF2 is indicated by red arrow. FOXF2 was reduced in 10 pairs of gastric cancer specimens compared with adjacent normal tissues by IHC analysis. Paired adjacent and normal samples from the same patient is assigned with same color.

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Figure 2. FOXF2 inhibits gastric cancer cell growth in vitro and in vivo. A, Overexpression of FOXF2 was confirmed by semiquantitative PCR and Western blot analysis. Middle, effect of FOXF2 overexpression on cell viability and colony formation. Right, quantitative analysis of colony formation efficiency (%). B, Knockdown of FOXF2 was confirmed by semiquantitative PCR and Western blot analysis. Middle, effect of FOXF2 knockdown on cell viability and colony formation. Right, quantitative analysis of colony formation efficiency (%). C, Effect of FOXF2 on metastatic ability of gastric cancer cells using in vitro migration and invasion transwell assays. Right, statistical analysis. D, Effect of FOXF2 on epithelial and mesenchymal markers. E, Expression of FOXF2 reduced the tumor growth rate and tumor weight in nude mice. F, The FOXF2 expression in the FOXF2-transfected tumors was confirmed by quantitative real- time PCR and Western blot analysis. Cell proliferation in tumors isolated from FOXF2-expressing or control nude mice xenografts was determined by Ki-67 staining. G, Effects of FOXF2 overexpression on cell cycle were determined by flow cytometry analysis. Bottom, quantitative analysis of cell proportion (%). H, Effects of FOXF2 overexpression on apoptosis was determined by flow cytometry analysis after dual staining with Annexin V-APC and 7-AAD. Bottom, quantitative analysis of apoptotic cells (%). I, Cell-cycle G1–S checkpoint signaling and apoptosis markers were analyzed by Western blot analysis.

of a truncating mutation (17). In contrast, silencing of FOXF2 in immunostaining (Fig. 2F). Taken together, our results suggest that HGC27 and MGC803 cells significantly promoted cell migration FOXF2 inhibits gastric cancer cell growth and functions as tumor and invasion (Fig. 2C), and suppressed epithelial markers (E- suppressor in gastric cancer cells. cadherin), increased mesenchymal markers (N-cadherin and Vimentin; Fig. 2D). These findings indicate that FOXF2 sup- FOXF2 induces cell-cycle arrest and apoptosis pressed the migration and invasion ability of gastric cancer. We next investigated the effect of FOXF2 on cell-cycle distri- In light of our in vitro findings, we examined the in vivo tumor- bution and apoptosis regulation by flow cytometry. FOXF2 sig- suppressive ability of FOXF2. Empty vector–transfected and nificantly increased G1 phase cells in AGS (64.1% 1.0% vs. FOXF2-transfected MKN45 cells were injected into the right flanks 69.0% 2.1%, P < 0.05) and MKN45 (54.5% 0.3% vs. 63.0% of nude mice, respectively. FOXF2 significantly suppressed the 3.2%, P < 0.01) but decreased S-phase cells in AGS (13.2% 1.5% growth of MKN45 xenografts in nude mice (Fig. 2E). The average vs. 8.2% 2.5%, P < 0.05) and in MKN45 (13.7% 0.5% vs. tumor weight of MKN45-FOXF2 tumors was reduced by 59% 8.7% 1.1%, P < 0.01; Fig. 2G). In keeping with this, FOXF2 compared with controls (0.179 0.136 g vs. 0.434 0.068 g, reduced the protein expression of the G1–S transition promoter P < 0.01, Fig. 2E). FOXF2 expression in the xenografts was cyclin D1, CDK4, and PCNA but enhanced the G1 gatekeepers confirmed by quantitative real-time PCR and Western blot anal- p27 and p21. In addition, ectopic expression of FOXF2 induced ysis (Fig. 2F). FOXF2-expressing xenografts displayed significantly cell apoptosis in AGS (2.73% 0.5% vs. 45.9% 8.2%, P < 0.01) reduced proliferative activity compared with controls by Ki-67 and MKN45 (35.7% 1.2% vs. 43.1% 2.7%, P < 0.05) by flow

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cytometry following Annexin V-APC and 7-aminoactinomycin 2–65). LEF1(DN) loses the b-catenin binding site but well pre- (7-AAD) double staining (Fig. 2H). This was confirmed by serves the DNA binding sequencing and therefore it cannot enhanced the protein levels of key cell apoptosis regulators response to Wnt upstream signaling. TOPflash luciferase activity cleaved caspase-3, cleaved caspase-7, and cleaved PARP (Fig. indicated that FOXF2 significantly suppressed TOPflash reporter 2I). These findings suggest that apoptosis in conjunction with activity, whereas overexpression of LEF1(DN) relieved this effect cell-cycle arrest, as induced by FOXF2, accounts for the growth (Supplementary Fig. S7). inhibition in FOXF2-expressing tumor cells. Given that FOXF2 suppressed Wnt downstream targets but failedtocauseanychangesonb-catenin at mRNA level, we next FOXF2 inhibits Wnt signaling pathway by reducing b-catenin investigated whether FOXF2 regulated the abundance of b-cate- protein nin protein. Ectopic expression of wild-type FOXF2 dose- FOXF2 is a transcription factor that contains a bipartite nuclear dependently decreased total b-catenin protein levels in AGS localization signal (NLS) within a DNA-binding domain known and 293ft cells, whereas DFOXF2 had little effect on b-catenin as the forkhead domain (18). To confirm the localization and protein (Fig. 4A). We also observed that FOXF2 significantly molecular mechanism of FOXF2, we generated the deletion of suppressed b-catenin protein in MKN45, whereas silencing of FOXF2 (DFOXF2), which lacks the 2 NLS in the N-terminal FOXF2 in HGC27 and MG803 upregulated b-catenin protein (amino acids 1–200; Fig. 3A). Western blot and immunocyto- (Fig. 2D). Given that FOXF2 suppressed Wnt signaling activity, chemistry showed that wild-type FOXF2 protein was located in we next examined whether FOXF2 could interrupt the interac- the nucleus, whereas DFOXF2 localized exclusively in cytoplasm tion between b-catenin and its cofactor LEF1 in the nucleus. We (Fig. 3A). coexpressed Flag-FOXF2, Flag-b-catenin, and hemagglutinin To understand the molecular basis of the tumor suppressive (HA)-LEF1 in 293ft cells, and immunoprecipitated HA-LEF1 property of FOXF2, we performed luciferase reporter screening in cell nuclear lysates with anti-HA antibodies. Western blot assays to assess the effect of FOXF2 on five signaling pathways analysis of the precipitates with an anti-FLAG antibody indi- including Wnt, p53/p21, STAT3, AP-1, and MAPK/ERK. Ectopic cated that FOXF2 reduced nuclear b-catenin binding with LEF1 expression of FOXF2 significantly repressed the activity of Wnt protein (Fig. 4B). We next analyzed the membrane, cytosolic, signaling as demonstrated by TOPflash luciferase reporter and nuclear amount of endogenous b-catenin in FOXF2 trans- (Fig. 3B), but not other pathways (Supplementary Fig. S4). The fected AGS and 293ft cells. Compared with the control cell inhibition effect of wild-type FOXF2 on TOPflash reporter became lines,themembraneamountofb-catenin was unchanged, more dramatically in the presence of WNT1 stimulation in AGS whereas the cytosolic and nuclear amount of b-catenin was and MKN45 (Fig. 3B). However, deletion of the forkhead domain substantially decreased in the FOXF2-transfected cell lines (Fig. of FOXF2 (DFOXF2) abolished the suppressive effect on TOPflash 4C). Moreover, immunofluorescence staining also showed that reporter in AGS (Fig. 3B), suggesting the nuclear localization of when cells were cotransfected with FOXF2 and b-catenin, FOXF2 was functional critical. Conversely, FOXF2 knockdown by FOXF2 reduced the b-catenin protein compared with untrans- two siRNAs in HCG27 and MGC803 increased the reporter fected cells (Supplementary Fig. S8). activity stimulated by WNT1 and b-catenin, respectively (Fig. 3B). Previous studies indicated that hypereactivated Wnt FOXF2 reduces b-catenin protein through ubiquitination– signaling is critical for gastric cancer cell growth (19, 20). We also proteasome pathway found that Porcn inhibitor IWP2 significantly inhibited cell Given that FOXF2 suppressed b-catenin at protein level rather growth in four gastric cancer cell lines (AGS, MKN45, HGC27, than at mRNA level, we then assessed whether FOXF2 regulated and MGC803) with IC50 3–4 mmol/L by MTT assay. On the other the b-catenin protein stability. We measured the half-life of Flag- hand, we measured the gastric cancer cell growth treated by Wnt- b-catenin following inhibition of new protein synthesis by cyclo- 3A conditioned medium (CM). Treatment of Wnt-3A CM signif- hexamide (CHX). Indeed, the half-life of b-catenin was dramat- icantly promoted the cell growth of AGS, MKN45, HGC27, and ically decreased in FOXF2-transfected cells within 3 hours com- MGC803 compared with the control group (Supplementary pared with the control cells (Fig. 4D). The ubiquitin–proteasome Fig. S5A and S5B). Moreover, FOXF2 significantly inhibited Wnt system and autophagy are two major mechanisms that mediate target genes cyclin D1 and c-myc in both mRNA and protein protein degradation. To further determine the molecular mech- levels, but not change b-catenin mRNA level (Fig. 3C), suggesting anism of FOXF2-induced downregulation of b-catenin, we FOXF2 negatively regulated Wnt signaling pathway in gastric detected b-catenin protein level in the absence or presence of the cancer cells at posttranscriptional level. In addition, we performed proteasome inhibitor MG132 and autophagy inhibitor chloro- Wnt signaling pathway RT2 Profiler PCR array and identified a quine. We treated AGS and 293ft cells stably expressing FOXF2 or group of Wnt target genes that were also downregulated in gastric control vector with MG132 (5 mmol/L and 10 mmol/L) and cancer cell line AGS upon FOXF2 overexpression (Supplementary chloroquine (15 mmol/L and 30 mmol/L) for 12 hours, respec- Fig. S6). We further validated these genes in AGS and MKN45 by tively. MG132 blocked FOXF2-induced degradation of b-catenin. real-time PCR upon stimulated with Wnt3a conditioned medium In contrast, we did not observe restoration of b-catenin in cells or L control medium. Overexpression of FOXF2 significantly treated with chloroquine (Fig. 4E). These results suggested that suppressed mRNA expression of Wnt target genes (Axin2, c-myc, FOXF2-induced degradation of b-catenin is mediated mainly cyclin D1, LEF1, and MET). Treatment with Wnt3a for 2 and through a proteasome pathway. 4 hours significantly stimulated the expression of these Wnt To further support the idea that FOXF2 induced b-catenin targets, whereas these gene inductions were abrogated by FOXF2, degradation via an ubiquitin–proteasome pathway, we tran- suggesting that these genes are direct Wnt targets regulated by siently transfected cells with FOXF2 along with HA-tagged FOXF2 (Fig. 3D). To prove the effect of FOXF2 is LEF1 dependent, wild-type ubiquitin or control vector. As speculated, we constructed an N terminal LEF1 deletion (D amino acids FOXF2 increased the level of ubiquitination of b-catenin

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Figure 3. FOXF2 inhibits Wnt signaling pathway. A, Schematic figure summarizing the forkhead domains of FOXF2 and deletion of FOXF2 (DFOXF2). DFOXF2 lacks the 2 nuclear localization signal (NLS) in the N-terminal. The cytosolic and nuclear quantities of FOXF2 and DFOXF2 in AGS cells were determined by Western blot analysis and immunocytochemistry. Cells were transfected with Flag-FOXF2 and stained with anti-Flag (green). B, Effect of FOXF2 overexpression on TOPflash luciferase reporter activity was determined in the presence or absence of WNT1 stimulation. Effect of FOXF2 knockdown by two types of FOXF2-siRNA on TOPflash luciferase reporter activity was determined in the presence or absence of WNT1 and b-catenin stimulation, respectively. C, Expression levels of b-catenin, cyclin D1, and c-myc in FOXF2-transfected cells were determined by quantitative real-time PCR and Western blot analysis. D, Overexpression of FOXF2 significantly suppressed Wnt target genes (Axin2, c-myc, cyclin D1, LEF1, and MET). CM, conditioned medium. detected by immunoblot after immunoprecipitation of ubiqui- b-catenin degradation by FOXF2 is independent of GSK-3b tin (Fig. 4F), which suggest that FOXF2 leads to b-catenin In the canonical Wnt pathway, phosphorylation of b-catenin polyubiquitylation. at the N-terminal aminol acids (Thr41, Ser37, and Ser33) by

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Figure 4. FOXF2 promotes b-catenin degradation via ubiquitin–proteasome pathway. A, Effect of FOXF2 and DFOXF2 overexpression on b-catenin protein was determined by Western blot analysis. Cells were cotransfected with indicated plasmid. B, The interaction with LEF1/b-catenin transcriptional complex after FOXF2 overexpression was determined by coimmunoprecipitation. Cells were cotransfected with indicated plasmid and immunoprecipitated nuclear cell lysates with anti-HA antibodies. C, Amount of endogenous b-catenin in membrane, cytosolic, and nuclear fractions was determined in FOXF2transfectedcellsbyWestern blot analysis. Na-K-ATPase, b-actin, and lamin A/C served as loading controls and cell fraction markers. Right, quantitative analysis of Western blot signal. M, membrane; C, cytosol; N, nucleus. D, The half-life of Flag-b-catenin was determined using the new protein synthesis inhibiter, cyclohexamide (CHX). s.e., short-time exposure; l.e., long-time exposure. E, MG132 blocked FOXF2-induced degradation of b-catenin. Cells were treated with MG132 or chloroquine for 12 hours. F, FOXF2 induced b-catenin degradation via an ubiquitin–proteasome pathway. Cells were transiently transfected with control vector or FOXF2 along with haemagglutinin (HA)-tagged ubiquitin. The level of b-catenin after immunoprecipitation of ubiquitin was detected by immunoblotting. Cells were treated with MG132 for 12 hours. G, FOXF2 overexpression suppressed the TOPﬂash luciferase activities in the presence of wild-type b-catenin and b-catenin S33Y mutant. H, Effect of FOXF2 on b-catenin S33Y mutant and b-catenin N-terminal deletion (D1–133) degradation was determined by Western blot analysis.

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Role of FOXF2 in Gastric Cancer

GSK-3b results in b-catenin proteasome-dependent degrada- effector downstream of FOXF2 in regulating of b-catenin tion. We then asked whether FOXF2-promoted b-catenin deg- (Fig. 5F). IRF2BPL overexpression suppressed the activity of radation required GSK-3b.WeperformedtheTOPflash lucif- Wnt signaling as demonstrated by TOPflash luciferase reporter erasereporterassayinthepresenceofwild-typeb-catenin or the (Fig.5G).Moreover,IRF2BPLsignificantly repressed the LEF1 constitutively active b-catenin S33Y mutant (b-catenin S33Y), promoter luciferase reporter activity and c-myc mRNA expres- which is insensitive to GSK-3b–mediated phosphorylation and sion (Fig. 5G). Taken together, our data revealed that FOXF2 subsequent proteasomal degradation. The transcriptional activ- induces E3 ligase IRF2BPL transcription, which in turn increas- ities of both wild-type b-catenin and b-catenin S33Y were ing b-catenin ubiquitination and degradation. significantly suppressed by FOXF2 (Fig. 4G), suggesting that We then examined whether FOXF2 directly induced IRF2BPL FOXF2 induced GSK-3b–independent degradation of b-cate- transcription. FOXF2 binding motif with a core sequence nin. This was confirmed by Western blot analysis that b-catenin "AAACA" was identified using transcription factor motif predic- S33Y was degraded by FOXF2 dose-dependently (Fig. 4H, left). tion analysis jaspar (http://jaspar.genereg.net; Fig. 5H). We then Consistent with this observation, b-catenin (D1–133), a stabi- performed a chromatin immunoprecipitation-PCR assay (ChIP- lized form of b-catenin owing to deletion degradation domain PCR) by using 10 sets of primers coving the promoter region of in the N terminal, was significantly reduced in the presence IRF2BPL form 3000 to þ1500 (Fig. 5H, left). ChIP-PCR dem- of FOXF2 (Fig. 4H, right). To further validate the observation onstrated that FOXF2 could bind to the IRF2BPL promoter region that FOXF2 degraded b-catenin independent of GSK-3b activity, at least four sites (Fig. 5H, right). Meanwhile, we also performed GSK-3b was silenced by two siRNAs (Supplementary Fig. S9A). ChIP-PCR analysis and found that FOXF2 could bind to the Knockdown of GSK-3b did not change the effect of FOXF2 on promoter region of KLHL22 and ASB2, suggesting that FOXF2 b-catenin protein degradation in AGS and 293ft (Supplemen- transcriptional activates KLHL22 and ASB2 (Supplementary Fig. tary Fig. S9A). Similar results were observed when cells were S12; Supplementary Table S1). Furthermore, we cloned the pro- treated with selective GSK3 inhibitor Lithium chloride (LiCl; moter region (2700 to TSS) into pGL3-basic plasmid and Supplementary Fig. S9B). Collectively, these findings suggested performed a luciferase activity assay. Wild-type FOXF2 but not that FOXF2-induced b-catenin degradation is independent of the mutant DFOXF2 significantly activated the luciferase reporter, GSK-3b. suggesting that FOXF2 activated IRF2BPL transcription (Fig. 5I). This was further confirmed by using the pGL3-basic plasmid FOXF2 directly upregulates E3 ligase IRF2BPL transcription harboring the 12-bp core "AAACA" motifs (Supplementary Fig. for b-catenin ubiquitination degradation S13). H3K27Ac has been identified as an active regulatory histone Given FOXF2 is a transcriptional factor and lacks of the modification marker. To further confirm FOXF2 binds and acti- ability to directly ubiquitinize b-catenin, we hypothesize that vates IRF2BPL transcription, we carried out H3K27Ac ChIP-qPCR FOXF2 transcriptional upregulates an E3 ligase that targets to evaluate IRF2BPL promoter activity. FOXF2 significantly b-catenin for proteasome-dependent degradation. Previous increased the level of H3K27Ac in the promoter of IRF2BPL, studies suggested that CBL, BTRC, JADE1, and TRIM33 were suggesting that FOXF2 positively regulated IRF2BPL gene tran- ubiquitin E3 ligases targeting b-catenin (21–24). However, scription (Fig. 5J, left). Western blot analysis also showed that none of the genes were upregulated upon FOXF2 overexpres- ectopic expression of FOXF2 increased IRF2BPL protein level (Fig. sion (Supplementary Fig. S10). To identify the E3 ligase reg- 5J, right). In addition, IRF2BPL mRNA expression was signifi- ulated by FOXF2 and responsible for b-catenin degradation, we cantly downregulated in human gastric cancer tissues compared performed a Human Ubiquitin Ligases RT2 Profiler PCR Array with the adjacent normal tissues (Fig. 5K). IRF2BPL mRNA covering 370 ubiquitin ligase genes. With a cut-off value of showed a positive correlation with FOXF2 in gastric cancer in 1.5-fold change, 36 genes were upregulated and 5 genes were our Chinese cohort (Spearman r ¼ 0.42, P < 0.05) and TCGA downregulated upon FOXF2 overexpression (Fig. 5A). The PCR cohort (Spearman r ¼ 0.38, P < 0.001; Fig. 5K). These findings array result was further validated by real-time PCR and three collectively suggested that FOXF2 directly upregulates E3 ligase genes (IRF2BPL, KLHL22,andASB2)wereconfirmed to be IRF2BPL transcription for b-catenin ubiquitination degradation upregulated at mRNA level upon FOXF2 overexpression (Fig. 5L). (Fig. 5A). Although all the 3 E3 ligases suppressed TOPflash luciferase activity, only IRF2BPL, but not ASB2 or KLHL22, FOXF2 methylation is associated with poor survival of gastric significantly reduced the b-catenin protein level (Fig. 5B; Sup- cancer patients plementary Fig. S11A and S11B). FOXF2 significantly induced We evaluated FOXF2 methylation in 103 primary gastric cancer IRF2BPL mRNA in AGS and MKN45, whereas silence of FOXF2 tissues by BGS. Methylated FOXF2 was detected in 50.5% of in HGC27 and MG803 suppressed IRF2BPL mRNA (Fig. 5A). primary GCs (52 of 103). We next determined the association Ectopic expression of IRF2BPL increased endogenous b-catenin between FOXF2 methylation and clinicopathologic features such ubiquitination (Fig. 5C). Conversely, knockdown of IRF2BPL as age, gender, differentiation, lymph node metastasis, and significantly reduced endogenous b-catenin ubiquitination lev- tumor–node–metastasis (TNM) stage, but no correlations were el (Fig. 5D). Moreover, immunoprecipitation assay showed found (Supplementary Table S4). However, FOXF2 methylation that IRF2BPL directly interact with b-catenin (Fig. 5E). To was associated with an increased risk of cancer-related death by further confirm this, we performed a rescue assay by using two univariate Cox regression (RR, 2.47 (95% CI, 1.39–4.47), P ¼ siRNAs targeting IRF2BPL in two gastric cancer cell lines to rule 0.012; Table 1). As expected, the TNM stage was also a significant out the off-target effect and cell-specific effect. Overexpression prognostic factor (P < 0.01). In particular, after adjustment for of FOXF2 significantly downregulated b-catenin protein and potential confounding factors, FOXF2 methylation was found to suppressed its activation, whereas depletion of IRF2BPL abro- be an independent risk factor for shortened survival in patients gated the effect, suggesting that IRF2BPL is an important with gastric cancer by multivariate Cox regression analysis

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Role of FOXF2 in Gastric Cancer

Table 1. Univariate and multivariate Cox regression analysis of potential poor prognostic factors in 103 gastric cancer patients from China cohort and 243 recurrence-free gastric cancer patients from TCGA cohort China cohort Univariate Multivariate Variable RR (95% CI) P RR (95% CI) P Age 1.00 (0.98–1.02) 0.998 0.99 (0.97–1.02) 0.780 Gender Male 1.41 (0.66–3.02) 0.372 1.81 (0.82–4.05) 0.144 Female 1.00 1.00 Differentiation Poor 0.93 (0.62–1.41) 0.843 0.96 (0.52–1.78) 0.898 Moderate/well 1.00 1.00 TNM stage I 0.05 (0.01–0.39) 0.001 0.05 (0.01–0.39) 0.005 II 0.15 (0.06–0.34) 0.004 0.13 (0.05–0.32) 0.001 III 0.32 (0.14–0.73) 0.007 0.25 (0.10–0.61) 0.002 IV 1.00 1.00 FOXF2 methylation Methylated 2.47 (1.39–4.47) 0.012 1.90 (1.05–3.48) 0.035 Unmethylated 1.00 1.00 TCGA cohort Univariate Multivariate Variable RR (95% CI) P RR (95% CI) P Age 1.04 (0.99–1.08) 0.067 1.06 (1.01–1.10) 0.016 Gender Male 0.98 (0.45–2.15) 0.966 0.76 (0.34–1.69) 0.495 Female 1.00 1.00 Differentiation Poor 5.16 (1.21–21.9) 0.026 4.61 (1.05–20.1) 0.042 Moderate/well 1.00 1.00 TNM stage I 0.11 (0.22–0.54) 0.007 0.08 (0.01–0.41) 0.003 II 0.12 (0.03–0.44) 0.001 0.09 (0.02–0.35) 0.001 III 0.33 (0.13–0.86) 0.023 0.22 (0.08–0.65) 0.006 IV 1.00 1.00 FOXF2 methylation Methylated 1.99 (1.22–3.28) 0.006 4.57 (1.66–12.6) 0.003 Unmethylated 1.00 1.00

[RR, 1.90 (95% CI, 1.05–3.48), P ¼ 0.035; Table 1)]. Kaplan– stage of gastric cancer patients from TCGA cohort (based on Meier survival curves showed that gastric cancer patients with average value of probe cg06005891, cg04187121, cg12611423, FOXF2 methylation had significantly poorer overall survival than and cg19519310; Fig. 6B; Table 1). These findings indicated that patients without methylation based on the log-rank test (P < FOXF2 promoter hypermethylation predicts a poor prognosis 0.05). After stratification by TNM stage, FOXF2 methylated in patients with gastric cancer, especially in the early stages. patients had significantly shorter survival in stages I–II (P < 0.05) but not in stages III–IV (P < 0.05; Fig. 6A). TCGA cohort of 243 recurrence-free gastric cancre patients verified the prog- Discussion nostic significance of FOXF2. FOXF2 promoter methylation In this study, we first demonstrated that FOXF2 was generally was also an independent predictor of poor survival at the early expressed in normal human stomach tissues, but frequently

Figure 5. IRF2BPL is transcriptionally upregulated by FOXF2 and targets b-catenin for degradation. A, Screening and validation of potential b-catenin E3 ligases by Human Ubiquitin Ligases RT2 Profiler PCR Array and real-time PCR. B, Overexpression of IRF2BPL did not affect b-catenin mRNA level but significantly suppressed b-catenin protein level. C, b-catenin ubiquitination level upon IRF2BPL overexpression was detected by immunoprecipitation. Indicated plasmid was transfected into AGS cells. D, siIRF2BPL knockdown efficiency was analyzed by real-time PCR. Silence of IRF2BPL did not affect b-catenin mRNA level. b-Catenin ubiquitination level upon knockdown of IRF2BPL was detected by immunoprecipitation. Indicated plasmid and siRNA were transfected into AGS cells. E, Flag-b-catenin interacted with endogenous IRF2BPL. Flag-tagged b-catenin was expressed in AGS cells. Cell lyses were immunoprecipitated by an anti-FLAG antibody antibody. The immunoprecipitates were subjected to Western blots using the indicated antibodies. F, Depletion of IRF2BPL abrogated the effect of FOXF2 in AGS and MKN45. G, Overexpression of IRF2BPL suppressed TOPflash and LEF1-luciferase activity, and suppressed c-myc mRNA level. H, Schematic figure summarizing FOXF2 ChIP-PCR primer sets and H3K27Ac ChIP-PCR primer sets in the IRF2BPL promoter region. FOXF2 bind on IRF2BPL promoter region (region #3, #4, #6, and #7). Putative binding motif of FOXF2 was generated using online software (http://jaspar.genereg.net/cgi- bin/jaspar_db.pl). I, Wild-typeFOXF2butnotthemutantDFOXF2 activated the IRF2BPL promoter luciferase reporter activity. J, H3K27Ac level was increased in the promoter region of IRF2BPL upon FOXF2 overexpression. Overexpression of FOXF2 upregulated IRF2BPL protein level. K, IRF2BPL was significantly downregulated in human gastric cancer tissues compared with the adjacent normal tissues. FOXF2 mRNA level positively correlated with IRF2BPL mRNA level in 30 gastric cancer patients and in TCGA dataset. L, Proposed mechanistic scheme of FOXF2 inducing b-catenin degradation and suppressing the Wnt signaling pathway in gastric cancer.

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Figure 6. FOXF2 methylation is an independent predictor of poor survival at the early stage of gastric cancer patients. A, Kaplan–Meier curves of gastric cancer patients, in TNM stages I–II, and in TNM stages III–IV based on the log-rank test. B, Kaplan–Meier curves of TCGA cohort, in TNM stages I–II, and in TNM stages III–IV based on the log-rank test.

silenced in gastric cancer cell lines and primary gastric cancer previous study also indicated that FOXF2 deficiency induced the tumor tissues. We showed that the silencing of FOXF2 was epithelial–mesenchymal transition (EMT) of basal-like breast regulated by promoter hypermethylation. In keeping out our cells (29). These results strongly suggest that FOXF2 acts as a identification, hypermethylated CpG island of FOXF2 promoter novel tumor suppressor in gastric cancer. was also reported in leukemia, breast cancer, colorectal cancer, Luciferase reporter assay demonstrated that FOXF2 could and kidney cancer (5, 25). Demethylation treatment by DNA suppress the TOPflash activity in gastric cancer cells (Fig. 3). methyltransferase inhibitor 5-Aza successfully restored the ex- Formation of LEF1/b-catenin complex is a prerequisite for the pression of FOXF2. 5-Aza (decitabine) incorporates into DNA transcription of Wnt target gene and its formation is mainly strands upon replication and irreversibly blocks DNA methyl- regulated by b-catenin protein levels (30). b-Catenin was greatly transferases function. 5-Aza has been under clinical study as an reduced by FOXF2 without alteration of b-catenin mRNA anticancer treatment since 1960s. However, the effect of 5-aza expression. These results indicated that FOXF2 reduces b-cate- alone on solid tumor is lower than conventional therapy. Recent nin protein expression through posttranscriptional mecha- clinical trials are conducted to test the role of 5-aza in various nism. In the absence of Wnt stimulation, cytoplasmic b-catenin combinations for intensification chemotherapy (epirubicin, cis- is constitutively phosphorylated via GSK-3b. Phosphorylated platin, fluorouracil) and Immunotherapy (Nivolumab) in solid b-catenin is ubiquitinated and degraded by the proteasome (30, tumors (https://clinicaltrials.gov/). TSA is a selective inhibitor of 31). Our results showed that proteasome inhibitor MG132 the class I and II histone deacetylase (HDAC) families. Previous could block FOXF2-induced degradation of b-catenin and studies indicated that TSA showed an antiproliferation effect in ubiquitination assay confirmed that FOXF2 caused b-catenin gastric cancer cells and xenograft nude mice model (26, 27). polyubiquitylation. Thus, FOXF2 induced b-catenin degrada- Although TSA has not been tested in a clinical setting, vorinostat, tion through ubiquitin–proteasome pathway. Moreover, we a pan-HDAC inhibitor structurally similar to TSA, has been examined whether FOXF2-induced b-catenin degradation was proved by FDA for the treatment of relapsed and refractory GSK-3b independent. Overexpression of FOXF2 increased cutaneous T-cell lymphoma (28). b-catenin S33Y degradation and suppressed the activity of Wnt A series of in vitro and in vivo functional experiments revealed signaling induced by b-catenin S33Y, which is GSK-3b degra- that FOXF2 possesses a tumor-suppressive function in gastric dation-resistant form of b-catenin. We also validated our con- cancer. The overexpression of FOXF2 could suppress the cell clusion by using GSK-3b siRNA and inhibitor. Therefore, these growth in two gastric cancer cell lines in vitro and in nude mice findings suggested that FOXF2 interrupted the interaction tumorigenesis in vivo; while the knockdown of FOXF2 promoted between LEF1 and b-catenin in the nucleus. FOXF2 promoted the cell growth. FOXF2 suppressed gastric cancer cell growth was b-catenin degradation via ubiquitin–proteasome pathway in mediated by inhibiting G1–S cell-cycle transition and inducing GSK-3b–independent fashion. cell apoptosis (Fig. 2). FOXF2 suppressed the migration and Previous studies suggested that CBL, BTRC, JADE1, and TRIM33 invasion ability of gastric cancer. In keeping with our finding, were ubiquitin E3 ligases targeting b-catenin. However, none of

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Role of FOXF2 in Gastric Cancer

these E3 ligases were upregulated upon FOXF2 expression, sug- patients with TNM stage I/II gastric cancer, its role remains gesting a novel mechanism contributed to b-catenin degradation. controversial due to the lack of data showing a definite benefit On the basis of Human Ubiquitin Ligases RT2 Profiler PCR Array in this group of patients. Thus, additional prognostic biomar- and subsequent validation assays, IRF2BPL was identified as the kers may provide better risk assessment that can guide person- most promising E3 candidate for regulating the abundance of alized chemotherapy. Promoter methylation has been reported b-catenin. Indeed, overexpression of IRF2BPL significantly as a promising predictive biomarker in gastric cancers (32–34). reduced b-catenin protein without alteration of its mRNA expres- Our results suggest that FOXF2 hypermethylation may serve as sion. Moreover IRF2BPL interacted with b-catenin and signifi- a new prognostic marker for patients with early gastric cancer. cantly increase b-catenin ubiquitination. Future researches are In conclusion, we have identified a novel tumor suppressor needed to decipher the physical interplay of IRF2BPL and b-cate- FOXF2, which is commonly inactivated by promoter methylation nin and other protein factors potentially being involved in the in gastric cancer. FOXF2 suppresses gastric cancer growth by regulation process. As FOXF2 is a nuclear transcriptional factor, inhibiting Wnt signaling through FOXF2–IRF2BPL–b-catenin the direct relationship between FOXF2 and IRF2BPL was exam- axis. FOXF2 directly binds to E3 ligate IRF2BPL promoter and ined. FOXF2 binding motif was predicted and eight sites with upregulates IRF2BPL. IRF2BPL then interacts with b-catenin for its DNA binding sequence of "AAACA" were identified in the ubiquitination and degradation. FOXF2 methylation is associated IRF2BPL promoter region. ChIP-PCR assay confirmed FOXF2 with shorter survival in gastric cancer patients and may serve as a could bind to at least four IRF2BPL promoter region (#3, #4, prognostic biomarker especially for early stage of gastric cancer #6, #7). IRF2BPL promoter luciferase assay and H3K27Ac ChIP patients. assay further indicated that FOXF2 positively regulated IRF2BPL expression. Of note, IRF2BPL may not be the only E3 ligase or Disclosure of Potential Conflicts of Interest pathway that mediating b-catenin inhibition. KLHL22 and ASB2 No potential conflicts of interest were disclosed. significantly suppressed TOPflash reporter activity, suggesting that at least these two E3 ligases may also played a negative role Authors' Contributions in regulating of Wnt signaling pathway. Conception and design: Y. Dong, J. Yu Taken together, we revealed a novel FOXF2-IRF2BPL-b-catenin Development of methodology: Y. Dong, Y. Zhang signaling axis in suppressing Wnt signaling activity in gastric Acquisition of data (provided animals, acquired and managed patients, cancer. FOXF2 transcriptionally binds and upregulates E3 ligase provided facilities, etc.): A. Higashimori, Y. Dong, Y. Zhang, W. Kang, J. Yu b Analysis and interpretation of data (e.g., statistical analysis, biostatistics, IRF2BPL, which in turn interacts with -catenin for its ubiquitina- computational analysis): A. Higashimori, Y. Dong, G. Nakatsu tion and degradation. FOXF2 also interrupts the interaction Writing, review, and/or revision of the manuscript: A. Higashimori, Y. Dong, between LEF1 and b-catenin in the nucleus. These effects of FOXF2 Y. Zhang, S.S.M. Ng, J. Yu contribute to the inhibition of Wnt signaling activity to suppress Administrative, technical, or material support (i.e., reporting or organizing gastric cancer cell growth (Fig. 5L). data, constructing databases): Y. Dong, J.J.Y. Sung, J. Yu We finally investigated the clinical importance of FOXF2 Study supervision: T. Arakawa, J.J.Y. Sung, F.K.L. Chan, J. Yu methylation in 103 gastric cancer patients and found that Acknowledgments 50.5% of them had FOXF2 promoter hypermethylation. FOXF2 This project was supported by research funds from RGC GRF Hong Kong methylation was an independent risk factor of poor survival in (472613, 14106415, 14111216 to J. Yu), HMRF Hong Kong (1195728 to J. Yu), patients with gastric cancer by multivariate Cox regression 135 program project (2016YFC1303200 to F.K.L. Chan), Shenzhen Virtual analysis (Table 1). The disease-free survival of patients with University Park Support Scheme to CUHK-SZRI (to J. Yu), and National Natural FOXF2 methylation was significantly shorter than that of other Science Foundation of China (81502436 to Y.J. Dong). patients with gastric cancer (Fig. 6A) by Kaplan–Meier survival curve analysis. In particular, methylation was significantly The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked associated with shorter survival for patients with stage I/II advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate gastric cancer (Fig. 6A). The clinical outcome of gastric cancer this fact. varies greatly depending on the aggressiveness of individual tumors. Many patients experience disease recurrence following Received August 10, 2017; revised December 26, 2017; accepted January 22, radical surgery. Although adjuvant chemotherapy may benefit 2018; published OnlineFirst January 26, 2018.

References 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, 4. Zhao J, Liang Q, Cheung KF, Kang W, Lung RW, Tong JH, et al. Genome- et al. Cancer incidence and mortality worldwide: sources, methods wide identification of Epstein-Barr virus-driven promoter methylation and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136: profiles of human genes in gastric cancer cells. Cancer 2013;119:304–12. E359–86. 5. Dunwell T, Hesson L, Rauch TA, Wang L, Clark RE, Dallol A, et al. A 2. Allemani C, Weir HK, Carreira H, Harewood R, Spika D, Wang XS, genome-wide screen identifies frequently methylated genes in hae- et al. Global surveillance of cancer survival 1995–2009: analysis matological and epithelial cancers. Mol Cancer 2010;9:44. of individual data for 25,676,887 patients from 279 population- 6. Myatt SS, Lam EW. The emerging roles of forkhead box (Fox) proteins in based registries in 67 countries (CONCORD-2). Lancet 2015;385: cancer. Nat Rev Cancer 2007;7:847–59. 977–1010. 7. Hellqvist M, Mahlapuu M, Blixt A, Enerb€ack S, Carlsson P. The human 3. Otani K, Li X, Arakawa T, Yu J. Epigenetic-mediated tumor suppressor genes forkhead protein FREAC-2 contains two functionally redundant activa- as diagnostic or prognostic biomarkers in gastric cancer. Expert Rev Mol tion domains and interacts with TBP and TFIIB. J Biol Chem 1998; Diagn 2013;13:445–55. 273:23335–43.

www.aacrjournals.org Cancer Res; 78(7) April 1, 2018 1655

Higashimori et al.

8.WangT,TamakoshiT,UezatoT,ShuF,Kanzaki-KatoN,FuY,etal. 21. Chitalia V, Shivanna S, Martorell J, Meyer R, Edelman E, Rahimi N. c-Cbl, a Forkhead transcription factor Foxf2 (LUN)-deficient mice exhibit ubiquitin E3 ligase that targets active b-catenin: a novel layer of Wnt abnormal development of secondary palate. Dev Biol 2003;259: signaling regulation. J Biol Chem 2013;288:23505–17 83–94. 22. Winston JT, Strack P, Beer-Romero P, Chu CY, Elledge SJ, Harper JW. The 9. Homayounfar N, Park SS, Afsharinejad Z, Bammler TK, MacDonald SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phos- JW, Farin FM, et al. Transcriptional analysis of human cranial com- phorylated destruction motifs in IkappaBalpha and beta-catenin and partments with different embryonic origins. Arch Oral Biol 2015; stimulates IkappaBalpha ubiquitination in vitro. Genes Dev 1999;13: 60:1450–60. 270–83. 10. Ormestad M, Astorga J, Landgren H, Wang T, Johansson BR, Miura N, 23. Chitalia VC, Foy RL, Bachschmid MM, Zeng L, Panchenko MV, Zhou MI, et al. Foxf1 and Foxf2 control murine gut development by limiting et al. Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mesenchymal Wnt signaling and promoting extracellular matrix pro- mediates Wnt pathway inhibition by pVHL. Nat Cell Biol 2008;10: duction. Development 2006;133:833–43. 1208–16. 11. Aitola M, Carlsson P, Mahlapuu M, Enerb€ackS,Pelto-HuikkoM,etal. 24. Xue J, Chen Y, Wu Y, Wang Z, Zhou A, Zhang S, et al. Tumour suppressor Forkhead transcription factor FoxF2 is expressed in mesodermal tissues TRIM33 targets nuclear b-catenin degradation. Nat Commun. 2015; 6. doi: involved in epithelio-mesenchymal interactions. Dev Dyn 2000;218: 10.1038/ncomms7156. 136–49. 25. Tian HP, Lun SM, Huang HJ, He R, Kong PZ, Wang QS, et al. DNA 12. van der Heul-Nieuwenhuijsen L, Dits N, Van Ijcken W, de Lange D, Jenster methylation affects the SP1-regulated transcription of FOXF2 in breast G. The FOXF2 pathway in the human prostate stroma. Prostate 2009; cancer cells. J Biol Chem 2015,290:19173–83. 69:1538–47. 26. Suzuki T, Yokozaki H, Kuniyasu H, Hayashi K, Naka K, Ono S, et al. Effect of 13.KongPZ,YangF,LiL,LiXQ,FengYM.DecreasedFOXF2mRNA trichostatin A on cell growth and expression of cell cycle- and apoptosis- expression indicates early-onset metastasis and poor prognosis for related molecules in human gastric and oral carcinoma cell lines. Int J breast cancer patients with histological grade II tumor. PLoS One 2013; Cancer 2000;88:992–7. 8:e61591. 27. Touma SE, Goldberg JS, Moench P, Guo X, Tickoo SK, Gudas LJ, et al. 14. Shi Z, Liu J, Yu X, Huang J, Shen S, Zhang Y, et al. Loss of FOXF2 expression Retinoic acid and the histone deacetylase inhibitor trichostatin a inhibit the predicts poor prognosis in hepatocellular carcinoma patients. Ann Surg proliferation of human renal cell carcinoma in a xenograft tumor model. Oncol 2015;23:211–7. Clin Cancer Res 2005;11:3558–66. 15. Zheng YZ, Wen J, Cao X, Yang H, Luo KJ, Liu QW, et al. Decreased mRNA 28. Duvic M, Talpur R, Ni X, Zhang C, Hazarika P, Kelly C, et al. Phase 2 trial of expression of transcription factor forkhead box F2 is an indicator of poor oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory prognosis in patients with resected esophageal squamous cell carcinoma. cutaneous T-cell lymphoma (CTCL). Blood 2007;109:31–9 Mol Clin Oncol 2015;3:713–9. 29. Wang QS, Kong PZ, Li XQ, Yang F, Feng YM. FOXF2 deficiency promotes 16. Otani K, Dong Y, Li X, Lu J, Zhang N, Xu L, et al. Odd-skipped related 1 is a epithelial-mesenchymal transition and metastasis of basal-like breast novel tumour suppressor gene and a potential prognostic biomarker in cancer. Breast Cancer Res 2015;17:30. gastric cancer. J Pathol 2014;234:302–15. 30. Kim SE, Huang H, Zhao M, Zhang X, Zhang A, Semonov MV, et al. Wnt 17. Oliveira MJ, Costa AM, Costa AC, Ferreira RM, Sampaio P, Machado JC, stabilization of beta-catenin reveals principles for morphogen receptor- et al. CagA associates with c-Met, E-cadherin, and p120-catenin in a scaffold assemblies. Science 2013;340:867–70. multiproteic complex that suppresses Helicobacter pylori-induced cell- 31. MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, invasive phenotype. J Infect Dis 2009;200:745–55. mechanisms, and diseases. Dev Cell 2009;17:9–26. 18. Hellqvist M, Mahlapuu M, Samuelsson L, Enerb€ack S, Carlsson P. Differ- 32. Yu J, Cheng YY, Tao Q, Cheung KF, Lam CN, Geng H, et al. Methylation of ential activation of lung-specific genes by two forkhead proteins, FREAC-1 protocadherin 10, a novel tumor suppressor, is associated with poor and FREAC-2. J Biol Chem 1996;271:4482–90. prognosis in patients with gastric cancer. Gastroenterology 2009;136: 19. Oshima H, Matsunaga A, Fujimura T, Tsukamoto T, Taketo MM, Oshima 640–51. M. Carcinogenesis in mouse stomach by simultaneous activation of the 33. Wang S, Cheng Y, Du W, Lu L, Zhou L, Wang H, et al. Zinc-finger protein Wnt signaling and prostaglandin E2 pathway. Gastroenterology 2006; 545 is a novel tumour suppressor that acts by inhibiting ribosomal RNA 131:1086–95 transcription in gastric cancer. Gut 2013;62:833–41. 20. Mao J, Fan S, Ma W, Fan P, Wang B, Zhang J, et al. Roles of Wnt/b-catenin 34. Xu L, Li X, Chu ES, Zhao G, Go MY, Tao Q, et al. Epigenetic inactivation of signaling in the gastric cancer stem cells proliferation and salinomycin BCL6B, a novel functional tumour suppressor for gastric cancer, is asso- treatment. Cell Death Dis 2014;5:e1039. ciated with poor survival. Gut 2012;61:977–85.

1656 Cancer Res; 78(7) April 1, 2018 Cancer Research

Forkhead Box F2 Suppresses Gastric Cancer through a Novel FOXF2−IRF2BPL β− -Catenin Signaling Axis

Akira Higashimori, Yujuan Dong, Yanquan Zhang, et al.

Cancer Res 2018;78:1643-1656. Published OnlineFirst January 26, 2018.

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