Zic1 suppresses gastric cancer metastasis by regulating Wnt/β‐catenin signaling and epithelial‐mesenchymal transition

The FASEB JournalVolume 34, Issue 2 p. 2161-2172 RESEARCH ARTICLEOpen Access Zic1 suppresses gastric cancer metastasis by regulating Wnt/β-catenin signaling and epithelial-mesenchymal transition Qiwei Ge, Qiwei Ge Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorYingying Hu, Yingying Hu Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorJiamin He, Jiamin He Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorFei Chen, Fei Chen Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorLunpo Wu, Lunpo Wu Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorXintao Tu, Xintao Tu Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USASearch for more papers by this authorYadong Qi, Yadong Qi Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorZizhen Zhang, Zizhen Zhang Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorMeng Xue, Meng Xue Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorShujie Chen, Shujie Chen Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorJing Zhong, Corresponding Author Jing Zhong 2512022@zju.edu.cn Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, China Correspondence Liangjing Wang and Jing Zhong, Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China. Email: wangljzju@zju.edu.cn (L. W.) and 2512022@zju.edu.cn (J. Z.)Search for more papers by this authorLiangjing Wang, Corresponding Author Liangjing Wang wangljzju@zju.edu.cn Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, China Correspondence Liangjing Wang and Jing Zhong, Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China. Email: wangljzju@zju.edu.cn (L. W.) and 2512022@zju.edu.cn (J. Z.)Search for more papers by this author Qiwei Ge, Qiwei Ge Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorYingying Hu, Yingying Hu Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorJiamin He, Jiamin He Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorFei Chen, Fei Chen Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorLunpo Wu, Lunpo Wu Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorXintao Tu, Xintao Tu Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USASearch for more papers by this authorYadong Qi, Yadong Qi Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorZizhen Zhang, Zizhen Zhang Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorMeng Xue, Meng Xue Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorShujie Chen, Shujie Chen Institution of Gastroenterology, Zhejiang University, Hangzhou, China Department of Gastroenterology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, ChinaSearch for more papers by this authorJing Zhong, Corresponding Author Jing Zhong 2512022@zju.edu.cn Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, China Correspondence Liangjing Wang and Jing Zhong, Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China. Email: wangljzju@zju.edu.cn (L. W.) and 2512022@zju.edu.cn (J. Z.)Search for more papers by this authorLiangjing Wang, Corresponding Author Liangjing Wang wangljzju@zju.edu.cn Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China Institution of Gastroenterology, Zhejiang University, Hangzhou, China Correspondence Liangjing Wang and Jing Zhong, Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China. Email: wangljzju@zju.edu.cn (L. W.) and 2512022@zju.edu.cn (J. Z.)Search for more papers by this author First published: 02 January 2020 https://doi.org/10.1096/fj.201901372RR Qiwei Ge, Yingying Hu, and Jiamin He contributed equally to this study. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Gastric cancer (GC) patients with metastasis had limited treatment options and dismal outcome. We have previously reported the aberrant expression of Zic family member 1 (Zic1) in GC. However, the functional roles and underlying mechanism of Zic1 in GC metastasis remain unknown. Here, we demonstrate that lower expression of Zic1 was correlated with more lymph node metastasis and poor outcome of GC patients. Ectopic expression of Zic1 suppressed both lung metastasis and peritoneal tumor dissemination of GC in mice. The metastatic suppressing ability of Zic1 was mediated by regulating the process of cell invasion, adhesion and epithelial-mesenchymal transition (EMT). Mechanistically, Zic1 could downregulate Wnt targets including c-Myc and Cyclin D1 by inhibiting LEF transcriptional activity in GC cells. Notably, Zic1 was inversely related to the expression of Cyclin D1 in GC tissues tested. In addition, Zic1 could physically interact with β-catenin/transcription factor 4 (TCF4) and disrupt their complex formation, while not affecting β-catenin nuclear localization. Collectively, our study indicated that Zic1 suppressed GC metastasis through attenuating Wnt/β-catenin signaling and the EMT process. Our work may provide novel therapeutic strategies for the metastasis of GC. Abbreviations EMT epithelial-mesenchymal transition GC gastric cancer IHC immunohistochemistry qRT-PCR quantitative real-time PCR TCF4 transcription factor 4 Zic1 Zic family member 1 1 INTRODUCTION Despite marked improvements in the diagnosis and treatments of gastric cancer (GC), GC remains one of the most common cancers and the third leading cause of cancer-related mortality globally.1, 2 Over the past decades, the 5-year survival rate for early stage GC patients has been improved. However, patients at a late stage still have a very dismal outcome due to the rapid metastasis.3 Treatment options for patients with advanced tumors were extremely limited. A comprehensive understanding of the key molecular events in tumor metastasis is lacking.4 Thus, uncovering new therapeutic targets may help to improve the overall survival of GC patients. As the first reported gene in the Zic family, Zic family member 1 (Zic1) is located on chromosome 3q25.1, which encodes a zinc finger transcription factor,5 and also serves as transcriptional cofactors.6, 7 Zic1 plays important roles in early embryogenesis and organogenesis, particularly in the central nervous system.5, 8 Accumulating evidence revealed that Zic1 could exhibit antitumorigenic effect in numerous epithelial cancers including endometrial cancer,9 colon cancer,10 thyroid cancer,11 and breast cancer.12 However, Zic1 could function as an oncogene in tumors originated from stroma, such as liposarcoma13 and pleural mesothelioma.14 We have previously identified that Zic1 was downregulated through promoter DNA methylation and acted as a tumor suppressor by regulating MAPK signaling and modulating cell-cycle distributions in GC.15, 16 Detection of Zic1 promoter hypermethylation in plasma DNA can be a new strategy for the early diagnosis of GC.17 Zic1 has been shown to be involved in several key signaling pathways during early embryogenesis, including Wnt/β-catenin, TGF-β and Shh signaling.18 Other Zic family proteins, such as Zic219 and Zic320 have been shown to suppress β-catenin-induced axis duplication in Xenopus embryos. Zic5 played an oncogenic role in hepatocellular carcinoma through activating Wnt/β-catenin.21 Particularly, Zic1 is coexpressed with β-catenin in the myofibroblast of Dupuytren disease.22 Wnt/β-catenin signaling is closely associated with colorectal cancer and indisputably linked to many other cancers including GC.23, 24 The Wnt/β-catenin signaling pathway is implicated in the maintenance of stem cell properties and serves as an important regulator of epithelial-mesenchymal transition (EMT) in cancer cells as well.25 Nevertheless, whether Zic1 regulates Wnt signaling in human cancers remains largely unknown. In this study, we found that the lower expression of Zic1 was associated with more lymph node metastasis as well as a poor prognosis of GC patients. Zic1 was shown to suppress the mobility of GC cells both in vitro and in vivo through modulating the EMT process. Zic1 could disrupt the interaction of β-catenin and transcription factor 4 (TCF4), hence inhibiting Wnt/β-catenin signaling. Thus, our study demonstrated the prognostic relevance of Zic1 in GC and may shed light on novel targeted therapy for metastatic GC patients. 2 METHODS 2.1 Cell lines and cell culture Gastric cancer cell lines (AGS, BGC-823, and SGC-7901) were purchased from Riken Gene Bank (Tsukuba, Japan) and American Type Culture Collection (ATCC, MA, USA), and cultured with RPMI1640 medium and F12K medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS, Sijiqing, China). Human embryonic kidney epithelial cell line 293T (HEK293T) was purchased from Invitrogen. Cells were kept at 37°C in a 5% CO2-containing atmosphere. 2.2 Plasmids and cell transfection The full-length Zic1 open reading frame was cloned into mammalian expression vector pcDNA3.1 as previously described15 and then subcloned into the pFLAG-CMV4 vector with an N-terminal Flag. The pGL3 LEF-Luc reporter was kindly provided by Prof. Tianhua Zhou (Zhejiang University, China). Cells were transfected with FuGENE HD (Promega, Madison, WI) in accordance with the manufacturer's protocol. To generate cell lines with stably overexpression of Zic1, BGC-823, and SGC-7901 cells were transduced with CMV-GFP lentivirus encoding Zic1 with puromycin selection cassette. The expression level of Zic1 was confirmed by quantitative real-time PCR (qRT-PCR) and Western blot, and the expression of GFP was confirmed by the inverted fluorescence microscope. 2.3 RNA extraction and quantitative real-time PCR Total RNAs were extracted according to the protocol of TRIzol reagent (Invitrogen, Carlsbad, USA). Complementary DNA (cDNA) was synthesized using RT reagent kit with gDNA Eraser (TaKaRa, Otsu, Japan). SYBR Green Master Mix Kit (Takara) was used to perform qRT-PCR on a LightCycler 480 Instrument (Roche, Penzberg, Germany). U6 and β-actin were used as endogenous controls. Primers used for PCR reactions are listed in the Supporting Informations. 2.4 Western blot Protein samples were loaded on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Bedford, USA). The following antibodies were used for Western blot: Zic1 from Abcam (1:1000, Shanghai, China); E-cadherin, N-cadherin, ZO-1, MMP2, Vimentin, β-catenin, c-Myc, Cyclin D1, TCF4, Histone H2A, GAPDH, Vinculin, and β-actin from Cell Signaling Technology (1:1000, Shanghai, China). An HRP-linked Goat anti-Rabbit IgG (1:3000, Baoke, China) was used as the secondary antibody. Light-chain specific Mouse anti-Rabbit IgG (1:1000, CST, Shanghai, China) was used as the secondary antibody in Co-IP assay. The blots were visualized by chemiluminescence using the Amersham Imager 600 System (GE Healthcare Life Sciences, Pittsburgh, USA). 2.5 Cell migration, invasion, and adhesion assays In vitro cell migration and invasion were measured by Transwell chambers (Corning Inc, Corning, USA) and the chambers were coated with Matrigel (BD Biosciences, Franklin Lakes, USA), respectively. Briefly, cells transfected with indicated plasmids were cultured in serum-free medium for 24 hours and then 1 × 105 cells were plated to the upper chamber in 250 μL medium with 1% fetal bovine serum (FBS), while the lower chamber was filled with 600 μL medium with 15% FBS. After 24 hours incubation, cells on the outer surface of the insert bottom were fixed, stained with DAPI and then counted under an optical microscope in five predetermined fields. Cells invading through membrane were incubated with 0.1% crystal violet. Then crystal violet was washed out from the cells with 10% acetic acid and quantified on a microplate reader (540 nm). For adhesion assay, cells (1 × 105/well) were seeded into 96-well plates. The medium was removed after 30 minutes, and cells stained with 0.1% crystal violet were washed with 10% acetic acid and the amount of crystal violet was measured using a microplate reader. 2.6 Dual-luciferase reporter assay BGC-823, AGS and HEK293T cells transiently expressing pFlag-CMV4-Zic1 or pFlag-CMV4-Vector were seeded in 12-well plates (2.5 × 104 cells per well) and transfected with LEF-Luc (200 ng) and pRL-TK vectors (10 ng, Promega) for 24 hours. Then cells were washed twice with phosphate buffer saline (PBS) and lysed in 200 μL passive lysis buffer (Promega). The luciferase activities were measured using dual luciferase reporter system (Promega). Firefly luciferase activities were normalized to Renilla luciferase activities. 2.7 Immunofluorescence assay Cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 1% BSA. Then cells were incubated with the primary antibody (β-catenin, Cell signaling, 1:200) at 4°C overnight. After three washes using PBS-T, cells were incubated with AF488-conjugated secondary antibody (Thermo Scientific, Waltham, MA). The nucleus was counterstained with DAPI. Images were captured using fluorescence microscope MODEL BX51TRF (Olympus Corporation, Tokyo, Japan). 2.8 Coimmunoprecipitation assay BGC-823 or HEK293T cells were transfected with indicated plasmids. Nucleoprotein Extraction Kit (Sangon Biotech, Shanghai, China) was used for nuclear protein isolation according to the manufacturer's instructions. The lysates were mixed with protein A/G magnetic beads (Thermo Scientific) and then incubated with anti-Flag or specific antibody at 4°C overnight. Rabbit normal IgG was used as a negative control. The magnetic beads were collected by magnetic separator and washed with IP buffer three times to remove all the nonspecific binding proteins. The bounded proteins were eluted with the elution buffer for western blot analysis. 2.9 In vivo mouse models For the lung metastasis model, 5 × 106 BGC-823 cells were intravenously injected through the tail vein of each 4-week-old female BALB/c nude mouse. For the peritoneal implantation metastasis model, 3 × 106 BGC-823 cells were injected into abdominal cavity of nude mice. All mice for metastasis assays were sacrificed at day 30 after injection. For tumor xenograft model, 1 × 107 SGC-7901 cells were subcutaneously injected into 4-week-old female BALB/c nude mice. All experimental procedures were approved by the Animal Ethics Committee of School of Medicine Zhejiang University. 2.10 Clinical samples Ninety patients with complete clinicopathologic characteristics and follow-up data who underwent surgery at the Second Affiliated Hospital, School of Medicine Zhejiang University and histologically diagnosed with GC were enrolled. Histological cancer types were evaluated by two independent pathologists in accordance with the TNM staging guide (2016) released by The American Joint Committee on cancer (AJCC). Tissue microarrays were made by paraffin-embedded consecutive sections. In addition, 50 surgically resected GC and matched adjacent noncancerous specimens were obtained from Sir Run Run Shaw Hospital, School of Medicine Zhejiang University. Specimens were immediately frozen in liquid nitrogen and stored in −80°C. All patients were provided with informed written consents for obtaining study specimens. Experiments were approved by the Ethics Committee of the Second Affiliated Hospital, School of Medicine Zhejiang University. 2.11 Immunohistochemistry Tissue arrays were subjected to immunohistochemical staining for the expression of Zic1 and Cyclin D1, with the same primary antibodies as those used in western blot analysis, at a dilution of 1:100 in PBS. A pathologist and an investigator independently evaluated the scores of Zic1 and Cyclin D1 expression with an immunoreactivity score (IRS), which was combined by a score of the percentage of positive cells (1, 0%~25%; 2, 25%~50%; 3, 50%~75%; and 4, 75%~100%) and a score of the staining intensity (1, no staining of cells; 2, mild staining; 3, moderate staining; and 4, marked staining). We used an average score of stain in the cytoplasm and nucleus when different intensities were detected. The total score ranged from 0 to 16. 2.12 Statistical analysis All experiments were performed at least three times. All data are presented as the Mean ± SD. Statistical analyses were performed in IBM SPSS Statistics (Version 22.0, IBM, Armonk, NY, USA) and a P value of less than .05 was considered statistically significant. The Mann–Whitney U-test was used to analyze the nonparametric data in clinical samples. χ2 test was used to assess the association of Zic1 expression with the clinicopathological parameters. Kaplan–Meier plots were used for overall survival rates, then compared with the log-rank test. Spearman's rank correlation test was used to analyze the correlation between expression level of different proteins in clinical samples. Significant differences between groups were determined using the Student's t test. 3 RESULTS 3.1 Zic1 is downregulated in GC and correlated with poor prognosis We first assessed the expression of Zic1 in 50 primary GC tissues as well as their matched adjacent noncancerous tissues (Cohort 1) and showed that the expression of Zic1 was downregulated in the GC tissues at both mRNA level (P < .001, Figure 1A) and protein level (Figure 1B). Further immunohistochemistry (IHC) staining illustrated a lower expression of Zic1 in GC tissues in another independent cohort with 90 GC patients (Cohort 2). Zic1 is widely expressed in normal gastric epithelial tissues, and is mainly expressed in the nucleus of surface mucous foveolar cells. Quantitative analysis indicated that Zic1 was significantly lower expressed in GC tissues comparing to the noncancerous tissues (P < .001, Figure 1C,D). Figure 1Open in figure viewer Downregulation of Zic1 is associated with lymph node metastasis and poor prognosis in gastric cancer (GC). A, Relative expression of Zic1 mRNA in human gastric tissues and matched nontumor tissues in Cohort 1 was detected by qRT-PCR. The relative expression of Zic1 in tumor sample is normalized to paired nontumor tissue as differential expression values (T/N) (n = 50, P < .001). B, Protein expression level of Zic1 was detected by Western blot analysis in 10 randomly selected GC and noncancerous tissues. GAPDH was used as loading controls. C, Quantification of Zic1 protein expression by scoring Immunohistochemistry (IHC) staining in gastric tissues of Cohort 2 (n = 90, P < .001). D, Representative images of IHC staining of Zic1 in GC and adjacent normal tissues. Scale bar, 100 μm. E, The expression of Zic1 was inversely correlated with lymph node metastasis in Cohort 2 (n = 90, P = .0014). F, The association of Zic1 expression and overall survival of gastric cancer patients was explored by the Kaplan–Meier survival analysis (n = 90, P = .0092) We then evaluated the association between the expression of Zic1 and several clinicopathological parameters as well as the patient prognosis. Patients with lymph node metastasis showed a significantly lower expression of Zic1 in the cancer tissues comparing to those patients without metastasis (Figures 1E and S1). Downregulation of Zic1 was also observed in GC patients with late TNM stage (P = .011, Table 1). Kaplan–Meier analysis showed that patients with lower expression level of Zic1 in GC tissues had a poor prognosis (P = .0092, Figure 1F). Collectively, these results indicated that Zic1 was downregulated in GC and correlated with poor prognosis in the patients. Table 1. The relationship between Zic1 expression and clinicopathological characteristics in patients with GC Clinicopathological characters Zic1 expression χ 2 P valuea High level (53) Low level (37) Age (year) 60 23 17 0.0574 .811 ≥60 30 20 Gender Male 38 22 1.4686 .226 Female 15 15 Tumor size (cm) <5.0 40 21 3.4943 .062 ≥5.0 13 16 Invasive depth T1-T2 21 11 0.9307 .335 T3-T4 32 26 Lymph node metastasis N = 0 26 9 5.6081 .017* N = 1-3 27 28 TNM stage I-II 33 13 6.4177 .011* III-IV 20 24 a Chi-square test. * P < .05. 3.2 Zic1 inhibits GC cells invasion and epithelial mesenchymal transition To investigate the mechanism underlying Zic1 mediated metastasis, we applied in vitro studies to explore the functional roles of Zic1. As Zic1 is silenced in most GC cell lines,15 we ectopically expressed Zic1 in GC cells (BGC-823 and AGS) with pFlag-CMV4-Zic1 plasmid, and overexpression of Zic1 markedly inhibited cell migration and cell invasion in BGC-823 and AGS cells (Figure 2A,B), but promoted cell adhesion in both parental cells (Figure 2C). Figure 2Open in figure viewer Zic1 regulates GC cell migration, invasion and adhesion. A, Cell migration was assessed by transwell assays. Cells which migrated to the bottom of the membrane were stained with DAPI. The mean number of visible migratory cells was counted in five random high-power fields. Scale bar, 100 μm. B, Cell invasion was assessed by transwell invasion assay. Invaded cells at the bottom of the membrane were stained with crystal violet, washed with 10% acetic acid and the amount of crystal violet was detected on a microplate reader (540 nm). Scale bar, 100 μm. C, Cell adhesion was quantified by cell adhesion assay. Cells adhered to the plates were stained with crystal violet. The crystal violet was dissolved in 10% acetic acid and the absorbance was measured at 540 nm. Scale bar, 100 μm. D-E, Western blot analysis (D) and qRT-PCR (E) were performed to measure the expression level of EMT markers after ectopic expression of Zic1 in GC cell lines. *P < .05, **P < .01, ***P < .001 Epithelial mesenchymal transition (EMT) has been recognized as a key step in the progression of cancer metastasis.26 Thus, we hypothesized that EMT might be involved in the process of GC metastasis. The epithelial markers, E-cadherin and ZO-1, were upregulated in both cell lines upon ectopic expression of Zic1, whereas the expression of mesenchymal markers, including N-cadherin, Vimentin, and MMP2 were reduced (Figure 2D). These effects were further confirmed by qRT-PCR analysis of EMT markers (Figure 2E), suggesting that Zic1 could suppress GC cells metastatic ability by regulating the process of cell invasion, adhesion, and EMT. 3.3 Zic1 suppresses GC lung and peritoneal metastasis in mice We next investigated whether Zic1 could play a role in GC metastasis in vivo. BGC-823 cells transduced with lentivirus carrying either GFP or Zic1-GFP were injected into abdominal cavity of nude mice. Mice received cells with Zic1 overexpression exhibited less peritoneal metastasis than the control group (Figure 3A,B). We also measured the effect of Zic1 on lung metastasis by intravenous injection of these GFP-positive cells to the nude mice. Fewer numbers of lung metastatic nodules were observed in the Zic1 overexpression group demonstrated by the lower green fluorescence in the lung as well as fewer lung nodules indicated by H&E staining (Figure 3C,D). Additionally, overexpression of Zic1 in tumor cells significantly prolonged survival of the mice (P < .05, Figure 3E). Since EMT process is critical in lung metastasis, we measured the expression of several key EMT markers in the metastatic tissues. We found that the epithelial marker E-cadherin was upregulated in tumors, while the mesenchymal markers N-cadherin and Vimentin were downregulated in Zic1 overexpression group (Figure 3F). We also examined the role of Zic1 in tumor growth in vivo. Nude mice subcutaneously transplanted with SGC-7901 cells expressing either empty vector or Zic1 vector (Figure S2A,B) developed solid tumors in 20 days. There was no significant difference of tumor weight between two groups (Figure S2C,D), indicating that ectopic expression of Zic1 may have no effect on tumor growth in a mouse xenograft model. Taken together, these results showed that Zic1 could suppress the EMT process and GC metastasis in vivo. Figure 3Open in figure viewer Overexpression of Zic1 suppresses gastric cancer metastasis in vivo. A, Zic1 overexpression group showed less peritoneal implantation metastasis nodules macroscopically. B, Statistical results of metastasis nodules weight from Zic1 overexpression and control groups (n = 5 each, P < .05). C-D, Zic1 overexpression group showed less lung metastasis with smaller range and lower intensity of the green fluorescence (C), less metastasis nodules on hematoxylin-and eosin-stained section (D). Scale bar, 100 μm. E, Zic1 overexpression group showed prolonged survival of mice (n = 6 each, P < .05). F, The expression of EMT makers in lung metastasis nodules was analyzed by Western blot analysis. GAPDH was used as loading controls 3.4 Zic1 regulates Wnt/β-catenin signaling pathway in GC Previous reports have showed that Zic family proteins regulate Wnt/β-catenin signaling during embryogenesis.6, 19 In this regard, we proposed that Zic1 might act on the Wnt/β-catenin pathway to suppress tumor metastasis. Indeed, overexpression of Zic1 significantly reduced the expression level of Wnt/β-catenin downstream genes, including Cyclin D1, c-Myc, and Axin2 (Figure 4A). Interestingly, we found that ectopically expression of Zic1 downregulated Cyclin D1 and c-Myc with or without LiCl (20 mM) stimulation, which could stabilize β-catenin through GSK-3β (Figure 4B). Consistently, we observed that Cyclin D1 and c-Myc were downregulated in lung metastasis tissues from nude mice in Zic1 overexpression group in vivo (Figure 4C). To investigate whether Wnt/β-catenin signaling pathway was inhibited by Zic1, we performed dual-luciferase reporter assays with LEF reporter. Overexpression of Zic1 drastically inhibited the LEF luciferase activity in all cell lines tested (BGC-823, AGS and HEK293T cells), indicating that Wnt/β-catenin signaling was attenuated by Zic1 (Figure 4D). Figure 4Open in figure viewer Zic1 regulates Wnt/β-catenin signaling pathway in gastric cancer. A, qRT-PCR revealed that