Smurf1 promotes gastric cancer growth and metastasis by regulating Axin2- dependent Wnt/β-catenin signaling

Smurf1 promotes gastric cancer growth and metastasis by regulating Axin2- dependent Wnt/β-catenin signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Smurf1 promotes gastric cancer growth and metastasis by regulating Axin2- dependent Wnt/β-catenin signaling Jinling Yu, Jiachen Jing, Zhen Feng, Zhonghua Chen, Beina Ji, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6694191/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) is involved in various biological processes through targeting specific proteins for ubiquitin-dependent degradation. Emerging evidence has shown that Smurf1 functions as an oncogene in many types of human tumours, including gastric cancer (GC). We aimed to investigate the role of Smurf1 in regulating GC progression and reveal its underlying mechanism. Smurf1 expression was analyzed in two datasets that are publicly accessible. Additionally, it was assayed in 29 pairs of GC tissues and para-cancerous tissues using quantitative reverse transcriptase PCR (qRT-PCR). The biological role of Smurf1 in GC cells was assessed in vitro and in a moue Xenograft model. Smurf1 levels were significantly up-regulated in GC tissues compared with normal tissues, and high Smurf1 expression was significantly correlated with worse disease-free survival (DFS). Forced expression of Smurf1 accelerated AGS cell growth, proliferation, and invasion in vitro and in vivo . Mechanistically, Smurf1 directly engaged with axis inhibition protein 2 (Axin2) and diminished the stability of the Axin2 protein by promoting its ubiquitination and subsequent degradation. As a result, Smurf1 promoted the activation of Wnt/β-catenin signaling. Importantly, IWR-1, a specific inhibitor of the Wnt pathway, effectively inhibited Smurf1-induced GC cell proliferation and invasion. These data suggest that upregulated Smurf1 facilitates GC progression through degrading Axin2 and activating Wnt/β-catenin signaling. Biological sciences/Cell biology Biological sciences/Chemical biology Biological sciences/Molecular biology gastric cancer Smurf1 Axin2 Wnt β-catenin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Gastric cancer (GC) is a common cancer globally. In 2020, there were approximately 1,100,000 new cases and 769,000 deaths ( 1 , 2 ), ranking fifth for morbidity and fourth for death rate in the world ( 3 , 4 ). Although GC morbidity and mortality have generally declined throughout the years in most populations, they are increasing in young adults ( 1 ). At present, surgical excision remains the only opportunity to cure GC. Chemotherapy and radiotherapy following radical surgery are beneficial to improve the overall survival (OS) rate, but the prognosis remains disappointing ( 5 ). Revealing the mechanisms underlying GC progression and chemoradiotherapy resistance is indispensable to improving disease outcomes. SMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) was initially discovered as a modulator of Smad protein stability ( 6 ). Under inflammatory stress, upregulated Smurf1 results in bone loss by facilitating Smad1 protein ubiquitination and subsequent degradation ( 7 ). Subsequent to this discovery, numerous target proteins regulated by Smurf1 have been identified across various biological processes. For instance, Smurf1 inhibits the osteogenic activity of osteoblasts and bone homeostasis by directly binding to mitogen-activated protein kinase kinase kinase 2 (MEKK2) and accelerating its degradation ( 8 ). Smurf1 functions as a limiting factor in regulating integrin activation and subsequent cell function by controlling Kindlin 2 protein degradation ( 9 ). Smurf1 facilitates breast cancer metastasis through physically interacting with Ras homolog family member A, leading to its ubiquitination and degradation ( 10 ). Although Smurf1 has been reported as a potential oncogenic factor in GC ( 11 ), the underlying mechanism remains unclear. Wnt/β-catenin pathway is crucial for tissue development and homeostasis ( 12 , 13 ). However, its over-activation leads to various types of diseases ( 14 – 18 ), especially in cancer initiation and progression ( 19 – 21 ). For example, Wnt signaling plays a vital role in transcription factor EB (TFEB)-triggered GC cell invasion and cancer metastasis ( 19 ). High TFEB expression is correlated with worse OS and disease-free survival (DFS) ( 19 ). Matsumoto et al. demonstrated that Wnt signaling activation accelerates hepatocellular carcinoma progression by increasing the expression of growth regulation by estrogen in breast cancer 1 (GREB1) ( 20 ). The Wnt pathway comprises four key components: ligands, receptors, cytoplasmic components like β-catenin and its “destruction complex” (GSK-3β, Axin, CK1, and APC), and nuclear components (TCF/LEF transcription factors) ( 12 , 22 , 23 ). The hallmark of Wnt pathway activation is the transfer of β-catenin into the nucleus, which initiates the transcription of TCF/LEF target genes ( 12 , 24 , 25 ). In a previous study, we demonstrated that histone lysine demethylase 4B (KDM4B) facilitates GC cell invasion through accelerating miR-125b-dependent nuclear translocation of β-catenin ( 26 ). Axis inhibition protein (Axin) directly interacts with GSK-3β, APC, CK1, and β-catenin, serving as a scaffold protein for the “destruction complex” in the Wnt pathway ( 27 ). Axin exists in two subtypes: Axin1 and Axin2. Previous research has indicated that Smurf1 regulates Wnt pathway activation through binding to the Axin1 protein and accelerating its ubiquitination ( 10 , 28 ). However, the association of Smurf1 with Axin2 is not well understood. The study primarily aimed to explore how Smurf1 regulates Wnt pathway activation and GC progression. Materials and Methods Data analysis Transcriptomic and clinical data of 408 GC specimens and 211 normal specimens were obtained from the Cancer Genome Atlas-Stomach Adenocarcinoma (TCGA-STAD) database and data analysis was carried out with GEPIA tool ( http://gepia.cancer-pku.cn/detail.php?gene ). Smurf1 levels were analyzed in GC specimens and normal specimens. The correlations between Smurf1 level and both OS and DFS in GC patients were analyzed through the TCGA dataset. In addition, the Smurf1 levels were analyzed in the GSE13911 dataset. Clinical specimens and cell culture Twenty-nine pairs of tumor tissues and para-carcinoma tissues were collected from Xu Hui District Center Hospital. Informed consent was obtained from all individual participants. Three cell lines, AGS, MKN-45, and Ges-1, were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in DMEM/F12 media (TIANHANG, Hangzhou, China) containing 10% fetal bovine serum (FBS, TIANHANG) in a cell incubator. The recombinant plasmid encoding the complete coding region of Smurf1, pcDNA-Smurf1, was constructed in our laboratory and transfected into GC cells to overexpress Smurf1 using PEI max (MineBio Life Sciences, Shanghai, China). Cell counting kit 8 (CCK-8) After overexpression with Smurf1 or treatment with 10 µM of IWR-1 (MCE, NJ, USA), AGS cells were seeded into 96-well plates and incubated for varying durations. After that, 10 µL of CCK-8 (MCE) were applied to treat the cells. Sixty minutes later, optical density (OD) values were recorded at 450 nm with a ReadMax 1200 microplate reader (Shanpu, Shanghai, China). Colony formation assay After Smurf1 overexpression, AGS cells were seeded into 12-well plates (approximately 350 cells per well) for two weeks until visible colonies occurred. After that, cells were rinsed thrice using phosphate-buffered saline (PBS), immobilized in methyl alcohol for 10 min, and stained in crystal violet for approximately 10 min. Cell colonies were calculated with an XDS-900C inverted microscope (Caikon, Shanghai, China). Transwell invasion assay Matrigel-coated transwell chambers with an 8-µm pore size (Corning, NY, USA) were applied to measure cell invasion. After Smurf1 overexpression or treatment with 10 µM of IWR-1, AGS cells were seeded into the upper chamber in DMEM/F12 medium without serum (10000 cells per well). The lower chamber was filled with 500 µL of DMEM/F12 complete medium. After 36 h, noninvasive cells were removed with cotton swabs. Cells that invaded the lower membrane surface were fixed in methyl alcohol for 15 min, stained in crystal violet for 15 min, and then photographed using an inverted microscope. Quantitative real-time PCR (qRT-PCR) Total RNA was isolated with TRIzol reagent (Beyotime, Shanghai, China), treated with DNase I (TaKaRa, Tokyo, Japan), quantified using a Bioanalyzer 2100 (Agilent, CA, USA), and then reverse-transcribed into first-strand cDNA in a 20 µl solution containing M-MLV (TaKaRa) and Oligo (dT) primers. qRT-PCR was performed on an Ariazmx real-time PCR system (Agilent) in a 10 µl solution containing cDNA, primers (Supplementary Table S1 ), and 2×T5 Fast qPCR Mix (TSINGKE, Beijing, China). β-actin was used as a reference gene, and mRNA levels were calculated by the 2 −ΔΔCT formula ( 29 ). Western blot Western blot Total protein was isolated with RIPA lysis buffer (Thermo Fisher Scientific, MA, USA). After quantitating by the BCA method (Abcam, CA, USA), protein was measured through a western blot assay as previously described ( 26 ). The primary antibodies were listed as follows: Axin1 (1.5 µg/mL, PA5-21042, Thermo Fisher), Axin2 (1:1500, ab109307, Abcam), ubiquitin (1:3000, ab140601, Abcam), β-catenin (1:2000, 71-2700, Thermo Fisher), and β-actin (1:10000, ab6276, Abcam). The second antibody used in the study was ab7090 (1:10000, Abcam). Protein bands were visualized by a BeyoECL Plus (Beyotime). Cycloheximide (Chx) chase assay After Smurf1 overexpression for 24 h, AGS cells were incubated with 40 µM of Chx (Merck, MA, USA) for 0, 2, 4, or 8 h. Subsequently, total protein was isolated with RIPA lysis buffer to assess Axin2 protein through a western blot assay. For the ubiquitination analysis, western blot was carried out to assess Axin2 protein in AGS cells following Smurf1 overexpression and MG132 (20 µM, MCE) treatment for 24 h. Co-immunoprecipitation (Co-IP) Briefly, total protein was collected from AGS cells and incubated with the Smurf1 antibody (1:150, ab57573, Abcam) for 15 h ( 30 ). Protein A/G magnetic beads (MCE) were placed into IP mixtures to bind antibodies and proteins. Subsequently, Axin2 protein in immunoprecipitates was measured using western blot analysis. Luciferase reporter assay After Smurf1 overexpression, AGS cells were co-transfected with TOPflash construct (80 ng, Merck) or FOPflash construct (Merck) and pRL-TK plasmid (8 ng, Promega, WI, USA) using PEI max transfection reagent. The pRL-TK plasmid served as a control for transfection efficiency. After treatment with Wnt3a for 48 h, dual luciferase activity was assessed using the Dual-Luciferase® Reporter Assay System (Promega) ( 31 ). Data were shown as a normalized TOPflash/FOPflash activity ratio. Immunofluorescence (IF) AGS cells on glass slides were immobilized with methyl alcohol, perforated with Triton X-100, and blocked with fluorescent blocking buffer (Thermo Fisher Scientific). Subsequently, cells were treated with an antibody targeting β-catenin (1:400, 71-2700, Thermo Fisher Scientific), followed by an Alexa Fluor® 647-labeled second antibody. The fluorescence signal was recorded with an FCK-50C fluorescence microscope (Caikon). Mouse xenograft model The study was carried out with the approval of the Experimental Animal Committee of Xu Hui District Center Hospital (No. 2022052) adherence to the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. Six-week-old BALB/c nude mice (Charles River, Shanghai, China) were raised in a pathogen-free room maintained at 24–26 o C. AGS cells and Smurf1-overexpressed AGS cells were collected to construct AGS xenograft model. In brief, the mouse xenograft model was created by subcutaneously injecting 1×10 7 AGS cells into the left flank of mice (n = 5). Subcutaneous (s.c.) tumours growth was monitored with a vernier calliper at days 7, 14, 21, and 35 and calculated by the formula length × width 2 × 0.5 ( 32 ). Immunohistochemical (IHC) analysis of Ki67 was carried out to assess in vivo cell proliferation, as previously described ( 33 ). For the experimental metastasis assay, AGS cells or Smurf1-overexpressed AGS cells (1×10 7 cells, 100 µl PBS) were injected into mice by tail vein. Nine weeks later, mice were euthanized by pentobarbital overdose, and lung tissues were harvested. Hematoxylin-eosin (H&E) staining was performed to quantify the tumour nodules in lung tissues, as previously described ( 34 ). Statistics Each experiment was repeated at least three times, and data were presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism 7.0 (CA, USA). Normality of all data was evaluated by the Shapiro-Wilk test (Supplementary Table S2 ). Homogeneity of variance was evaluated by the F test or Brown-Forsythe test (Supplementary Table S3 ). Data were analyzed with the Welch’s t-test (Fig. 1 B), Mann Whitney test (Fig. 1 C, 2 D, 2 F, 3 D, 3 F, 4 A, 5 A, and 5 D), Kruskal-Wallis test followed by Dunnett’s post hoc test (Fig. 2 A and 6 B), and two-way ANOVA followed by Sidak’s multiple comparisons test (Fig. 2 B, 3 B, 4 E, and 6 A), respectively. The statistical analyses of TCGA-STAD data (Fig. 1 A, 1 D, and 1 E) were carried out with GEPIA tool ( http://gepia.cancer-pku.cn/detail.php?gene ). A comprehensive summary of the statistical methods and corresponding p values was shown in Supplementary Table S4 . A p value of < 0.05 was considered significant. Results Smurf1 expression was increased in GC tissues To explore the biological effect of Smurf1 on GC progression, Smurf1 expression was first analyzed in two publicly available datasets, TCGA and GSE13911. Figure 1 A revealed that Smurf1 expression was prominently increased in GC tissues compared to normal controls in the TCGA-STAD database. In the GSE13911 dataset, Smurf1 expression was also upregulated in GC tissues (Fig. 1 B). In addition, 29 pairs of GC tissues and para-cancerous tissues were used to assay the Smurf1 level. Figure 1 C displayed that Smurf1 expression was prominently enhanced in tumour tissues compared with para-cancerous tissues. The correlation between Smurf1 levels and the prognosis of GC patients was further investigated. Although high Smurf1 levels were not linked to worse OS (n = 384, p = 0.82, Fig. 1 D), they were significantly associated with DFS (n = 384, p = 0.00087, Fig. 1 E). The frequent increase of Smurf1 indicates that Smurf1 might exert a tumor activator in GC. Smurf1 overexpression accelerated GC cell growth and invasion The biological effect of Smurf1 on GC cell growth, proliferation, and invasion was next assessed. To this end, Smurf1 expression was first assayed in GC cells and a normal gastric epithelial cell. Figure 2 A showed that Smurf1 expression was increased in AGS and MKN-45 cells compared with Ges-1 cells. Smurf1 overexpression was carried out in AGS cells, and its biological role was assessed in AGS cells because the endogenous Smurf1 levels in AGS cells were lower than those in MKN-45 cells. As shown in Supplementary Figure S1 A and Fig. 2 B, Smurf1 overexpression significantly accelerated AGS cell proliferation. Smurf1 overexpression also markedly facilitated AGS cell growth (Fig. 2 C and D). Moreover, Smurf1 overexpression prominently accelerated AGS cell invasion (Fig. 2 E and F). Furthermore, the roles of Smurf1 overexpression in tumour growth and metastasis were investigated in a mouse xenograft model. As shown in Fig. 3 A and B, Smurf1 overexpression significantly promoted tumor growth. Ki67 staining from tumor tissues revealed that Smurf1 overexpression accelerated cell proliferation in vivo (Fig. 3 C and D). The roles of Smurf1 in facilitating cancer lung metastasis were measured by histological analysis. H&E staining showed that Smurf1 overexpression caused the formation of larger and more lung metastatic nodules (Fig. 3 E and F). Smurf1 promoted Axin2 ubiquitination and proteasome-dependent degradation We next predicted the potential target proteins regulated by Smurf1 using the UbiBrowser tool ( http://ubibrowser.ncpsb.org.cn/ubibrowser/ ) , and a total of 359 proteins were identified (Supplementary Table S5 ). Here, Axin1 and Axin2 were selected for further validation based on the following three reasons: i) Previous studies have demonstrated that Smurf1 mediates Axin1 ubiquitination and regulates Wnt/β-catenin signalling in murine embryonic fibroblasts ( 10 , 28 ), ii) Axin2 is a potential target protein regulated by Smurf1 (Supplementary Table S5 , Supplementary Figure S1 B), iii) abnormal activation of the Wnt pathway is vital for cancer progression ( 35 , 36 ). As shown in Fig. 4 A and B, although Smurf1 overexpression did not affect Axin1 and Axin2 mRNA expression in AGS cells, Smurf1 markedly decreased Axin2 protein expression, indicating that Smurf1 might directly interact with Axin2 and promote Axin2 protein degradation in GC cells. To explore whether Smurf1 facilitates Axin2 ubiquitination and subsequent proteasome-dependent degradation, the direct combination of Smurf1 with Axin2 was assayed through Co-IP, and results revealed that Smurf1 directly combined with Axin2 protein in AGS cells (Fig. 4 C). To assess whether Smurf1 accelerated Axin2 protein degradation, AGS cells were incubated with Chx to prevent protein synthesis. Chx chase analysis showed that forced expression of Smurf1 markedly reduced the Axin2 protein levels in AGS cells (Fig. 4 D and E). To reveal whether Smurf1 decreased Axin2 protein expression through ubiquitin-dependent degradation, Smurf1 was overexpressed in AGS cells, and then cell extracts were subjected to Co-IP analysis with an antibody targeting Axin2, followed by western blot assay using an anti-ubiquitin antibody. Figure 4 F revealed that Smurf1 overexpression obviously promoted Axin2 ubiquitination. As expected, Smurf1 overexpression inhibited Axin2 protein level, whereas the effects were reversed by MG132, a proteasome inhibitor (Fig. 4 G), indicating that Smurf1 accelerates Axin2 ubiquitination and results in proteasome-dependent degradation. Smurf1 accelerated cell proliferation and invasion through regulating the Wnt pathway The regulatory effects of Smurf1 on Wnt pathway activation were next assessed. Here, TOPflash/FOPflash, western blot, and IF analysis were carried out to determine Wnt activation or nuclear translocation of β-catenin. Smurf1-overexpressed AGS cells were transfected with TOPflash or FOPflash. Figure 5 A showed that there was an increased TOPflash/FOPflash activity ratio in Smurf1-overexpressed AGS cells following Wnt3a treatment, suggesting that the Wnt pathway was activated due to Smurf1 overexpression. Western blot assay showed that Smurf1 overexpression accelerated the transfer of β-catenin into the nucleus (Fig. 5 B-D). IF assay also displayed the promoting role of Smurf1 in the nuclear translocation of β-catenin (Fig. 5 E). Furthermore, the roles of Smurf1 in AGS cell proliferation and invasion (Fig. 6 A and B) were suppressed by IWR-1, a Wnt pathway inhibitor. Collectively, these data demonstrate that Smurf1 accelerates GC cell proliferation and invasion by activating the Wnt pathway. Discussion GC is usually diagnosed at advanced stages because of the absence of early typical symptoms and predictive biomarkers, which results in high mortality and a poor prognosis. Although chemoradiotherapy can effectively improve the prognosis of such patients, drug resistance is a main obstacle to successful therapy for advanced GC ( 37 , 38 ). Smurf1 exhibits pro-oncogenic activity and is correlated with drug resistance in many types of tumours ( 39 – 42 ). In the study, the mechanism by which Smurf1 accelerates GC progression was further explored. We demonstrated that, i) Smurf1 expression was upregulated in GC tissues, and Smurf1 overexpression predicted a poor DFS, ii) up-regulated Smurf1 facilitated GC growth and metastasis, iii) Smurf1 promoted Axin2 ubiquitination and proteasomal degradation, iv) Smurf1 accelerated GC cell growth and invasion by activating the Wnt pathway. Hyper-activation of the Wnt pathway is closely correlated with tumour progression in multiple pivotal aspects, such as cancer cell proliferation ( 43 ), epithelial-mesenchymal transition ( 44 ), invasion ( 45 ), and stemness maintenance ( 44 , 46 ). As the scaffold protein for the “destruction complex” in the Wnt pathway, Axin is essential for inhibiting the activation of the Wnt pathway through the assembly the “destruction complex” ( 47 ). Mutations or aberrant expressions of Axin were frequently observed in various types of tumours. Many studies have revealed a nonsense mutation of Axin1 and a frameshift mutation of Axin2 in epithelial ovarian cancer (EOC) ( 48 , 49 ). Kim et al. identified nine frameshift mutations in Axin2 in 32 patients with GC ( 50 ). AlkB homolog 5 (ALKBH5) accelerates colorectal cancer (CRC) progression by promoting Axin2 mRNA degradation and thus over-activating Wnt/β-catenin signaling ( 51 ). Caudal-related homeobox transcription factor 2 (CDX2) suppresses colon cancer cell growth through upregulating Axin2 expression and inactivating Wnt signaling ( 52 ). Axin1 inhibition causes the acquired resistance of CRC cells to Wnt pathway blockade ( 53 ). Axin2 protein degradation, regulated by E3 ubiquitin ligase E3C (UBE3C), causes the activation of the Wnt pathway and subsequent GC cell proliferation ( 54 ). Unlike Axin1, Axin2 is a downstream gene of the TCF/LEF transcription factor. Given the positive regulation of Axin2 by the Wnt pathway and the fact that Axin2 is an indispensable member of the “destruction complex”, Axin2 might be a critical negative feedback regulator for the Wnt pathway in cancer cells ( 55 , 56 ). For example, Axin2 overexpression inactivates the Wnt pathway and represses colon cancer cell proliferation ( 52 ). Although the underlying mechanisms remain unclear, the pro-oncogenic effect of Smurf1 has been revealed in GC. MicroRNA-mediated inhibition of Smurf1 suppresses GC cell proliferation and invasion and alleviates the resistance of GC cells to cisplatin ( 39 , 40 ). In the study, Smurf1 expression was analysed in publicly available datasets, and its biological role was assessed. Smurf1 levels were significantly increased in GC tissues compared to para-carcinoma tissues in the TCGA and GSE13911 databases. High Smurf1 levels predicted worse OS and DFS. Functionally, Smurf1 overexpression accelerated GC cell growth, proliferation, and invasion. Unexpectedly, two previous studies demonstrated that although Smurf1 promotes Axin1 protein ubiquitination, Smurf1 does not accelerate ubiquitin-dependent Axin1 degradation ( 10 , 28 ). Moreover, Smurf1 decreases the Wnt pathway activation through disturbing the combination of Axin1 with LRP5/6 ( 10 , 28 ). Given the important roles of Smurf1 and the Wnt pathway in cancer progression, we explored the effect of Smurf1 on regulating the Wnt/β-catenin signaling activation in GC cells. Inconsistent with previous results, Smurf1 overexpression cannot affect Axin1 protein level on GC cells but decreases Axin2 protein expression through binding to Axin2 for its ubiquitination and proteasomal degradation and results in subsequent activation of Wnt pathway. Limitations Although Smurf1 expression was shown to be increased in the TCGA and GSE13911 datasets, it is essential to validate the increase in a larger clinical sample size. Furthermore, the correlation between Smurf1 level and OS and DFS in GC needs further investigation in a larger sample size and an extended follow-up duration. In addition, it is meaningful to identify whether Smurf1 activates Wnt signaling and thereby promotes cancer cell growth and migration in other types of tumors besides GC. Conclusion These data demonstrate that Smurf1 accelerates GC growth and metastasis through regulating Axin2-dependent Wnt pathway activation, providing a promising target for treating this disease. Declarations Author Contributions: Conception and design: JJ, JY, ZF Provision of study materials or patients: All authors. Collection and assembly of data: JY, BJ, JH, ZF, ZC, BJ, JH, JG. Data analysis and interpretation: ZF, ZC, BJ, JH, JG. Manuscript writing: All authors. Final approval of manuscript: All authors. Funding This work was supported by the key project of the Project of Shanghai Municipal Health Commission [Grant No.202140472]. Availability of data and materials All data in our study are available upon request to Zhen Feng. Ethical approval and consent to participate The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by committee for the Protection of Human Subjects board of XuHui District Center Hospital in the approval (NO. 2022052) and informed consent was obtained from all individual participants in accordance with the ARRIVE guiding to minimize animal suffering. Nine weeks later, mice were euthanized by pentobarbital overdose. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Patient consent for publication Informed consent was obtained from all individual participants. Conflict of Interest All authors have completed the ICMJE uniform disclosure form. The authors have no conflicts of interest to declare. Reporting Checklist The authors have completed the CONSANT reporting checklist. References Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 71 , 209–249 (2021). Yang, K. et al. Real-world outcomes of regorafenib combined with immune checkpoint inhibitors in patients with advanced or metastatic microsatellite stable colorectal cancer: A multicenter study (CII, 2021). Herszenyi, L. & Tulassay, Z. Epidemiology of gastrointestinal and liver tumors. Eur. Rev. Med. Pharmacol. Sci. 14 , 249–258 (2010). Brenner, H., Rothenbacher, D. & Arndt, V. Epidemiology of stomach cancer. Methods Mol. Biol. 472 , 467–477 (2009). Naumann, M. & Crabtree, J. E. Helicobacter pylori-induced epithelial cell signalling in gastric carcinogenesis. Trends Microbiol. 12 , 29–36 (2004). Fu, L., Cui, C. P., Zhang, X. & Zhang, L. The functions and regulation of Smurfs in cancers. Sem. Cancer Biol. 67 , 102–116 (2020). Guo, R. et al. Ubiquitin ligase Smurf1 mediates tumor necrosis factor-induced systemic bone loss by promoting proteasomal degradation of bone morphogenetic signaling proteins. J. Biol. Chem. 283 , 23084–23092 (2008). Yamashita, M. et al. Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation. Cell 121 , 101–113 (2005). Wei, X. et al. Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation. J. Cell. Biol. 216 , 1455–1471 (2017). Fei, C. et al. Smurf1-mediated Lys29-linked nonproteolytic polyubiquitination of axin negatively regulates Wnt/beta-catenin signaling. Mol. Cell. Biol. 33 , 4095–4105 (2013). Tao, Y., Sun, C., Zhang, T. & Song, Y. SMURF1 promotes the proliferation, migration and invasion of gastric cancer cells. Oncol. Rep. 38 , 1806–1814 (2017). Liu, J. et al. Wnt/beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal. Transduct. Target. therapy . 7 , 3 (2022). Koelman, E. M. R., Yeste-Vazquez, A. & Grossmann, T. N. Targeting the interaction of beta-catenin and TCF/LEF transcription factors to inhibit oncogenic Wnt signaling. Bioorg. Med. Chem. 70 , 116920 (2022). Skronska-Wasek, W., Gosens, R., Konigshoff, M. & Baarsma, H. A. WNT receptor signalling in lung physiology and pathology. Pharmacol. Ther. 187 , 150–166 (2018). Dejana, E. The role of wnt signaling in physiological and pathological angiogenesis. Circul. Res. 107 , 943–952 (2010). Baron, R. & Kneissel, M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat. Med. 19 , 179–192 (2013). Yu, P. et al. Deep Targeted Sequencing and Its Potential Implication for Cancer Therapy in Chinese Patients with Gastric Adenocarcinoma. oncologist 26 , e756–e768 (2021). Galluzzi, L., Spranger, S., Fuchs, E. & Lopez-Soto, A. WNT Signaling in Cancer Immunosurveillance. Trends Cell Biol. 29 , 44–65 (2019). Li, S. et al. Wnt/beta-Catenin Signaling Axis Is Required for TFEB-Mediated Gastric Cancer Metastasis and Epithelial-Mesenchymal Transition. Mol. Cancer Res. 18 , 1650–1659 (2020). Matsumoto, S. et al. Wnt Signaling Stimulates Cooperation between GREB1 and HNF4alpha to Promote Proliferation in Hepatocellular Carcinoma. Cancer Res. 83 , 2312–2327 (2023). Isik, A. & Firat, D. Letter to the editor concerning Most cited 100 articles from Turkey on abdominal wall hernias: a bibliometric study. Turk. J. Surg. 37 , 193–194 (2021). Foord, S. M. et al. International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol. Rev. 57 , 279–288 (2005). Reyes, M., Flores, T., Betancur, D., Pena-Oyarzun, D. & Torres, V. A. Wnt/beta-Catenin Signaling in Oral Carcinogenesis. Int. J. Mol. Sci. 21 , (2020). Clevers, H. Wnt/beta-catenin signaling in development and disease. Cell 127 , 469–480 (2006). Shah, K. & Kazi, J. U. Phosphorylation-Dependent Regulation of WNT/Beta-Catenin Signaling. Front. Oncol. 12 , 858782 (2022). Jing, J. C. et al. KDM4B promotes gastric cancer metastasis by regulating miR-125b-mediated activation of Wnt signaling. J. Cell. Biochem. , (2018). Gammons, M. V., Renko, M., Johnson, C. M., Rutherford, T. J. & Bienz, M. Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled. Mol. Cell . 64 , 92–104 (2016). Fei, C. et al. Smurf1-mediated axin ubiquitination requires Smurf1 C2 domain and is cell cycle-dependent. J. Biol. Chem. 289 , 14170–14177 (2014). Winer, J., Jung, C. K., Shackel, I. & Williams, P. M. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal. Biochem. 270 , 41–49 (1999). Zhang, W. et al. A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation. PLoS Biol. 19 , e3001361 (2021). Tano, K. et al. MALAT-1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility-related genes. FEBS Lett. 584 , 4575–4580 (2010). Feng, W. et al. Kruppel-like factor 4 promotes c-Met amplification-mediated gefitinib resistance in non-small-cell lung cancer. Cancer Sci. 109 , 1775–1786 (2018). Badary, D. M., Abdel-Wanis, M. E., Hafez, M. Z. & Aboulhagag, N. A. Immunohistochemical analysis of PTEN, HER2/neu, and ki67 expression in patients with gastric cancer and their association with survival. Pathophysiology 24 , 99–106 (2017). Liu, J. et al. Human papillomavirus type 16 E7 oncoprotein-induced upregulation of lysine-specific demethylase 5A promotes cervical cancer progression by regulating the microRNA-424-5p/suppressor of zeste 12 pathway. Exp. Cell. Res. 396 , 112277 (2020). Yu, Z. et al. A novel UBE2T inhibitor suppresses Wnt/beta-catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination. Oncogene 40 , 1027–1042 (2021). Flanagan, D. J., Vincan, E. & Phesse, T. J. Wnt Signaling in Cancer: Not a Binary ON:OFF Switch. Cancer Res. 79 , 5901–5906 (2019). Sun, J. et al. The Sensitivity Prediction of Neoadjuvant Chemotherapy for Gastric Cancer. Front. Oncol. 11 , 641304 (2021). Chen, J. et al. Neoadjuvant chemoradiotherapy for resectable gastric cancer: A meta-analysis. Front. Oncol. 12 , 927119 (2022). Jiang, M. et al. miR-1254 inhibits cell proliferation, migration, and invasion by down-regulating Smurf1 in gastric cancer. Cell Death Dis. 10 , 32 (2019). Lu, L. et al. MicroRNA-424 regulates cisplatin resistance of gastric cancer by targeting SMURF1 based on GEO database and primary validation in human gastric cancer tissues. OncoTargets therapy . 12 , 7623–7636 (2019). Du, M. G. et al. Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis. J. Biol. Chem. 297 , 101307 (2021). Xia, Q., Li, Y., Han, D. & Dong, L. SMURF1, a promoter of tumor cell progression? Cancer Gene Ther. 28 , 551–565 (2021). Chen, J. et al. Effects of the Wnt/beta-Catenin Signaling Pathway on Proliferation and Apoptosis of Gastric Cancer Cells. Contrast media & molecular imaging. : : 5132691, 2022. (2022). Qi, J. et al. Targeting Wnt/beta-Catenin Signaling by TET1/FOXO4 Inhibits Metastatic Spreading and Self-Renewal of Cancer Stem Cells in Gastric Cancer. Cancers 14 , (2022). Tang, W. et al. The miR-3648/FRAT1-FRAT2/c-Myc negative feedback loop modulates the metastasis and invasion of gastric cancer cells. Oncogene , (2022). Liang, W., Zhang, T., Huo, J. & Yang, J. MARCH1 promotes the growth and maintaining of stem cell-like characteristics of gastric cancer cells by activating the Wnt/beta-catenin signaling pathway. Tissue cell. 78 , 101895 (2022). Nong, J. et al. Phase separation of Axin organizes the beta-catenin destruction complex. J. Cell Biol. 220 , (2021). Wu, R., Zhai, Y., Fearon, E. R. & Cho, K. R. Diverse mechanisms of beta-catenin deregulation in ovarian endometrioid adenocarcinomas. Cancer Res. 61 , 8247–8255 (2001). Nguyen, V. H. L., Hough, R., Bernaudo, S. & Peng, C. Wnt/beta-catenin signalling in ovarian cancer: Insights into its hyperactivation and function in tumorigenesis. J. ovarian Res. 12 , 122 (2019). Kim, M. S., Kim, S. S., Ahn, C. H., Yoo, N. J. & Lee, S. H. Frameshift mutations of Wnt pathway genes AXIN2 and TCF7L2 in gastric carcinomas with high microsatellite instability. Hum. Pathol. 40 , 58–64 (2009). Zhai, J. et al. ALKBH5 Drives Immune Suppression Via Targeting AXIN2 to Promote Colorectal Cancer and Is a Target for Boosting Immunotherapy. Gastroenterology 165 , 445–462 (2023). Yu, J. et al. CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/beta-catenin signaling via transactivation of GSK-3beta and Axin2 expression. Cell. Death Dis. 10 , 26 (2019). Picco, G. et al. Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions. EMBO Mol. Med. 9 , 293–303 (2017). Zhang, Y. et al. UBE3C promotes proliferation and inhibits apoptosis by activating the beta-catenin signaling via degradation of AXIN1 in gastric cancer. Carcinogenesis 42 , 285–293 (2021). Lee, E., Salic, A., Kruger, R., Heinrich, R. & Kirschner, M. W. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS biology 1: E10, (2003). Mazzoni, S. M. & Fearon, E. R. AXIN1 and AXIN2 variants in gastrointestinal cancers. Cancer Lett. 355 , 1–8 (2014). Supplementary Figures Supplementary Figures S1 is not available with this version. Additional Declarations No competing interests reported. Supplementary Files SupplementarytableS1.docx SupplementarytableS2.docx SupplementarytableS3.docx SupplementarytableS4.docx SupplementarytableS5.xlsx Rawwbimages.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Aug, 2025 Reviews received at journal 25 Jul, 2025 Reviews received at journal 18 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers invited by journal 23 Jun, 2025 Editor assigned by journal 18 Jun, 2025 Editor invited by journal 09 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 First submitted to journal 18 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6694191","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475450576,"identity":"71c33d96-40bd-4b09-b521-eea9de3e94d8","order_by":0,"name":"Jinling Yu","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jinling","middleName":"","lastName":"Yu","suffix":""},{"id":475450577,"identity":"18d7ac97-0ef1-45a5-b14a-ecbc49caf185","order_by":1,"name":"Jiachen Jing","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiachen","middleName":"","lastName":"Jing","suffix":""},{"id":475450578,"identity":"5b301baf-5796-4a11-bac8-8485934e6884","order_by":2,"name":"Zhen Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIie3RMUvEMBTA8ZZAuzzJmoDgV3hyUBXK3Vd54aBTcXFxjBTionv9IuJmStZyrsItvVFwaHHqolZwEkxvFMxveEN4/+GRKAqCPyjV04inwRlzHV3mwLn2J2C/E3ltCuza4lDWds8E2xblzrgcNc0k6a17GR/y8+iZECl5Aoxs3A+lJ4FNcSrb4iKuiTqCLZwwzeTd/e/JSpQZHhunKkEWSWzhTNuEHXgSOHrNUJkPZYTSgnADaGkmEbDoGmPVDbhIENk9Eiiz+MqsVZ2aBMmuQdZN5b0F0nYxjGapHh1/243vyxXnVdMPnmSSiB8PX9/kx/q5jSAIgn/uEyTeWDiLG3+PAAAAAElFTkSuQmCC","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":true,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Feng","suffix":""},{"id":475450579,"identity":"45b6c539-2ac2-4db6-853b-a31fc3e3bbf4","order_by":3,"name":"Zhonghua Chen","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhonghua","middleName":"","lastName":"Chen","suffix":""},{"id":475450580,"identity":"99f1f829-061d-4dac-a9d5-8ea798a67c1c","order_by":4,"name":"Beina Ji","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Beina","middleName":"","lastName":"Ji","suffix":""},{"id":475450581,"identity":"f5890575-1bcb-4280-830b-113fcb2de348","order_by":5,"name":"Jing Hong","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Hong","suffix":""},{"id":475450582,"identity":"78689e10-27ca-42ee-9dc6-07830a308414","order_by":6,"name":"Jing Guo","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Guo","suffix":""},{"id":475450583,"identity":"099a9b69-945d-4a1e-a378-3eef7ca92df4","order_by":7,"name":"Nan Tang","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Tang","suffix":""},{"id":475450584,"identity":"99bcee07-4700-4b0f-ae96-96cfd3d5b6f1","order_by":8,"name":"Shuo Gu","email":"","orcid":"","institution":"Xu Hui District Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2025-05-19 01:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6694191/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6694191/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-23707-3","type":"published","date":"2025-11-19T15:58:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85565920,"identity":"24044a0d-0347-4b22-a100-9aceb39d2669","added_by":"auto","created_at":"2025-06-27 14:32:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2308140,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 expression was increased in GC tissues.\u003c/p\u003e\n\u003cp\u003e(A) Smurf1\u003cstrong\u003e \u003c/strong\u003elevels in the TCGA-STAD database (408 STAD tissues and 211 normal tissues) were analyzed using the GEPIA toolaccording to the following criteria: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and fold change (FC) \u0026lt; 1.5. (B) Smurf1\u003cstrong\u003e \u003c/strong\u003elevels were significantly increased in GC tissues (n = 26) compared with normal tissues (n = 31) in GSE13911 dataset. \u003cem\u003ep\u003c/em\u003e = 0.001. (C) qRT-PCR analysis of Smurf1 mRNA levels in GC tissues (n = 29) and matched para-cancerous tissues. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (D) The OS was analyzed using the GEPIA tool and TCGA-LUAD data according to Smurf1\u003cstrong\u003e \u003c/strong\u003eexpression. \u003cem\u003ep\u003c/em\u003e = 0.82. (E) The DFS was analyzed using the GEPIA tool and TCGA-LUAD data according to Smurf1\u003cstrong\u003e \u003c/strong\u003eexpression. \u003cem\u003ep\u003c/em\u003e = 0.00087.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/fed3b4e8c785a5c2cd11706b.jpg"},{"id":85565573,"identity":"b3e84a26-c4f1-4a6f-b799-879579e9c987","added_by":"auto","created_at":"2025-06-27 14:24:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1552015,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 accelerated GC cell growth, proliferation, and invasion.\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of Smurf1 mRNA levels in different GC cell lines (AGS and MKN-45) and Ges-1 cells. (B) AGS cell proliferation was assessed using the CCK-8 assay at different time points after Smurf1 overexpression. (C and D) After Smurf1 overexpression, AGS cell growth was measured via colony formation assay after two weeks. (E and F) After Smurf1 overexpression, AGS cell invasion was measured via transwell invasion assay after 36 h. The results were shown as the median (1\u003csup\u003est\u003c/sup\u003e quartile and 3\u003csup\u003erd\u003c/sup\u003e quartile). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/cdb10cc755208bf1b3e91d3d.jpg"},{"id":85565921,"identity":"a1cd779c-6b76-4a33-adae-167b3aa6b5e6","added_by":"auto","created_at":"2025-06-27 14:32:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2909135,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 accelerated GC growth and metastasis in mouse xenograft model.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(A and B) AGS and Smurf1-overexpressed AGS cells were subcutaneously injected\u0026nbsp;into the left flank of nude mice, and tumour growth was monitored with a vernier calliper at days 7, 14, 21, and 35 and calculated by the formula length × width\u003csup\u003e2 \u003c/sup\u003e× 0.5. (C and D) Tumour tissues were used to carry out IHC analysis of Ki67 to assess \u003cem\u003ein vivo\u003c/em\u003e cell proliferation. Lung metastases\u0026nbsp;were assessed by\u0026nbsp;H\u0026amp;E\u0026nbsp;staining\u0026nbsp;(E) and then calculated (F) in\u0026nbsp;lung\u0026nbsp;tissues. The results were shown as the median (1\u003csup\u003est\u003c/sup\u003e quartile and 3\u003csup\u003erd\u003c/sup\u003e quartile). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/d5f53c4c885fe0a566fb316b.jpg"},{"id":85565923,"identity":"0e742f4f-b017-46c1-9fe5-4e842d905999","added_by":"auto","created_at":"2025-06-27 14:32:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":811753,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 promoted Axin2 ubiquitination and ubiquitin-dependent degradation.\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR analysis of Axin1 and Axin2 mRNA levels in AGS cells after Smurf1 overexpression. (B) Western blot analysis of Axin1 and Axin2 protein levels in AGS cells after Smurf1 overexpression. (C) Co-IP assay of the direct combination of Smurf1 with Axin2 in AGS cells using the Smurf1 antibody, followed by western blot analysis using the Axin2 antibody. (D and E) After Smurf1 overexpression, \u003cem\u003ede novo\u003c/em\u003e protein synthesis was inhibited in AGS cells using 40 µM of Chx, and then Axin2 protein levels were measured using western blot assays at different time points. (F) After Smurf1 overexpression in AGS cells, a Co-IP assay was carried out using the Axin2 antibody, followed by western blot analysis using the ubiquitin antibody. (G) After Smurf1 overexpression and treatment with 20 µM of MG132 in AGS cells, Axin2 protein levels were measured using the western blot assay. The results were shown as the median (1\u003csup\u003est\u003c/sup\u003e quartile and 3\u003csup\u003erd\u003c/sup\u003e quartile). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/e95eaecebb2fd1f9f96d031e.jpg"},{"id":85566600,"identity":"c10f0e6c-0929-4033-97cc-d5f9c0718c2a","added_by":"auto","created_at":"2025-06-27 14:40:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":558711,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 promoted Wnt/β-catenin signaling activation.\u003c/p\u003e\n\u003cp\u003e(A) After Smurf1 overexpression in AGS cells, Wnt/β-catenin signaling activation was assessed using the TOPflash/FOPflash dual luciferase reporter system. Relative luciferase units (RLU) were calculated to assess β-catenin-triggered transcription. After Smurf1 overexpression in AGS cells, cytoplasmic (B and D) and nuclear (C and D) β-catenin protein levels were measured using the western blot assay. β-actin and Lamin B1 served as cytoplasmic and nuclear\u0026nbsp;markers, respectively. (E) After Smurf1 overexpression in AGS cells, β-catenin nuclear translocation was assessed using IF analysis. Red fluorescence indicated β-catenin, and blue fluorescence indicated the nucleus. The results were shown as the median (1\u003csup\u003est\u003c/sup\u003e quartile and 3\u003csup\u003erd\u003c/sup\u003e quartile). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/1825e2cf42a2dabaebefcd51.jpg"},{"id":85565578,"identity":"8b3cca28-1af9-4f1b-8b4d-edd33d9eaca4","added_by":"auto","created_at":"2025-06-27 14:24:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":135789,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 promoted GC cell proliferation and invasion through activating the Wnt/β-catenin pathway.\u003c/p\u003e\n\u003cp\u003e(A) AGS cells were overexpressed with Smurf1 and treated with 10µM of IWR-1, and then cell proliferation was assessed using the CCK-8 assay at the different time points. (B) AGS cells were overexpressed with Smurf1 and treated with 10 µM of IWR-1, and cell invasion was assessed via transwell invasion assay after 36 h. The results were shown as the median (1\u003csup\u003est\u003c/sup\u003e quartile and 3\u003csup\u003erd\u003c/sup\u003e quartile). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/5806053a067a282f243f4658.jpg"},{"id":96650216,"identity":"f218c3f3-1d24-446b-a3fe-32f48fa6bcb2","added_by":"auto","created_at":"2025-11-24 16:09:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9199735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/b028cf9e-9084-445f-8fec-d4a09c2e9255.pdf"},{"id":85565570,"identity":"936fa8e1-f3a8-4726-998a-56b05cf4f7a4","added_by":"auto","created_at":"2025-06-27 14:24:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13484,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/ad70f3270b3b18acc6a34924.docx"},{"id":85565572,"identity":"f667f774-db5e-403f-a7f3-16beadedc271","added_by":"auto","created_at":"2025-06-27 14:24:12","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25160,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/e75d4e564ad7dbee7e7e6f34.docx"},{"id":85565579,"identity":"9bd045c5-6a43-41f3-b53e-7c1bc833c5b5","added_by":"auto","created_at":"2025-06-27 14:24:12","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19745,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/2e1dd404d0593eefbd0bade6.docx"},{"id":85565574,"identity":"7a240be8-f27d-481e-adaa-28864f95dc5a","added_by":"auto","created_at":"2025-06-27 14:24:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":23278,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/8d380a29a978d2a4f981be12.docx"},{"id":85565924,"identity":"9e6b1720-31b0-4973-a249-df676cd031b0","added_by":"auto","created_at":"2025-06-27 14:32:13","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":41161,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarytableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/a54879c698e9233ea82dd0fa.xlsx"},{"id":85565589,"identity":"1a165dd6-75bc-41fb-a37f-1b96d5562a50","added_by":"auto","created_at":"2025-06-27 14:24:13","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1542657,"visible":true,"origin":"","legend":"","description":"","filename":"Rawwbimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6694191/v1/29035fdef488067b766bd158.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Smurf1 promotes gastric cancer growth and metastasis by regulating Axin2- dependent Wnt/β-catenin signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is a common cancer globally. In 2020, there were approximately 1,100,000 new cases and 769,000 deaths (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), ranking fifth for morbidity and fourth for death rate in the world (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Although GC morbidity and mortality have generally declined throughout the years in most populations, they are increasing in young adults (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). At present, surgical excision remains the only opportunity to cure GC. Chemotherapy and radiotherapy following radical surgery are beneficial to improve the overall survival (OS) rate, but the prognosis remains disappointing (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Revealing the mechanisms underlying GC progression and chemoradiotherapy resistance is indispensable to improving disease outcomes.\u003c/p\u003e \u003cp\u003eSMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) was initially discovered as a modulator of Smad protein stability (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Under inflammatory stress, upregulated Smurf1 results in bone loss by facilitating Smad1 protein ubiquitination and subsequent degradation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Subsequent to this discovery, numerous target proteins regulated by Smurf1 have been identified across various biological processes. For instance, Smurf1 inhibits the osteogenic activity of osteoblasts and bone homeostasis by directly binding to mitogen-activated protein kinase kinase kinase 2 (MEKK2) and accelerating its degradation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Smurf1 functions as a limiting factor in regulating integrin activation and subsequent cell function by controlling Kindlin 2 protein degradation (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Smurf1 facilitates breast cancer metastasis through physically interacting with Ras homolog family member A, leading to its ubiquitination and degradation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Although Smurf1 has been reported as a potential oncogenic factor in GC (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), the underlying mechanism remains unclear.\u003c/p\u003e \u003cp\u003eWnt/β-catenin pathway is crucial for tissue development and homeostasis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, its over-activation leads to various types of diseases (\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), especially in cancer initiation and progression (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). For example, Wnt signaling plays a vital role in transcription factor EB (TFEB)-triggered GC cell invasion and cancer metastasis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). High TFEB expression is correlated with worse OS and disease-free survival (DFS) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Matsumoto et al. demonstrated that Wnt signaling activation accelerates hepatocellular carcinoma progression by increasing the expression of growth regulation by estrogen in breast cancer 1 (GREB1) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The Wnt pathway comprises four key components: ligands, receptors, cytoplasmic components like β-catenin and its \u0026ldquo;destruction complex\u0026rdquo; (GSK-3β, Axin, CK1, and APC), and nuclear components (TCF/LEF transcription factors) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The hallmark of Wnt pathway activation is the transfer of β-catenin into the nucleus, which initiates the transcription of TCF/LEF target genes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In a previous study, we demonstrated that histone lysine demethylase 4B (KDM4B) facilitates GC cell invasion through accelerating miR-125b-dependent nuclear translocation of β-catenin (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAxis inhibition protein (Axin) directly interacts with GSK-3β, APC, CK1, and β-catenin, serving as a scaffold protein for the \u0026ldquo;destruction complex\u0026rdquo; in the Wnt pathway (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Axin exists in two subtypes: Axin1 and Axin2. Previous research has indicated that Smurf1 regulates Wnt pathway activation through binding to the Axin1 protein and accelerating its ubiquitination (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, the association of Smurf1 with Axin2 is not well understood. The study primarily aimed to explore how Smurf1 regulates Wnt pathway activation and GC progression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eTranscriptomic and clinical data of 408 GC specimens and 211 normal specimens were obtained from the Cancer Genome Atlas-Stomach Adenocarcinoma (TCGA-STAD) database and data analysis was carried out with GEPIA tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/detail.php?gene\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/detail.php?gene\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Smurf1 levels were analyzed in GC specimens and normal specimens. The correlations between Smurf1 level and both OS and DFS in GC patients were analyzed through the TCGA dataset. In addition, the Smurf1 levels were analyzed in the GSE13911 dataset.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinical specimens and cell culture\u003c/h3\u003e\n\u003cp\u003eTwenty-nine pairs of tumor tissues and para-carcinoma tissues were collected from Xu Hui District Center Hospital. Informed consent was obtained from all individual participants.\u003c/p\u003e \u003cp\u003eThree cell lines, AGS, MKN-45, and Ges-1, were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in DMEM/F12 media (TIANHANG, Hangzhou, China) containing 10% fetal bovine serum (FBS, TIANHANG) in a cell incubator. The recombinant plasmid encoding the complete coding region of Smurf1, pcDNA-Smurf1, was constructed in our laboratory and transfected into GC cells to overexpress Smurf1 using PEI max (MineBio Life Sciences, Shanghai, China).\u003c/p\u003e\n\u003ch3\u003eCell counting kit 8 (CCK-8)\u003c/h3\u003e\n\u003cp\u003eAfter overexpression with Smurf1 or treatment with 10 \u0026micro;M of IWR-1 (MCE, NJ, USA), AGS cells were seeded into 96-well plates and incubated for varying durations. After that, 10 \u0026micro;L of CCK-8 (MCE) were applied to treat the cells. Sixty minutes later, optical density (OD) values were recorded at 450 nm with a ReadMax 1200 microplate reader (Shanpu, Shanghai, China).\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eAfter Smurf1 overexpression, AGS cells were seeded into 12-well plates (approximately 350 cells per well) for two weeks until visible colonies occurred. After that, cells were rinsed thrice using phosphate-buffered saline (PBS), immobilized in methyl alcohol for 10 min, and stained in crystal violet for approximately 10 min. Cell colonies were calculated with an XDS-900C inverted microscope (Caikon, Shanghai, China).\u003c/p\u003e\n\u003ch3\u003eTranswell invasion assay\u003c/h3\u003e\n\u003cp\u003eMatrigel-coated transwell chambers with an 8-\u0026micro;m pore size (Corning, NY, USA) were applied to measure cell invasion. After Smurf1 overexpression or treatment with 10 \u0026micro;M of IWR-1, AGS cells were seeded into the upper chamber in DMEM/F12 medium without serum (10000 cells per well). The lower chamber was filled with 500 \u0026micro;L of DMEM/F12 complete medium. After 36 h, noninvasive cells were removed with cotton swabs. Cells that invaded the lower membrane surface were fixed in methyl alcohol for 15 min, stained in crystal violet for 15 min, and then photographed using an inverted microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated with TRIzol reagent (Beyotime, Shanghai, China), treated with DNase I (TaKaRa, Tokyo, Japan), quantified using a Bioanalyzer 2100 (Agilent, CA, USA), and then reverse-transcribed into first-strand cDNA in a 20 \u0026micro;l solution containing M-MLV (TaKaRa) and Oligo (dT) primers. qRT-PCR was performed on an Ariazmx real-time PCR system (Agilent) in a 10 \u0026micro;l solution containing cDNA, primers (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and 2\u0026times;T5 Fast qPCR Mix (TSINGKE, Beijing, China). β-actin was used as a reference gene, and mRNA levels were calculated by the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e formula (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot\u003c/div\u003e \u003cp\u003eTotal protein was isolated with RIPA lysis buffer (Thermo Fisher Scientific, MA, USA). After quantitating by the BCA method (Abcam, CA, USA), protein was measured through a western blot assay as previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The primary antibodies were listed as follows: Axin1 (1.5 \u0026micro;g/mL, PA5-21042, Thermo Fisher), Axin2 (1:1500, ab109307, Abcam), ubiquitin (1:3000, ab140601, Abcam), β-catenin (1:2000, 71-2700, Thermo Fisher), and β-actin (1:10000, ab6276, Abcam). The second antibody used in the study was ab7090 (1:10000, Abcam). Protein bands were visualized by a BeyoECL Plus (Beyotime).\u003c/p\u003e\n\u003ch3\u003eCycloheximide (Chx) chase assay\u003c/h3\u003e\n\u003cp\u003eAfter Smurf1 overexpression for 24 h, AGS cells were incubated with 40 \u0026micro;M of Chx (Merck, MA, USA) for 0, 2, 4, or 8 h. Subsequently, total protein was isolated with RIPA lysis buffer to assess Axin2 protein through a western blot assay. For the ubiquitination analysis, western blot was carried out to assess Axin2 protein in AGS cells following Smurf1 overexpression and MG132 (20 \u0026micro;M, MCE) treatment for 24 h.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eBriefly, total protein was collected from AGS cells and incubated with the Smurf1 antibody (1:150, ab57573, Abcam) for 15 h (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Protein A/G magnetic beads (MCE) were placed into IP mixtures to bind antibodies and proteins. Subsequently, Axin2 protein in immunoprecipitates was measured using western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eAfter Smurf1 overexpression, AGS cells were co-transfected with TOPflash construct (80 ng, Merck) or FOPflash construct (Merck) and pRL-TK plasmid (8 ng, Promega, WI, USA) using PEI max transfection reagent. The pRL-TK plasmid served as a control for transfection efficiency. After treatment with Wnt3a for 48 h, dual luciferase activity was assessed using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Data were shown as a normalized TOPflash/FOPflash activity ratio.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eAGS cells on glass slides were immobilized with methyl alcohol, perforated with Triton X-100, and blocked with fluorescent blocking buffer (Thermo Fisher Scientific). Subsequently, cells were treated with an antibody targeting β-catenin (1:400, 71-2700, Thermo Fisher Scientific), followed by an Alexa Fluor\u0026reg; 647-labeled second antibody. The fluorescence signal was recorded with an FCK-50C fluorescence microscope (Caikon).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMouse xenograft model\u003c/h2\u003e \u003cp\u003eThe study was carried out with the approval of the Experimental Animal Committee of Xu Hui District Center Hospital (No. 2022052) adherence to the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. Six-week-old BALB/c nude mice (Charles River, Shanghai, China) were raised in a pathogen-free room maintained at 24\u0026ndash;26 \u003csup\u003eo\u003c/sup\u003eC. AGS cells and Smurf1-overexpressed AGS cells were collected to construct AGS xenograft model. In brief, the mouse xenograft model was created by subcutaneously injecting 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e AGS cells into the left flank of mice (n\u0026thinsp;=\u0026thinsp;5). Subcutaneous (s.c.) tumours growth was monitored with a vernier calliper at days 7, 14, 21, and 35 and calculated by the formula length \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.5 (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Immunohistochemical (IHC) analysis of Ki67 was carried out to assess \u003cem\u003ein vivo\u003c/em\u003e cell proliferation, as previously described (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). For the experimental metastasis assay, AGS cells or Smurf1-overexpressed AGS cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells, 100 \u0026micro;l PBS) were injected into mice by tail vein. Nine weeks later, mice were euthanized by pentobarbital overdose, and lung tissues were harvested. Hematoxylin-eosin (H\u0026amp;E) staining was performed to quantify the tumour nodules in lung tissues, as previously described (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eEach experiment was repeated at least three times, and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed using GraphPad Prism 7.0 (CA, USA). Normality of all data was evaluated by the Shapiro-Wilk test (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Homogeneity of variance was evaluated by the F test or Brown-Forsythe test (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Data were analyzed with the Welch\u0026rsquo;s t-test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), Mann Whitney test (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), Kruskal-Wallis test followed by Dunnett\u0026rsquo;s post hoc test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and two-way ANOVA followed by Sidak\u0026rsquo;s multiple comparisons test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), respectively. The statistical analyses of TCGA-STAD data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) were carried out with GEPIA tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/detail.php?gene\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/detail.php?gene\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A comprehensive summary of the statistical methods and corresponding p values was shown in Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e. A p value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSmurf1 expression was increased in GC tissues\u003c/h2\u003e \u003cp\u003eTo explore the biological effect of Smurf1 on GC progression, Smurf1 expression was first analyzed in two publicly available datasets, TCGA and GSE13911. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA revealed that Smurf1 expression was prominently increased in GC tissues compared to normal controls in the TCGA-STAD database. In the GSE13911 dataset, Smurf1 expression was also upregulated in GC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, 29 pairs of GC tissues and para-cancerous tissues were used to assay the Smurf1 level. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC displayed that Smurf1 expression was prominently enhanced in tumour tissues compared with para-cancerous tissues. The correlation between Smurf1 levels and the prognosis of GC patients was further investigated. Although high Smurf1 levels were not linked to worse OS (n\u0026thinsp;=\u0026thinsp;384, p\u0026thinsp;=\u0026thinsp;0.82, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), they were significantly associated with DFS (n\u0026thinsp;=\u0026thinsp;384, p\u0026thinsp;=\u0026thinsp;0.00087, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The frequent increase of Smurf1 indicates that Smurf1 might exert a tumor activator in GC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSmurf1 overexpression accelerated GC cell growth and invasion\u003c/h2\u003e \u003cp\u003eThe biological effect of Smurf1 on GC cell growth, proliferation, and invasion was next assessed. To this end, Smurf1 expression was first assayed in GC cells and a normal gastric epithelial cell. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA showed that Smurf1 expression was increased in AGS and MKN-45 cells compared with Ges-1 cells. Smurf1 overexpression was carried out in AGS cells, and its biological role was assessed in AGS cells because the endogenous Smurf1 levels in AGS cells were lower than those in MKN-45 cells. As shown in Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Smurf1 overexpression significantly accelerated AGS cell proliferation. Smurf1 overexpression also markedly facilitated AGS cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Moreover, Smurf1 overexpression prominently accelerated AGS cell invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003eFurthermore, the roles of Smurf1 overexpression in tumour growth and metastasis were investigated in a mouse xenograft model. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B, Smurf1 overexpression significantly promoted tumor growth. Ki67 staining from tumor tissues revealed that Smurf1 overexpression accelerated cell proliferation \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). The roles of Smurf1 in facilitating cancer lung metastasis were measured by histological analysis. H\u0026amp;E staining showed that Smurf1 overexpression caused the formation of larger and more lung metastatic nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSmurf1 promoted Axin2 ubiquitination and proteasome-dependent degradation\u003c/h2\u003e \u003cp\u003eWe next predicted the potential target proteins regulated by Smurf1 using the UbiBrowser tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ubibrowser.ncpsb.org.cn/ubibrowser/\u003c/span\u003e\u003cspan address=\"http://ubibrowser.ncpsb.org.cn/ubibrowser/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, and a total of 359 proteins were identified (Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Here, Axin1 and Axin2 were selected for further validation based on the following three reasons: i) Previous studies have demonstrated that Smurf1 mediates Axin1 ubiquitination and regulates Wnt/β-catenin signalling in murine embryonic fibroblasts (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), ii) Axin2 is a potential target protein regulated by Smurf1 (Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e, Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), iii) abnormal activation of the Wnt pathway is vital for cancer progression (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B, although Smurf1 overexpression did not affect Axin1 and Axin2 mRNA expression in AGS cells, Smurf1 markedly decreased Axin2 protein expression, indicating that Smurf1 might directly interact with Axin2 and promote Axin2 protein degradation in GC cells.\u003c/p\u003e \u003cp\u003eTo explore whether Smurf1 facilitates Axin2 ubiquitination and subsequent proteasome-dependent degradation, the direct combination of Smurf1 with Axin2 was assayed through Co-IP, and results revealed that Smurf1 directly combined with Axin2 protein in AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To assess whether Smurf1 accelerated Axin2 protein degradation, AGS cells were incubated with Chx to prevent protein synthesis. Chx chase analysis showed that forced expression of Smurf1 markedly reduced the Axin2 protein levels in AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E). To reveal whether Smurf1 decreased Axin2 protein expression through ubiquitin-dependent degradation, Smurf1 was overexpressed in AGS cells, and then cell extracts were subjected to Co-IP analysis with an antibody targeting Axin2, followed by western blot assay using an anti-ubiquitin antibody. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF revealed that Smurf1 overexpression obviously promoted Axin2 ubiquitination. As expected, Smurf1 overexpression inhibited Axin2 protein level, whereas the effects were reversed by MG132, a proteasome inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), indicating that Smurf1 accelerates Axin2 ubiquitination and results in proteasome-dependent degradation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSmurf1 accelerated cell proliferation and invasion through regulating the Wnt pathway\u003c/h2\u003e \u003cp\u003eThe regulatory effects of Smurf1 on Wnt pathway activation were next assessed. Here, TOPflash/FOPflash, western blot, and IF analysis were carried out to determine Wnt activation or nuclear translocation of β-catenin. Smurf1-overexpressed AGS cells were transfected with TOPflash or FOPflash. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA showed that there was an increased TOPflash/FOPflash activity ratio in Smurf1-overexpressed AGS cells following Wnt3a treatment, suggesting that the Wnt pathway was activated due to Smurf1 overexpression. Western blot assay showed that Smurf1 overexpression accelerated the transfer of β-catenin into the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). IF assay also displayed the promoting role of Smurf1 in the nuclear translocation of β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, the roles of Smurf1 in AGS cell proliferation and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B) were suppressed by IWR-1, a Wnt pathway inhibitor. Collectively, these data demonstrate that Smurf1 accelerates GC cell proliferation and invasion by activating the Wnt pathway.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGC is usually diagnosed at advanced stages because of the absence of early typical symptoms and predictive biomarkers, which results in high mortality and a poor prognosis. Although chemoradiotherapy can effectively improve the prognosis of such patients, drug resistance is a main obstacle to successful therapy for advanced GC (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Smurf1 exhibits pro-oncogenic activity and is correlated with drug resistance in many types of tumours (\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e–\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). In the study, the mechanism by which Smurf1 accelerates GC progression was further explored. We demonstrated that, i) Smurf1 expression was upregulated in GC tissues, and Smurf1 overexpression predicted a poor DFS, ii) up-regulated Smurf1 facilitated GC growth and metastasis, iii) Smurf1 promoted Axin2 ubiquitination and proteasomal degradation, iv) Smurf1 accelerated GC cell growth and invasion by activating the Wnt pathway.\u003c/p\u003e \u003cp\u003eHyper-activation of the Wnt pathway is closely correlated with tumour progression in multiple pivotal aspects, such as cancer cell proliferation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), epithelial-mesenchymal transition (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), invasion (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), and stemness maintenance (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). As the scaffold protein for the “destruction complex” in the Wnt pathway, Axin is essential for inhibiting the activation of the Wnt pathway through the assembly the “destruction complex” (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Mutations or aberrant expressions of Axin were frequently observed in various types of tumours. Many studies have revealed a nonsense mutation of \u003cem\u003eAxin1\u003c/em\u003e and a frameshift mutation of \u003cem\u003eAxin2\u003c/em\u003e in epithelial ovarian cancer (EOC) (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Kim et al. identified nine frameshift mutations in \u003cem\u003eAxin2\u003c/em\u003e in 32 patients with GC (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). AlkB homolog 5 (ALKBH5) accelerates colorectal cancer (CRC) progression by promoting Axin2 mRNA degradation and thus over-activating Wnt/β-catenin signaling (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Caudal-related homeobox transcription factor 2 (CDX2) suppresses colon cancer cell growth through upregulating Axin2 expression and inactivating Wnt signaling (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Axin1 inhibition causes the acquired resistance of CRC cells to Wnt pathway blockade (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Axin2 protein degradation, regulated by E3 ubiquitin ligase E3C (UBE3C), causes the activation of the Wnt pathway and subsequent GC cell proliferation (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Unlike Axin1, Axin2 is a downstream gene of the TCF/LEF transcription factor. Given the positive regulation of Axin2 by the Wnt pathway and the fact that Axin2 is an indispensable member of the “destruction complex”, Axin2 might be a critical negative feedback regulator for the Wnt pathway in cancer cells (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). For example, Axin2 overexpression inactivates the Wnt pathway and represses colon cancer cell proliferation (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the underlying mechanisms remain unclear, the pro-oncogenic effect of Smurf1 has been revealed in GC. MicroRNA-mediated inhibition of Smurf1 suppresses GC cell proliferation and invasion and alleviates the resistance of GC cells to cisplatin (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). In the study, Smurf1 expression was analysed in publicly available datasets, and its biological role was assessed. Smurf1 levels were significantly increased in GC tissues compared to para-carcinoma tissues in the TCGA and GSE13911 databases. High Smurf1 levels predicted worse OS and DFS. Functionally, Smurf1 overexpression accelerated GC cell growth, proliferation, and invasion.\u003c/p\u003e \u003cp\u003eUnexpectedly, two previous studies demonstrated that although Smurf1 promotes Axin1 protein ubiquitination, Smurf1 does not accelerate ubiquitin-dependent Axin1 degradation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Moreover, Smurf1 decreases the Wnt pathway activation through disturbing the combination of Axin1 with LRP5/6 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Given the important roles of Smurf1 and the Wnt pathway in cancer progression, we explored the effect of Smurf1 on regulating the Wnt/β-catenin signaling activation in GC cells. Inconsistent with previous results, Smurf1 overexpression cannot affect Axin1 protein level on GC cells but decreases Axin2 protein expression through binding to Axin2 for its ubiquitination and proteasomal degradation and results in subsequent activation of Wnt pathway.\u003c/p\u003e "},{"header":"Limitations","content":"\u003cp\u003eAlthough Smurf1 expression was shown to be increased in the TCGA and GSE13911 datasets, it is essential to validate the increase in a larger clinical sample size. Furthermore, the correlation between Smurf1 level and OS and DFS in GC needs further investigation in a larger sample size and an extended follow-up duration. In addition, it is meaningful to identify whether Smurf1 activates Wnt signaling and thereby promotes cancer cell growth and migration in other types of tumors besides GC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThese data demonstrate that Smurf1 accelerates GC growth and metastasis through regulating Axin2-dependent Wnt pathway activation, providing a promising target for treating this disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design:\u0026nbsp;JJ, JY, ZF\u003c/p\u003e\n\u003cp\u003eProvision of study materials or patients: All authors.\u003c/p\u003e\n\u003cp\u003eCollection and assembly of data: JY, BJ, JH, ZF, ZC, BJ, JH, JG.\u003c/p\u003e\n\u003cp\u003eData analysis and interpretation:\u0026nbsp;ZF, ZC, BJ, JH, JG.\u003c/p\u003e\n\u003cp\u003eManuscript writing: All authors.\u003c/p\u003e\n\u003cp\u003eFinal approval of manuscript: All authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the key project of the Project of Shanghai Municipal Health Commission [Grant No.202140472].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data in our study are available upon request to\u0026nbsp;Zhen Feng.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by committee for the Protection of Human Subjects board of XuHui District Center Hospital in the approval (NO. 2022052) and informed consent was obtained from all individual participants in accordance with the ARRIVE guiding to minimize animal suffering. Nine weeks later, mice were euthanized by pentobarbital overdose. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have completed the ICMJE uniform disclosure form. The authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReporting Checklist\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have completed the CONSANT reporting checklist.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 209\u0026ndash;249 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, K. et al. \u003cem\u003eReal-world outcomes of regorafenib combined with immune checkpoint inhibitors in patients with advanced or metastatic microsatellite stable colorectal cancer: A multicenter study\u003c/em\u003e (CII, 2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerszenyi, L. \u0026amp; Tulassay, Z. Epidemiology of gastrointestinal and liver tumors. \u003cem\u003eEur. Rev. Med. Pharmacol. Sci.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 249\u0026ndash;258 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrenner, H., Rothenbacher, D. \u0026amp; Arndt, V. Epidemiology of stomach cancer. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e \u003cb\u003e472\u003c/b\u003e, 467\u0026ndash;477 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaumann, M. \u0026amp; Crabtree, J. E. Helicobacter pylori-induced epithelial cell signalling in gastric carcinogenesis. \u003cem\u003eTrends Microbiol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 29\u0026ndash;36 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, L., Cui, C. P., Zhang, X. \u0026amp; Zhang, L. The functions and regulation of Smurfs in cancers. \u003cem\u003eSem. Cancer Biol.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e, 102\u0026ndash;116 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo, R. et al. Ubiquitin ligase Smurf1 mediates tumor necrosis factor-induced systemic bone loss by promoting proteasomal degradation of bone morphogenetic signaling proteins. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e283\u003c/b\u003e, 23084\u0026ndash;23092 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamashita, M. et al. Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e, 101\u0026ndash;113 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei, X. et al. Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation. \u003cem\u003eJ. Cell. Biol.\u003c/em\u003e \u003cb\u003e216\u003c/b\u003e, 1455\u0026ndash;1471 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFei, C. et al. Smurf1-mediated Lys29-linked nonproteolytic polyubiquitination of axin negatively regulates Wnt/beta-catenin signaling. \u003cem\u003eMol. Cell. Biol.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 4095\u0026ndash;4105 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao, Y., Sun, C., Zhang, T. \u0026amp; Song, Y. SMURF1 promotes the proliferation, migration and invasion of gastric cancer cells. \u003cem\u003eOncol. Rep.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 1806\u0026ndash;1814 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, J. et al. Wnt/beta-catenin signalling: function, biological mechanisms, and therapeutic opportunities. \u003cem\u003eSignal. Transduct. Target. therapy\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, 3 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoelman, E. M. R., Yeste-Vazquez, A. \u0026amp; Grossmann, T. N. Targeting the interaction of beta-catenin and TCF/LEF transcription factors to inhibit oncogenic Wnt signaling. \u003cem\u003eBioorg. Med. Chem.\u003c/em\u003e \u003cb\u003e70\u003c/b\u003e, 116920 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkronska-Wasek, W., Gosens, R., Konigshoff, M. \u0026amp; Baarsma, H. A. WNT receptor signalling in lung physiology and pathology. \u003cem\u003ePharmacol. Ther.\u003c/em\u003e \u003cb\u003e187\u003c/b\u003e, 150\u0026ndash;166 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDejana, E. The role of wnt signaling in physiological and pathological angiogenesis. \u003cem\u003eCircul. Res.\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e, 943\u0026ndash;952 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaron, R. \u0026amp; Kneissel, M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 179\u0026ndash;192 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, P. et al. Deep Targeted Sequencing and Its Potential Implication for Cancer Therapy in Chinese Patients with Gastric Adenocarcinoma. \u003cem\u003eoncologist\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, e756\u0026ndash;e768 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalluzzi, L., Spranger, S., Fuchs, E. \u0026amp; Lopez-Soto, A. WNT Signaling in Cancer Immunosurveillance. \u003cem\u003eTrends Cell Biol.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 44\u0026ndash;65 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, S. et al. Wnt/beta-Catenin Signaling Axis Is Required for TFEB-Mediated Gastric Cancer Metastasis and Epithelial-Mesenchymal Transition. \u003cem\u003eMol. Cancer Res.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 1650\u0026ndash;1659 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto, S. et al. Wnt Signaling Stimulates Cooperation between GREB1 and HNF4alpha to Promote Proliferation in Hepatocellular Carcinoma. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e, 2312\u0026ndash;2327 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsik, A. \u0026amp; Firat, D. Letter to the editor concerning Most cited 100 articles from Turkey on abdominal wall hernias: a bibliometric study. \u003cem\u003eTurk. J. Surg.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 193\u0026ndash;194 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoord, S. M. et al. International Union of Pharmacology. XLVI. G protein-coupled receptor list. \u003cem\u003ePharmacol. Rev.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 279\u0026ndash;288 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReyes, M., Flores, T., Betancur, D., Pena-Oyarzun, D. \u0026amp; Torres, V. A. Wnt/beta-Catenin Signaling in Oral Carcinogenesis. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClevers, H. Wnt/beta-catenin signaling in development and disease. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e, 469\u0026ndash;480 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah, K. \u0026amp; Kazi, J. U. Phosphorylation-Dependent Regulation of WNT/Beta-Catenin Signaling. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 858782 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing, J. C. et al. KDM4B promotes gastric cancer metastasis by regulating miR-125b-mediated activation of Wnt signaling. \u003cem\u003eJ. Cell. Biochem.\u003c/em\u003e, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGammons, M. V., Renko, M., Johnson, C. M., Rutherford, T. J. \u0026amp; Bienz, M. Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled. \u003cem\u003eMol. Cell\u003c/em\u003e. \u003cb\u003e64\u003c/b\u003e, 92\u0026ndash;104 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFei, C. et al. Smurf1-mediated axin ubiquitination requires Smurf1 C2 domain and is cell cycle-dependent. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e289\u003c/b\u003e, 14170\u0026ndash;14177 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiner, J., Jung, C. K., Shackel, I. \u0026amp; Williams, P. M. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. \u003cem\u003eAnal. Biochem.\u003c/em\u003e \u003cb\u003e270\u003c/b\u003e, 41\u0026ndash;49 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, W. et al. A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation. \u003cem\u003ePLoS Biol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, e3001361 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTano, K. et al. MALAT-1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility-related genes. \u003cem\u003eFEBS Lett.\u003c/em\u003e \u003cb\u003e584\u003c/b\u003e, 4575\u0026ndash;4580 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng, W. et al. Kruppel-like factor 4 promotes c-Met amplification-mediated gefitinib resistance in non-small-cell lung cancer. \u003cem\u003eCancer Sci.\u003c/em\u003e \u003cb\u003e109\u003c/b\u003e, 1775\u0026ndash;1786 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadary, D. M., Abdel-Wanis, M. E., Hafez, M. Z. \u0026amp; Aboulhagag, N. A. Immunohistochemical analysis of PTEN, HER2/neu, and ki67 expression in patients with gastric cancer and their association with survival. \u003cem\u003ePathophysiology\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 99\u0026ndash;106 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, J. et al. Human papillomavirus type 16 E7 oncoprotein-induced upregulation of lysine-specific demethylase 5A promotes cervical cancer progression by regulating the microRNA-424-5p/suppressor of zeste 12 pathway. \u003cem\u003eExp. Cell. Res.\u003c/em\u003e \u003cb\u003e396\u003c/b\u003e, 112277 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, Z. et al. A novel UBE2T inhibitor suppresses Wnt/beta-catenin signaling hyperactivation and gastric cancer progression by blocking RACK1 ubiquitination. \u003cem\u003eOncogene\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 1027\u0026ndash;1042 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlanagan, D. J., Vincan, E. \u0026amp; Phesse, T. J. Wnt Signaling in Cancer: Not a Binary ON:OFF Switch. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e79\u003c/b\u003e, 5901\u0026ndash;5906 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, J. et al. The Sensitivity Prediction of Neoadjuvant Chemotherapy for Gastric Cancer. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 641304 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J. et al. Neoadjuvant chemoradiotherapy for resectable gastric cancer: A meta-analysis. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 927119 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, M. et al. miR-1254 inhibits cell proliferation, migration, and invasion by down-regulating Smurf1 in gastric cancer. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 32 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, L. et al. MicroRNA-424 regulates cisplatin resistance of gastric cancer by targeting SMURF1 based on GEO database and primary validation in human gastric cancer tissues. \u003cem\u003eOncoTargets therapy\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, 7623\u0026ndash;7636 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu, M. G. et al. Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e297\u003c/b\u003e, 101307 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, Q., Li, Y., Han, D. \u0026amp; Dong, L. SMURF1, a promoter of tumor cell progression? \u003cem\u003eCancer Gene Ther.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 551\u0026ndash;565 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J. et al. Effects of the Wnt/beta-Catenin Signaling Pathway on Proliferation and Apoptosis of Gastric Cancer Cells. Contrast media \u0026amp; molecular imaging. : : 5132691, 2022. (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi, J. et al. Targeting Wnt/beta-Catenin Signaling by TET1/FOXO4 Inhibits Metastatic Spreading and Self-Renewal of Cancer Stem Cells in Gastric Cancer. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, W. et al. The miR-3648/FRAT1-FRAT2/c-Myc negative feedback loop modulates the metastasis and invasion of gastric cancer cells. \u003cem\u003eOncogene\u003c/em\u003e, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang, W., Zhang, T., Huo, J. \u0026amp; Yang, J. MARCH1 promotes the growth and maintaining of stem cell-like characteristics of gastric cancer cells by activating the Wnt/beta-catenin signaling pathway. \u003cem\u003eTissue cell.\u003c/em\u003e \u003cb\u003e78\u003c/b\u003e, 101895 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNong, J. et al. Phase separation of Axin organizes the beta-catenin destruction complex. \u003cem\u003eJ. Cell Biol.\u003c/em\u003e \u003cb\u003e220\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, R., Zhai, Y., Fearon, E. R. \u0026amp; Cho, K. R. Diverse mechanisms of beta-catenin deregulation in ovarian endometrioid adenocarcinomas. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e, 8247\u0026ndash;8255 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen, V. H. L., Hough, R., Bernaudo, S. \u0026amp; Peng, C. Wnt/beta-catenin signalling in ovarian cancer: Insights into its hyperactivation and function in tumorigenesis. \u003cem\u003eJ. ovarian Res.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 122 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, M. S., Kim, S. S., Ahn, C. H., Yoo, N. J. \u0026amp; Lee, S. H. Frameshift mutations of Wnt pathway genes AXIN2 and TCF7L2 in gastric carcinomas with high microsatellite instability. \u003cem\u003eHum. Pathol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 58\u0026ndash;64 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhai, J. et al. ALKBH5 Drives Immune Suppression Via Targeting AXIN2 to Promote Colorectal Cancer and Is a Target for Boosting Immunotherapy. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cb\u003e165\u003c/b\u003e, 445\u0026ndash;462 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, J. et al. CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/beta-catenin signaling via transactivation of GSK-3beta and Axin2 expression. \u003cem\u003eCell. Death Dis.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 26 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePicco, G. et al. Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions. \u003cem\u003eEMBO Mol. Med.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 293\u0026ndash;303 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y. et al. UBE3C promotes proliferation and inhibits apoptosis by activating the beta-catenin signaling via degradation of AXIN1 in gastric cancer. \u003cem\u003eCarcinogenesis\u003c/em\u003e \u003cb\u003e42\u003c/b\u003e, 285\u0026ndash;293 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, E., Salic, A., Kruger, R., Heinrich, R. \u0026amp; Kirschner, M. W. The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS biology 1: E10, (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazzoni, S. M. \u0026amp; Fearon, E. R. AXIN1 and AXIN2 variants in gastrointestinal cancers. \u003cem\u003eCancer Lett.\u003c/em\u003e \u003cb\u003e355\u003c/b\u003e, 1\u0026ndash;8 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Figures","content":"\u003cp\u003eSupplementary Figures S1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"gastric cancer, Smurf1, Axin2, Wnt, β-catenin","lastPublishedDoi":"10.21203/rs.3.rs-6694191/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6694191/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSMAD-specific E3 ubiquitin protein ligase 1 (Smurf1) is involved in various biological processes through targeting specific proteins for ubiquitin-dependent degradation. Emerging evidence has shown that Smurf1 functions as an oncogene in many types of human tumours, including gastric cancer (GC). We aimed to investigate the role of Smurf1 in regulating GC progression and reveal its underlying mechanism. Smurf1 expression was analyzed in two datasets that are publicly accessible. Additionally, it was assayed in 29 pairs of GC tissues and para-cancerous tissues using quantitative reverse transcriptase PCR (qRT-PCR). The biological role of Smurf1 in GC cells was assessed \u003cem\u003ein vitro\u003c/em\u003e and in a moue Xenograft model. Smurf1 levels were significantly up-regulated in GC tissues compared with normal tissues, and high Smurf1 expression was significantly correlated with worse disease-free survival (DFS). Forced expression of Smurf1 accelerated AGS cell growth, proliferation, and invasion \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, Smurf1 directly engaged with axis inhibition protein 2 (Axin2) and diminished the stability of the Axin2 protein by promoting its ubiquitination and subsequent degradation. As a result, Smurf1 promoted the activation of Wnt/β-catenin signaling. Importantly, IWR-1, a specific inhibitor of the Wnt pathway, effectively inhibited Smurf1-induced GC cell proliferation and invasion. These data suggest that upregulated Smurf1 facilitates GC progression through degrading Axin2 and activating Wnt/β-catenin signaling.\u003c/p\u003e","manuscriptTitle":"Smurf1 promotes gastric cancer growth and metastasis by regulating Axin2- dependent Wnt/β-catenin signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 14:24:08","doi":"10.21203/rs.3.rs-6694191/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-08T13:36:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T08:49:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-19T03:20:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254838396094924288756395511482014133145","date":"2025-07-14T12:47:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118957382273690709950351121079930385968","date":"2025-07-02T00:43:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-23T12:53:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-18T13:08:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-09T16:32:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T06:05:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-19T01:47:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1b0ff3b4-5f78-4b84-8f2f-0dc5c81c1ea5","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50485403,"name":"Biological sciences/Cell biology"},{"id":50485404,"name":"Biological sciences/Chemical biology"},{"id":50485405,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-11-24T16:04:11+00:00","versionOfRecord":{"articleIdentity":"rs-6694191","link":"https://doi.org/10.1038/s41598-025-23707-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-19 15:58:46","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-06-27 14:24:08","video":"","vorDoi":"10.1038/s41598-025-23707-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-23707-3","workflowStages":[]},"version":"v1","identity":"rs-6694191","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6694191","identity":"rs-6694191","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}