A20 enhances migration and metastasis of gastric cancer cells by promoting occludin degradation

A20 enhances migration and metastasis of gastric cancer cells by promoting occludin degradation | 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 A20 enhances migration and metastasis of gastric cancer cells by promoting occludin degradation Yan-Shen Shan, Yu-Ting Kuo, Hao-Chen Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6780956/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2026 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Abstract Chronic inflammation is a well-established risk factor in the development of gastric cancer (GC). Tumor necrosis factor α-induced protein 3 (TNFAIP3, also known as A20) is an inflammation-associated protein that functions as an oncogene in various cancers, but the role of A20 in GC progression remains unclear. In this study, we found that overexpression of A20 significantly enhanced GC cell migration, whereas A20 knockdown suppressed this effect. A20 promoted epithelial-mesenchymal transition and led to the loss of the tight junction protein occludin. Mechanistically, A20 induced occludin endocytosis and lysosomal degradation via its ovarian tumor (OTU) domain. Pull-down assays revealed that A20 interacts with the migration-related protein RhoA, increasing its stability, and thereby sustaining ROCK2 phosphorylation, which contributes to occludin degradation. PLA further showed that mutation of the OTU domain disrupted the interaction between A20 and RhoA in AGS cells, indicating the necessity of the OTU domain for this interaction. In conclusion, our findings demonstrate that A20 promotes GC cell migration by stabilizing RhoA and facilitating occludin degradation, underscoring A20 as a potential therapeutic target to inhibit GC metastasis. Biological sciences/Cancer/Tumour biomarkers Health sciences/Diseases/Cancer/Cancer microenvironment gastric cancer tumor necrosis factor α induced protein 3 A20 occludin RhoA metastasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Gastric cancer (GC) is one of the deadliest malignancies worldwide, ranking 4th in cancer incidence and 5th in cancer-related mortality [ 1 ]. While early-stage GC is often associated with a favorable prognosis, more than 62% of patients are diagnosed with metastatic disease, which significantly worsens clinical outcomes [ 2 ]. Despite advancements in basic research and targeted therapies that have improved treatment efficacy, the prognosis for late-stage GC remains poor [ 3 ]. Therefore, unveiling the molecular mechanisms underlying GC progression and metastasis is critical for the development of effective therapeutic strategies. Previous studies have demonstrated that protumor inflammation can activate oncogenic signaling pathways that facilitate cancer progression and metastasis [ 4 , 5 ]. GC has been recognized as an inflammation-associated cancer [ 6 , 7 ], but the specific mechanisms by which inflammatory factors drive GC remain to be fully elucidated. One key player in inflammation signaling is TNF-α–induced protein 3 (TNFAIP3, also known as A20), which is rapidly induced by NF-κB upon stimulation with cytokines or environmental stressors via binding to the TNFAIP3 promoter [ 8 ]. A20 typically acts as a negative regulator of NF-κB signaling by targeting IκBα, thereby terminating the inflammatory response [ 9 ]. However, accumulating evidence has shown that high A20 expression may play a pro-oncogenic role in various cancers through NF-κB independent mechanisms [ 10 – 13 ]. Cancer cell migration is a prerequisite for metastasis, which involves disruption of cell-cell junctions, cytoskeletal rearrangement, extracellular matrix remodeling, and chemotactic responses [ 14 , 15 ]. Additionally, epithelial-mesenchymal transition (EMT) is also regarded as a pivotal biological process in tumor metastasis [ 16 ]. The metastatic cascade is orchestrated by a complex network of extracellular and intracellular signaling events [ 17 ]. Recent studies have indicated that elevated A20 expression promotes cancer progression and metastasis across several tumor types. For instance, in melanoma, A20 fosters tumor growth, metastasis, and resistance to BRAF-targeted therapies [ 18 ]. A20 also inhibits the cell surface translocation of the “eat-me” signal calreticulin via suppression of stanniocalcin 1 degradation to facilitate immune evasion [ 11 ]. In breast cancer and GC, A20 has been shown to promote EMT and malignant transformation [ 19 , 20 ]. However, the detailed mechanism by which A20 drives GC metastasis remains largely unknown. In this study, we identified a novel mechanism by which A20 facilitates gastric cancer cell migration through the degradation of occludin, an essential tight junction component. Our results demonstrate that A20 promotes GC cell migration by inducing endocytic lysosomal degradation of occludin, thereby disrupting cell-cell adhesion. These findings suggest that A20 serves as a key regulator of GC metastasis. Targeting the A20-mediated occludin degradation pathway could offer a promising therapeutic strategy to inhibit GC cell migration and metastatic spread. Materials and Methods Cell line and culture conditions Human gastric cancer (GC) cell lines AGS, SNU-1, MKN45, NCI-N87, and KATO III were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan), while the HR cell line was acquired from the Cell Bank of the Clinical Medicine Research Center, College of Medicine, National Cheng Kung University Hospital (NCKUH). All cell lines were cultured in PRMI medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) 1% glutamine and 1% antibiotic-antimycotic, and were incubated in a humidified atmosphere of 5% CO2 at 37℃. The short tandem repeat (STR) analysis was used for cell line authentication. Transformation and transfection in GC cell lines To establish stable transfected cell lines, GC cell lines were separately infected with the appropriate pLKO.1 lentiviral vectors containing TNFAIP3 (TRCN0000050961 and TRCN0000218517), RHOA (TRCN0000047710 and TRCN0000047712), and non-target Luciferin short hairpin RNA (shRNA) vectors (TRCN0000072243) that were purchased from the National RNAi Core Facility, Academia Sinica, Taipei, Taiwan. Briefly, GC cells were grown overnight with 0.5 mL of viral supernatant containing 8 µg/mL polybrene in medium. Fresh medium containing 2 µg/mL puromycin was added in the next day. Cells were maintained in the continuous presence of puromycin. Plasmids pCMV-flag (PS100001, Origene), pCMV-A20-Myc-flag (RC221337, Origene), occludin cDNA ORF Clone, Human, C-GFPSpark® tag (HG15134-ACG, SinoBiological), and RhoA cDNA ORF Clone, Human, C-OFPSpark® tag (HG12110-ACR, SinoBiological) were constructed and then were used to transfected AGS cells by lipofectamin 3000. Cells were maintained in the continuous presence of G418 (500 µg/mL, A1720, Sigma-Aldrich) or Hygromycin B (200 µg/mL, 31282-04-9, Sigma-Aldrich). The knockdown and overexpression efficiencies were checked by Western blotting. The mutagenesis of A20 was performed using the QuikChange Lightning- Mutagenesis Kit (Stratagene, USA). The primers for A20 plasmid mutagenesis and Sanger sequencing are shown in Supplementary Table S1 and S2. Transwell migration assay Cell migration assays were performed using 24-well inserts (Falcon cell culture inserts, 8-µm pore size; BD Biosciences). In brief, the lower chamber was filled with 600µL of growth medium, and 1×10 5 cells seeded into the upper chamber containing 100µL serum free medium were incubated at 37°C for 9–48 hours (depending on cell lines). Then cells attaching to the upper side of the membrane were removed gently with a cotton swab and rinsed. Cells that migrated through the membrane and attached to the bottom membrane were fixed in methanol for 10 minutes at room temperature and were stained with hematoxylin (MERCK). The number of migrating cells was quantified by counting 5 independent symmetrical visual fields under the microscope. Wound healing assay Culture-Inserts 2 Well (80209, ibidi) was placed on a 6 well plate, providing two cell culture reservoirs divided by a 500 µm wall. A total of 5×10 4 cells in 100 µL of serum-free medium were seeded in each well of the insert. Once the cells reached confluence, the cell density was monitored under the microscope until a 100% optically confluent cell layer was confirmed. The Culture-Insert was then gently removed using sterile tweezers. The cell layer was washed with growth medium to remove cell debris and non-adherent cells. Subsequently, 2 mL of growth medium was added to each well, and the cell migration process was monitored by capturing images at multiple time points over the following hours. The images were quantified and analyzed to determine the area covered by migrated cells. Protein degradation analysis After GC cells were collected and the supernatant was removed, the cell pellet was resuspended in serum-free medium. A total of 1×10 6 GC cells were then plated and incubated overnight at 37°C in serum-free medium. For occludin dynamics analysis, the serum-free medium was replaced with medium containing 10% FBS and incubated for 0, 2, 4, 6 or 8 hours. In RhoA degradation analysis, the serum-free medium was replaced with 10% FBS medium supplemented with cycloheximide (CHX, 20 µg/mL, 239763-M, Sigma-Aldrich) and incubated for 0, 15, 30, 60 or 90 minutes. All cell lysates were collected and stored at -20°C until further analysis. Animal model An orthotopic mouse model of human GC was used in this study. MKN45 cells (2×10 6 ) were suspended in 20 uL of Matrigel and were orthotopically injected into the stomach wall of NOD-SCID mice. After tumor development for 4 weeks, the body weight and survival rate of the mice were monitored. At week 12, the mice were sacrificed for tumor collection. Primary tumors, organs, and metastatic lesions were harvested from each mouse and weighed for subsequent analysis. Real-time PCR Total RNA was isolated from GC cell lines using TRIzol Reagent (Invitrogen, USA) and chloroform (Sigma, USA), and was reverse transcribed into cDNA. Expression of mRNA was measured using the ABI 7500 Fast Real-Time PCR System (ABI, USA). Results were normalized to GADPH using the 2 −ΔΔCT method. The primers used for mRNA expression analysis are listed in Supplementary Table S3 . Western blotting analysis Whole cell lysates were collected using RIPA lysis buffer, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). A mixture of 25 µg protein and 6× loading dye was heated at 100°C for 5 minutes and then placed on ice for an additional 5 minutes. Protein samples were loaded onto a 12% SDS-polyacrylamide gel for electrophoresis and subsequently transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk diluted in 0.05% TBST for 1 hour at room temperature. Following blocking, the membrane was incubated with primary antibodies overnight at 4°C and then with secondary antibodies for 1 hour at room temperature. Protein signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) according to manufacturer’s instruction and were visualized using the iBright FL1000 Imaging System. The antibodies used to detect protein levels are listed in Supplementary Table S4 . Immunofluorescence (IF) staining GC cells were seeded onto chamber slides at a density of 5×10 4 cells per well. After fixation with paraformaldehyde and permeabilization with Triton X-100, the samples were incubated with primary antibodies overnight at 4℃, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour. F-actin was stained with Rhodamine Phalloidin (PHDR1, Cytoskeleton), and nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI). IF images were acquired using a Confocal Laser Scanning Microscope FV3000 (Olympus, Tokyo, Japan). Fluorescence signals were quantified using ImageJ software (NIH, USA). Pull-down immunoprecipitation (IP) and IP-mass spectrometry (IP-MS) IP was performed using the Millipore Protein G Plus/Protein A Magnetic Beads (16–663, Millipore). Briefly, cells lysates from 5×10 6 cells were incubated with anti-DDK (1µg, 14793S, cell signaling) and anti-RhoA (1µg, sc‑418, Santa Cruz) antibodies. The immunoprecipitated proteins were either analyzed by western blotting or submitted to LC-MS/MS protein identification (Biotools). GTP-bound RhoA was pulled down using Rhotekin-RBD agarose beads (BK036-5, Cytoskeleton) and detected by western blotting. Statistical analysis Each experiment was performed independently at least three times under identical conditions. Data are expressed as the mean ± standard error of the mean (SEM). Statistical differences were analyzed using Student’s t -test and one-way ANOVA, as appropriate. All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA) and SigmaPlot 12.0 (Systat Software Inc., CA, USA). Statistical significance was defined as follows: *** P < 0.001, ** P < 0.01, and * P < 0.05. Results A20 enhances the migration and metastasis of GC cells We first performed Western blotting to screen A20 expression across various GC cell lines (Fig. 1 A). To study the role of A20 in GC, we established A20 overexpressing AGS cell lines (AO/WT1 and AO/WT2) and A20 knockdown cell lines in NCIN87 and MKN45 cells (shA20-1 and shA20-2) (Fig. 1 B). We found that expression levels of A20 were not associated with the proliferation of GC cells (Supplementary Fig. S1 A). However, wound healing assays showed that A20 overexpression increased the migratory ability of AGS cells, while A20 knockdown reduced the migration of NCIN87 and MKN45 cells (Fig. 1 C). Similar results were seen in transwell migration assays (Fig. 1 D). Additionally, compared with control cells, A20 overexpressing AGS cells exhibited a mesenchymal-like phenotype, whereas A20 knockdown led to a shrunken, less spread morphology in MKN45 cells (Supplementary Fig. S1 B). Collectively, these results indicate that A20 is able to increase GC cell migration. To further investigate whether A20 contributes to GC metastasis, GC cells were orthotopically inoculated into the stomachs of mice. A20 knockdown in MKN45 cells could increase survival compared to controls (p = 0.029) (Fig. 2 A). Although the difference in stomach tumor weight between control and A20 knockdown groups was marginal (p = 0.061), oA20 knockdown significantly reduced metastasis to the peritoneal cavity and distant organs (Fig. 2 B-D, P < 0.01). These results suggest that A20 exerts its oncogenic effects by promoting GC cell migration and metastasis. A20 downregulates the tight junction protein occludin in GC cells Previous studies have reported the involvement of A20 in EMT [ 19 , 20 ]. Accordingly, we sought to investigated the mechanism by which A20 regulates EMT in GC cells. In E-cadherin negative AGS cells, A20 overexpression reduced the expression of occludin (Supplementary Fig. S2 A). Conversely, A20 knockdown in NCIN87 cells increased occludin expression and decreased the levels of vimentin, snail, and twist1/2. Similarly, A20 knockdown in MKN45 cells also upregulated occludin and reduced N-cadherin, vimentin, snail, slug, and twist1/2 expression (Supplementary Fig. S2 B). These results were further confirmed by qPCR analysis in AGS and MKN45 cells (Fig. S2 C). Notably, occludin expression was inversely correlated with A20 expression across GC cell lines. As the disruption of cell-cell junction is a critical early step in cell migration during cancer metastasis, and loss of occludin contributes to increased aggressiveness in various cancers [ 21 – 23 ], we further examined this relationship in clinical specimens. IF staining revealed an inverse correlation between A20 and occludin in GC tissues (Fig. 3 A). A20 overexpression (AO/WT1) induced occludin translocation to the cytoplasm and co-localization with A20 in AGS cells, whereas knockdown of A20 (shA20-1) maintained occludin localization at the cell membrane of NCIN87 and MKN45 cells (Fig. 3 B). To evaluate the functional role of occludin in GC cell migration, we silenced occludin in AGS and MKN45 cells (Fig. 3 C). Depletion of occludin significantly enhanced the migratory capacity of both cell lines (Fig. 3 D and 3 E), suggesting that A20 promotes GC cell migration, at least in part, by downregulating occludin. Taken together, our findings demonstrate that A20 promotes EMT and facilitates the loss of occludin, consequently enhancing the migratory and metastatic potential of GC cells. A20 induces occludin degradation via endocytosis and lysosomal processing In response to environmental stimuli, occludin can translocate from the cell membrane to the cytoplasm, followed by endocytosis and subsequent proteasomal or lysosomal degradation [ 24 ]. Therefore, we sought to study whether A20 is involved in this process to regulate occludin degradation. After stimulation with FBS to induce EMT, occludin expression in AGS cells was decreased in a time-dependent manner, and this effect was further potentiated by A20 overexpression. Similarly, in MKN45 cells, occludin expression was also decreased over time, but knockdown of A20 attenuated this reduction (Fig. 4 A). To determine the proteolytic pathway in A20-mediated occludin degradation, we employed the proteasome inhibitor MG-132 in the presence of cycloheximide (CHX) that inhibits de novo protein synthesis. MG-132 restored occludin expression in vector control AGS cells and A20 knockdown MKN45 cells but had no effect in A20 overexpressing AGS cells and shLuc MKN45 cells., suggesting that A20 promotes occludin degradation through a proteasome independent mechanism (Fig. 4 B). Given that lysosomal degradation contributes to occludin loss during tight junction disruption [ 25 ], and that the endocytosis inhibitor wortmannin can prevent tight-junction disassembly in epithelial cells [ 26 ], we investigated the role of endocytic and lysosomal pathways in A20-mediated occludin degradation. Treatment with the lysosome inhibitors chloroquine (CQ) as well as wortmannin in the presence of cycloheximide (CHX) could significantly inhibit occludin degradation in AGS cells (Fig. 4 C). Under the stimulation of FBS, treatment with CQ restored occludin expression in both AGS and MKN45 cells (Fig. 4 D). Moreover, CQ or wortmannin strongly counteracted A20 overexpression-induced cell migration in AGS cells (Fig. 4 E), supporting the conclusion that A20 promotes GC cell migration by facilitating occludin degradation via enhanced endocytosis and lysosomal activity. The ovarian tumor (OTU) domain of A20 is critical to occludin endocytosis and lysosomal degradation Previous research has demonstrated that the A20 OTU domain possesses deubiquitinating activity [ 27 ], and the ZnF4 domain and ZnF7 domain have catalytic activity as a E3 ligase [ 19 , 28 ]. Mutations such as C103A in the OUT domain, C624A/C627A in the ZnF4 domain, and F770A/G771A in the ZnF7 domain can impair their enzymatic activity, indicating that these amino acid residues are critical for A20 biological functions. To determine which domain is essential for occludin degradation and cell migration, we generated A20 expression constructs harboring the C103A mutation in the OTU domain (OTU mut ), C624A/C627A in the ZnF4 domain (ZnF4 mut ), and F770A/G771A in the ZnF7 domain (ZnF7 mut ) (Supplementary Fig. S3 ). Wound healing assays revealed that, in AGS cells, the OTU mut construct blocked A20-induced cell migration, but Zn4 mut and Zn7 mut did not (Fig. 5 A). This inhibitory effect of OTU mut on cell migration was further confirmed by transwell migration assay (Fig. 5 B). To study the impact of OTU mut on occludin degradation, we performed Western blotting following FBS stimulation. Occludin degradation was enhanced in A20 overexpressing AGS cells (AO/WT1), but this effect was abolished in OTU mut cells (Fig. 5 C). Because occludin degradation involves endocytosis and cytosolic trafficking, we examined the co-localization of occludin with early endosomes using IF staining for EEA1. The co-localization signal in AO/WT1 AGS cells was stronger than in OTU mut or vector control cells (Fig. 5 D). Given our results that lysosomal degradation contributed to occludin loss, we performed dual IF staining for the lysosomal marker LAMP1 and occludin. The co-localization of occludin with LAMP1 was reduced in OTUmut cells compared to AO/WT1 AGS cells (Fig. 5 E). Additionally, both biotin-based endocytosis assays and membrane/cytosol fractionation showed that occludin internalization was promoted in AO/WT1 AGS cells but was decreased in OTU mut cells (Fig. 5 F). Taken together, these results demonstrate that the OTU domain of A20 is essential to promote occludin endocytosis and subsequent lysosomal degradation, thereby facilitating GC cell migration. A20-induced occludin degradation involves activation of the RhoA/ROCK2 signaling pathway To date, no studies have described how A20 regulates occludin degradation. To elucidate this mechanism, we conducted immunoprecipitation followed by mass spectrometry (IP-MS) to identify A20-interacting proteins potentially involved in occludin degradation and cancer metastasis. KEGG pathway analysis of proteins pulled down with Flag-tagged A20 revealed RhoA as a binding partner, known to be associated with tight junction assembly, actin cytoskeleton regulation, and endocytosis (Supplementary Fig. S4 ). In A20 knockdown MKN45 cells, RhoA expression was decreased and negatively correlated to occludin expression (Fig. 6 A). Conversely, A20 overexpression enhanced RhoA expression 24 hours after FBS stimulation and thus promoted AGS cell migration (Fig. 6 B). IF staining further demonstrated increased expression and co-localization of A20 and RhoA in AO/WT1 AGS cells compared to vector control cells (Fig. 6 C). RhoA knockdown increased occludin expression in both AO/WT1 and vector control AGS cells (Fig. 6 D and 6 E); similar effects were observed with a RhoA inhibitor (Rho i). These results suggest that RhoA acts downstream of A20 to drive GC cell migration. It has been reported that RhoA promotes occludin endocytosis by activating ERK or ROCK2 signaling [ 29 , 30 ]. In our study, we observed that only ROCK2 phosphorylation was prolonged in AO/WT1 AGS cells, and this phosphorylation was diminished in OTU mut cells (Fig. 6 G). Activation of RhoA/ROCK2 signaling promotes downstream F-actin assembly, a key indicator of cell migration. Phalloidin-Rhodamine staining revealed that AO/WT1 AGS cells exhibited increased expression of F-actin (red), which co-localized with endocytic occludin (green) (Supplementary Fig. S5 A). To further assess the involvement of ROCK2 phosphorylation in occludin degradation, AGS and MKN45 cells were treated with an endocytosis inhibitor dynole and a ROCK2 inhibitor Y-27632. The inhibition of either endocytosis or ROCK2 effectively reduced occludin degradation in AGS and MKN45 cells (Supplementary Fig. S5 B). Taken together, these results suggest that A20 promotes occludin degradation in GC cells through activation of RhoA/ROCK2 signaling. The OTU domain of A20 interacts with and stabilizes RhoA for occludin degradation To confirm the interaction between A20 and RhoA, we performed co-IP, which showed reciprocal binding between the two proteins (Fig. 7 A). Their interaction was significantly weakened in OTU mut AGS cells after FBS stimulation, indicating that the C103A mutation in A20 OTU domain disrupts the binding of A20 with RhoA (Fig. 7 B). The interaction was further validated in HEK293T cells co-transfected with A20 and RhoA (Supplementary Fig. S6 ). Proximity ligation assay (PLA) also showed a strong interaction between A20 and RhoA in AO/WT1 AGS cells but a marked reduction in OTU mut cells (Fig. 7 C), accompanied by increased F-actin expression, suggesting enhanced cell motility. Triple IF staining demonstrated cytoplasmic co-localization of A20, RhoA, and occludin in AO/WT1 AGS and shLuc MKN45 cells, whereas occludin remained membrane-bound in vector, OTU mut, and shA20 cells (Fig. 7 D and Supplementary Fig. S7 A). Rhotekin-RBD pull-down assays showed that A20 overexpression increased RhoA-GTP levels, while the OTU mutant impaired RhoA activity and disrupted its association with occludin (Fig. 7 E). Live-cell imaging using occludin-GFP and RhoA-OFP confirmed enhanced cytosolic mobility only in AO/WT1 AGS cells (Fig. 7 F and Supplementary Fig. S7 B). Collectively, these results demonstrate that A20 OTU domain facilitates occludin endocytosis and degradation by stabilizing and activating RhoA in GC cells. To determine whether the OTU domain of A20 is required for RhoA protein stabilization, GC cells were treated with CHX. After 90 minutes of CHX treatment, the degradation of RhoA was slower in AO/WT1 AGS cells than in vector control and OTU mut AGS cells (Fig. 7 G). Overall, these results suggest that the OTU domain of A20 protein can stabilize RhoA, thereby promoting occludin degradation and enhancing the migratory capacity of GC cells. Discussion In this study, we identify a novel mechanism in which A20 facilitates GC cell migration through its OTU domain by inducing occludin internalization and subsequent lysosomal degradation. While A20 has been previously implicated in epithelial-mesenchymal transition (EMT) and tumor progression, our findings clarify how A20 contributes to tight junction disassembly, a key step in cancer cell dissemination. Previous studies have associated A20 with the EMT in GC cells [ 20 ], aligns with our findings that expression of EMT markers was affected by A20 knockdown. Among the EMT markers, occludin, a critical component of tight junctions, plays a central role in maintaining epithelial barrier integrity. Its depletion contributes to increased cellular permeability and is commonly observed during cancer metastasis [ 31 ]. Occludin has been also linked to gastric epithelial hyperplasia and inflammation in animal models [ 32 ], and to cytosolic redistribution in GC cells following H. pylori infection [ 33 ]. Our study confirms occludin as a tumor suppressor, as its silencing increased GC cell migration, and identifies A20 as a promoter of occludin degradation. Although tight junction loss has been known as a facilitator of tumor invasion [ 34 ], the role of A20 in this process remains vague. In contrast to our findings, Kolodziej et al. reported that A20 maintained tight junctions in intestinal epithelial cells by preventing LPS-induced occludin loss via its N-terminal activity [ 35 ]. This discrepancy may be due to cell type specificity or different stimuli, suggesting the context-dependent regulatory roles of A20 in inflammation and cancer [ 10 , 36 ]. Despite earlier evidence has associated A20 with metastasis in inflammation-associated cancers [ 12 , 19 , 20 ], this is the first detailed demonstration of A20 driving GC cell migration by promoting lysosomal occludin degradation. Previous studies have reported that A20 is localized to endocytic compartments and interacts with lysosome-associated proteins LAMP1 and LAPTM5 [ 37 , 38 ]. In our study, biotin-labeling endocytosis assays revealed that A20 enhances occludin internalization, an important early event of GC cell migration. Pharmacological inhibition of lysosomes or endocytosis suppressed both occludin degradation and GC cell motility, indicating that targeting this pathway to prevent occludin degradation, for example, using CQ, might be a promising anti-metastatic strategy. Further investigation is needed to define the trafficking route and machinery that mediate the endocytic pathway of occludin. RhoA is a small GTPase protein belonging to the Rho family, which plays a well-established role in cancer progression, including tight-junction breakdown, EMT induction, and increased migration [ 39 – 41 ]. Tight junction components like occludin, ZO-1, and claudins form a barrier limiting cell dissemination, which can be disrupted by RhoA signaling [ 42 ]. Blockade of the RhoA signaling pathway effectively suppresses cancer aggressiveness [ 43 ]. A meta-analysis by Nam et al. linked RhoA expression to advanced tumor stage and poor differentiation in GC [ 44 ]. Despite its relevance, little is known about the regulation of RhoA protein expression. Our results show that A20 can stabilize RhoA and consequently facilitate occludin degradation, indicating that A20 is a critical upstream regulator of RhoA in GC metastasis. Notably, RhoA silencing or inhibition could significantly restore occludin levels in A20 overexpressing cells. Given the ubiquitin-editing enzymatic activity of A20, we explored the functional domains responsible for RhoA regulation. Prior studies have highlighted the oncogenic functions of its zinc finger domain in promoting EMT by mediating multiple mono-ubiquitin modification of Snail [ 19 ], but our study found that the OTU deubiquitinase domain is also crucial for GC cell migration. We identified that A20 binds and stabilizes RhoA through its OTU domain. Mutation of the catalytic cysteine residue (C103A) in the OTU domain abrogated ROCK2 phosphorylation, suggesting that A20 acts as a regulator of the RhoA/ROCK2 pathway. However, the precise mechanism by which the OTU domain modulates RhoA stabilization and the downstream signaling pathways remains to be elucidated. RhoA signaling has been reported to be essential for cytoskeletal reorganization, a prerequisite for cancer cell motility [ 45 , 46 ]. Activated RhoA drives F-actin polymerization and reassembly of myosin and tubulin filaments, facilitating cell adhesion and migration [ 46 , 47 ]. Live-cell imaging in this study showed co-localization and coordinated movement of A20, RhoA, and occludin, suggesting that A20 not only destabilizes tight junctions but also modulates cytoskeletal architecture to promote GC migration. Thus, A20, via its OTU domain, serves as a central modulator of both tight-junction integrity and cellular polarity. In conclusion, our study provided strong evidence that A20 stabilizes RhoA through its OTU domain to induce occludin internalization and lysosomal degradation, thus promoting GC cell migration and metastasis. This newly identified A20/RhoA/occludin signaling axis offers mechanistic insight into GC progression and highlights the A20 OTU domain as a promising therapeutic target against metastasis. Abbreviations GC gastric cancer EMT epithelial-mesenchymal transition TNF-α Tumor necrosis factorα TNFAIP3 Tumor necrosis factor α-induced protein 3 OTU ovarian tumor ZnF Zinc finger HRP horseradish peroxidase IP Immunoprecipitation IF Immunofluorescence PLA proximity ligation assay CQ chloroquine CHX cycloheximide Declarations Availability of data and materials The datasets supporting the conclusions of this article are included within this article (and its supplementary information files). Competing interests The authors declare no competing interests. Funding This study was supported by the Taiwan Ministry of Science and Technology (NSTC 109-2314-B-006-028-MY2). Authors’ contributions YTK: Methodology, investigation, writing-original draft. HCW: Conceptualization, writing-review and editing. YSS: Conceptualization, resources, visualization, supervision, funding acquisition, project administration, writing-review and editing. Ethics approval and consent to participate All procedures involving human tissues and data in this study were approved by the Institutional Review Board (IRB) of NCKUH (IRB No. B-ER-107-432). The requirement for informed consent was waived by the IRB. All methods were conducted in accordance with relevant guidelines and regulations. Consent for publication All authors have reviewed the manuscript and given consent for publication. References Siegel RL, Miller KD, Fuchs HE, Jemal A: Cancer Statistics, 2021 . CA: a cancer journal for clinicians 2021, 71 (1):7-33. Kinami S, Saito H, Takamura H: Significance of Lymph Node Metastasis in the Treatment of Gastric Cancer and Current Challenges in Determining the Extent of Metastasis . Frontiers in oncology 2021, 11 :806162. Guan WL, He Y, Xu RH: Gastric cancer treatment: recent progress and future perspectives . Journal of hematology & oncology 2023, 16 (1):57. Hibino S, Kawazoe T, Kasahara H, Itoh S, Ishimoto T, Sakata-Yanagimoto M, Taniguchi K: Inflammation-Induced Tumorigenesis and Metastasis . International journal of molecular sciences 2021, 22 (11). O'Brien VP, Kang Y, Shenoy MK, Finak G, Young WC, Dubrulle J, Koch L, Rodriguez Martinez AE, Williams J, Donato E et al : Single-cell Profiling Uncovers a Muc4-Expressing Metaplastic Gastric Cell Type Sustained by Helicobacter pylori-driven Inflammation . Cancer research communications 2023, 3 (9):1756-1769. Rihawi K, Ricci AD, Rizzo A, Brocchi S, Marasco G, Pastore LV, Llimpe FLR, Golfieri R, Renzulli M: Tumor-Associated Macrophages and Inflammatory Microenvironment in Gastric Cancer: Novel Translational Implications . International journal of molecular sciences 2021, 22 (8). Waldum H, Fossmark R: Inflammation and Digestive Cancer . International journal of molecular sciences 2023, 24 (17). Krikos A, Laherty CD, Dixit VM: Transcriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements . The Journal of biological chemistry 1992, 267 (25):17971-17976. Mothes J, Ipenberg I, Arslan S, Benary U, Scheidereit C, Wolf J: A Quantitative Modular Modeling Approach Reveals the Effects of Different A20 Feedback Implementations for the NF-kB Signaling Dynamics . Frontiers in physiology 2020, 11 :896. Peng X, Zhang C, Zhou ZM, Wang K, Gao JW, Qian ZY, Bao JP, Ji HY, Cabral VLF, Wu XT: A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics . Inflammation research : official journal of the European Histamine Research Society [et al] 2022, 71 (5-6):695-710. Luo M, Wang X, Wu S, Yang C, Su Q, Huang L, Fu K, An S, Xie F, To KKW et al : A20 promotes colorectal cancer immune evasion by upregulating STC1 expression to block "eat-me" signal . Signal transduction and targeted therapy 2023, 8 (1):312. Paul S, Bhardwaj M, Kang SC: GSTO1 confers drug resistance in HCT‑116 colon cancer cells through an interaction with TNFalphaIP3/A20 . International journal of oncology 2022, 61 (5). Liu J, Yang S, Wang Z, Chen X, Zhang Z: Ubiquitin ligase A20 regulates p53 protein in human colon epithelial cells . Journal of biomedical science 2013, 20 (1):74. Nehme Z, Roehlen N, Dhawan P, Baumert TF: Tight Junction Protein Signaling and Cancer Biology . Cells 2023, 12 (2). Bergers G, Fendt SM: The metabolism of cancer cells during metastasis . Nature reviews Cancer 2021, 21 (3):162-180. Huang Y, Hong W, Wei X: The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis . Journal of hematology & oncology 2022, 15 (1):129. Attiq A, Afzal S: Trinity of inflammation, innate immune cells and cross-talk of signalling pathways in tumour microenvironment . Frontiers in pharmacology 2023, 14 :1255727. Ma J, Wang H, Guo S, Yi X, Zhao T, Liu Y, Shi Q, Gao T, Li C, Guo W: A20 promotes melanoma progression via the activation of Akt pathway . Cell death & disease 2020, 11 (9):794. Lee JH, Jung SM, Yang KM, Bae E, Ahn SG, Park JS, Seo D, Kim M, Ha J, Lee J et al : A20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1 . Nature cell biology 2017, 19 (10):1260-1273. Du B, Liu M, Li C, Geng X, Zhang X, Ning D, Liu M: The potential role of TNFAIP3 in malignant transformation of gastric carcinoma . Pathology, research and practice 2019, 215 (8):152471. Osanai M, Murata M, Nishikiori N, Chiba H, Kojima T, Sawada N: Epigenetic silencing of occludin promotes tumorigenic and metastatic properties of cancer cells via modulations of unique sets of apoptosis-associated genes . Cancer research 2006, 66 (18):9125-9133. Wang M, Liu Y, Qian X, Wei N, Tang Y, Yang J: Downregulation of occludin affects the proliferation, apoptosis and metastatic properties of human lung carcinoma . Oncology reports 2018, 40 (1):454-462. Li Y, Yuan T, Zhang H, Liu S, Lun J, Guo J, Wang Y, Zhang Y, Fang J: PHD3 inhibits colon cancer cell metastasis through the occludin-p38 pathway . Acta biochimica et biophysica Sinica 2023, 55 (11):1749-1757. Mortell KH, Marmorstein AD, Cramer EB: Fetal bovine serum and other sera used in tissue culture increase epithelial permeability . In vitro cellular & developmental biology : journal of the Tissue Culture Association 1993, 29A (3 Pt 1):235-238. Kim KA, Kim D, Kim JH, Shin YJ, Kim ES, Akram M, Kim EH, Majid A, Baek SH, Bae ON: Autophagy-mediated occludin degradation contributes to blood-brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models . Fluids and barriers of the CNS 2020, 17 (1):21. Utech M, Mennigen R, Bruewer M: Endocytosis and recycling of tight junction proteins in inflammation . Journal of biomedicine & biotechnology 2010, 2010 :484987. Soni D, Wang DM, Regmi SC, Mittal M, Vogel SM, Schluter D, Tiruppathi C: Deubiquitinase function of A20 maintains and repairs endothelial barrier after lung vascular injury . Cell death discovery 2018, 4 (1):60. Yin H, Karayel O, Chao YY, Seeholzer T, Hamp I, Plettenburg O, Gehring T, Zielinski C, Mann M, Krappmann D: A20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-kappaB signaling in activated T cells . Cellular and molecular life sciences : CMLS 2022, 79 (2):112. Wang Y, Zhang J, Yi XJ, Yu FS: Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells . Experimental eye research 2004, 78 (1):125-136. Feng S, Zou L, Wang H, He R, Liu K, Zhu H: RhoA/ROCK-2 Pathway Inhibition and Tight Junction Protein Upregulation by Catalpol Suppresses Lipopolysaccaride-Induced Disruption of Blood-Brain Barrier Permeability . Molecules 2018, 23 (9). Martin TA, Jiang WG: Loss of tight junction barrier function and its role in cancer metastasis . Biochimica et biophysica acta 2009, 1788 (4):872-891. Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T, Tsukita S: Complex phenotype of mice lacking occludin, a component of tight junction strands . Molecular biology of the cell 2000, 11 (12):4131-4142. Wroblewski LE, Shen L, Ogden S, Romero-Gallo J, Lapierre LA, Israel DA, Turner JR, Peek RM, Jr.: Helicobacter pylori dysregulation of gastric epithelial tight junctions by urease-mediated myosin II activation . Gastroenterology 2009, 136 (1):236-246. Huang S, Zhang J, Li Y, Xu Y, Jia H, An L, Wang X, Yang Y: Downregulation of Claudin5 promotes malignant progression and radioresistance through Beclin1-mediated autophagy in esophageal squamous cell carcinoma . Journal of translational medicine 2023, 21 (1):379. Kolodziej LE, Lodolce JP, Chang JE, Schneider JR, Grimm WA, Bartulis SJ, Zhu X, Messer JS, Murphy SF, Reddy N et al : TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions . PloS one 2011, 6 (10):e26352. Yokoyama Y, Tamachi T, Iwata A, Maezawa Y, Meguro K, Yokota M, Takatori H, Suto A, Suzuki K, Hirose K et al : A20 (Tnfaip3) expressed in CD4(+) T cells suppresses Th2 cell-mediated allergic airway inflammation in mice . Biochemical and biophysical research communications 2022, 629 :47-53. Li L, Hailey DW, Soetandyo N, Li W, Lippincott-Schwartz J, Shu HB, Ye Y: Localization of A20 to a lysosome-associated compartment and its role in NFkappaB signaling . Biochimica et biophysica acta 2008, 1783 (6):1140-1149. Glowacka WK, Alberts P, Ouchida R, Wang JY, Rotin D: LAPTM5 protein is a positive regulator of proinflammatory signaling pathways in macrophages . The Journal of biological chemistry 2012, 287 (33):27691-27702. Sharif M, Baek YB, Naveed A, Stalin N, Kang MI, Park SI, Soliman M, Cho KO: Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of RhoA/ROCK/MLC Signaling Pathway . Journal of virology 2021, 95 (11). Hasan MK, Widhopf GF, 2nd, Zhang S, Lam SM, Shen Z, Briggs SP, Parker BA, Kipps TJ: Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis . NPJ breast cancer 2019, 5 :35. Xu Z, Gu C, Yao X, Guo W, Wang H, Lin T, Li F, Chen D, Wu J, Ye G et al : CD73 promotes tumor metastasis by modulating RICS/RhoA signaling and EMT in gastric cancer . Cell death & disease 2020, 11 (3):202. Terry S, Nie M, Matter K, Balda MS: Rho signaling and tight junction functions . Physiology 2010, 25 (1):16-26. Kim JH, Park S, Lim SM, Eom HJ, Balch C, Lee J, Kim GJ, Jeong JH, Nam S, Kim YH: Rational design of small molecule RHOA inhibitors for gastric cancer . The pharmacogenomics journal 2020, 20 (4):601-612. Nam S, Lee Y, Kim JH: RHOA protein expression correlates with clinical features in gastric cancer: a systematic review and meta-analysis . BMC cancer 2022, 22 (1):798. Chen Z, Liu S, Xia Y, Wu K: MiR-31 Regulates Rho-Associated Kinase-Myosin Light Chain (ROCK-MLC) Pathway and Inhibits Gastric Cancer Invasion: Roles of RhoA . Medical science monitor : international medical journal of experimental and clinical research 2016, 22 :4679-4691. Oh M, Batty S, Banerjee N, Kim TH: High extracellular glucose promotes cell motility by modulating cell deformability and contractility via the cAMP-RhoA-ROCK axis in human breast cancer cells . Molecular biology of the cell 2023, 34 (8):ar79. Xie L, Huang H, Zheng Z, Yang Q, Wang S, Chen Y, Yu J, Cui C: MYO1B enhances colorectal cancer metastasis by promoting the F-actin rearrangement and focal adhesion assembly via RhoA/ROCK/FAK signaling . Annals of translational medicine 2021, 9 (20):1543. Additional Declarations There is no duality of interest Supplementary Files SupplementaryTableS1.docx Supplementary Table S1. SupplementaryTableS2.docx Supplementary Table S2. SupplementaryTableS3.docx Supplementary Table S3. SupplementaryTableS4.docx Supplementary Table S4. SupplementalFigure1.docx Supplemental Figure S1 SupplementalFigure2.docx Supplemental Figure S2 SupplementalFigure3.docx Supplemental Figure S3 SupplementalFigure4.docx Supplemental Figure S4 SupplementalFigure5.docx Supplemental Figure S5 SupplementalFigure6.docx Supplemental Figure S6 SupplementalFigure7.docx Supplemental Figure S7 Cite Share Download PDF Status: Published Journal Publication published 28 Mar, 2026 Read the published version in Cell Death Discovery → Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6780956","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":465140090,"identity":"77c1ff20-cfea-4a88-8c7c-4b2b7e493d8a","order_by":0,"name":"Yan-Shen Shan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYHACNiC2YWBgZm4AMhJAIgbEaEkDamFE1pJAUMthICZWi8H5488e/NxxPpq/nbGBuaAmLbGBvXmbBOOPw7i13MgxN+w9czt3xmGglhnHchIbeI6VSTAk4NPCwybB23Y7twGkhbehIrFBIscMqOU2XodJ/m07lzsfrkX+DQEtBxLMpHnbDuRugGgBOkyCB78WyRs5ZtKybcm5G4FaDvMcSzNu40krtkhI+49TCx/IYW/b7HLnnT988DFPTbJsP/vhjTc+2KTh1KJwAIkDZoOiCW9MyjfgkRwFo2AUjIJRAAYAWalWsO8wR2cAAAAASUVORK5CYII=","orcid":"","institution":"College of Medicine, National Cheng Kung University","correspondingAuthor":true,"prefix":"","firstName":"Yan-Shen","middleName":"","lastName":"Shan","suffix":""},{"id":465140091,"identity":"28c7e130-f216-4716-a6d1-e9310dc85982","order_by":1,"name":"Yu-Ting Kuo","email":"","orcid":"","institution":"College of Medicine, National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Ting","middleName":"","lastName":"Kuo","suffix":""},{"id":465140092,"identity":"5b3a2369-f577-4491-9820-d5f62a3f1545","order_by":2,"name":"Hao-Chen Wang","email":"","orcid":"","institution":"National Cheng Kung University","correspondingAuthor":false,"prefix":"","firstName":"Hao-Chen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-30 04:40:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6780956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6780956/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-026-03082-2","type":"published","date":"2026-03-28T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92439624,"identity":"011246f7-f988-4c64-807b-7c973d7e563a","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"json","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5054,"visible":true,"origin":"","legend":"","description":"","filename":"CDD251850.json","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/d8a29ae10fb256dfe3ec9a6c.json"},{"id":92440436,"identity":"01a9befa-b606-45b0-8f0f-c652a5b3ac3a","added_by":"auto","created_at":"2025-09-29 18:20:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15904,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/1613dfb76f3be64b37ef3b58.docx"},{"id":92439627,"identity":"5dcf83bb-d52e-4c9e-91fb-dcdf20897038","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17770,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/05798bd310dfacebe6a9b120.docx"},{"id":92440438,"identity":"bea2eec6-cae8-46ee-a06d-81ffa9d668a3","added_by":"auto","created_at":"2025-09-29 18:20:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19208,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/42d49b5e24654b38b9a17fa8.docx"},{"id":92439634,"identity":"3a306b54-7a40-4a50-9f64-c4442f584f43","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":22882,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/4114e7b33175f2a897ad730f.docx"},{"id":92441277,"identity":"45e83971-45a9-4439-99c4-10e6aa695c7a","added_by":"auto","created_at":"2025-09-29 18:36:09","extension":"html","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153476,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/6a3d235aff7bd2149e75748f.html"},{"id":92440164,"identity":"d7e4ad64-7db5-4d43-aeb9-6b8a2efee9d0","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5059450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA20 enhances the migratory capacity of GC cells. A.\u003c/strong\u003e Western blot analysis of A20 expression across multiple GC cell lines. \u003cstrong\u003eB.\u003c/strong\u003e Validation of A20 overexpression in AGS cells (AO/WT1 and AO/WT2) and A20 knockdown in NCIN87 and MKN45 cells (shA20-1 and shA20-2) by Western blotting. \u003cstrong\u003eC.\u003c/strong\u003e Wound healing assays show that A20 overexpression increased migration in AGS cells, while A20 knockdown reduced migration in NCIN87 cells. \u003cstrong\u003eD.\u003c/strong\u003e Transwell migration assays confirmed that A20 overexpression promoted AGS cell migration, and A20 knockdown suppressed migration in NCIN87 and MKN45 cells. Data are presented as mean ± SEM from three independent experiments. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/101a96349ba2eeeab9116158.png"},{"id":92439637,"identity":"75d96817-7190-4411-afb5-c39e532c6170","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7148987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA20 promotes GC metastasis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Kaplan-Meier survival analysis showed that A20 knockdown significantly prolonged survival of orthotopic MKN45 tumor-bearing mice (\u003cem\u003eP\u003c/em\u003e= 0.029). \u003cstrong\u003eB.\u003c/strong\u003e A20 knockdown did not significantly affect total stomach weight (\u003cem\u003eP\u003c/em\u003e = 0.063) but markedly reduced peritoneal metastatic tumor burden. \u003cstrong\u003eC.\u003c/strong\u003e IHC staining for cytokeratin 19 (CK19) revealed decreased distant organ metastasis in mice injected with A20 knockdown MKN45 cells compared to parental cells. \u003cstrong\u003eD.\u003c/strong\u003e The number of metastatic tumor nodules was reduced per mouse following knockdown of A20. Data are presented as mean ± SEM (n = 5). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/4b46286122c9f3c690f923fd.png"},{"id":92440439,"identity":"a6cdfc21-8cef-47d1-83f6-c6ec59536071","added_by":"auto","created_at":"2025-09-29 18:20:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9317018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA20 induces occludin loss and disrupts tight junctions in GC tissue and cells. A.\u003c/strong\u003e Occludin expression was inversely correlated with A20 expression in human GC tissue samples. \u003cstrong\u003eB.\u003c/strong\u003eA20 overexpression downregulated cell membrane-localized occludin in AGS cells, whereas A20 knockdown upregulated cell membrane-localized occludin in NCIN87 and MKN45 cells. \u003cstrong\u003eC.\u003c/strong\u003e Knockdown efficiency of occludin in AGS and MKN45 cells was confirmed by Western blotting. \u003cstrong\u003eD and E.\u003c/strong\u003e Wound healing and transwell migration assays demonstrated that silencing occludin significantly enhanced the migratory capacity of AGS and MKN45 cells. Data are presented as mean ± SEM (n = 3 independent experiments). Significant differences are indicated (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/38d66047657edb1247e4e940.png"},{"id":92441058,"identity":"456713a1-90a9-4934-b9ff-f04071c4a376","added_by":"auto","created_at":"2025-09-29 18:28:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2517308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh A20 expression promotes occludin degradation via the lysosomal pathway. A. \u003c/strong\u003eWestern blotting showed that occludin degradation was accelerated by A20 overexpression in AGS cells (AO/WT1), but occludin expression was preserved by A20 knockdown in MKN45 cells. Occludin expression was quantified relative to vector or shLuc controls (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eB. \u003c/strong\u003eTreatment with the\u003cstrong\u003e \u003c/strong\u003eproteasome inhibitor MG132 could not prevent occludin degradation in AO/WT1 AGS cells and MKN45 cells with high A20 expression. \u003cstrong\u003eC. \u003c/strong\u003eTreatment with the\u003cstrong\u003e \u003c/strong\u003elysosome inhibitor CQ effectively inhibited occludin degradation in AGS cells. \u003cstrong\u003eD. \u003c/strong\u003eA20-induced occludin degradation was suppressed after 24 hours of CQ treatment in both AGS and MKN45 cells. \u003cstrong\u003eE. \u003c/strong\u003eTranswell migration assays showed that treatment with CQ or Wortmannin (Wort) significantly inhibited A20-induced migration in AGS cells. Data are presented as mean ± SEM (n = 3 independent experiments). Significant upregulation: #\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; significant downregulation: ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/0aaff45a541fe21ee66208ed.png"},{"id":92441062,"identity":"9801af0f-51e7-4217-ab46-fdb9549afaea","added_by":"auto","created_at":"2025-09-29 18:28:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3709142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA20 regulates occludin endocytosis, translocation, and degradation via its OTU domain.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eC103A mutation on the A20 OTU domain (OTU\u003csup\u003emut\u003c/sup\u003e) reduced the migratory capacity of AGS cells (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.). \u003cstrong\u003eB. \u003c/strong\u003eOTU\u003csup\u003emut\u003c/sup\u003e suppressed A20-induced migration in AGS cells (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.). \u003cstrong\u003eC. \u003c/strong\u003eA20-induced occludin degradation (AO/WT1) was attenuated by OTU\u003csup\u003emut\u003c/sup\u003e in AGS cells. Occludin expression at different time points was quantified relative to 0 hour (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003eD. \u003c/strong\u003eIF staining showed co-localization of occludin (green) with EEA1 (red) in vector control, AO/WT1, and OTU\u003csup\u003emut\u003c/sup\u003e AGS cells. Scale bar = 10 mm.\u003cstrong\u003e E. \u003c/strong\u003eCo-localization of occludin (green) with LAMP1 (red) was observed by IF staining. Scale bar = 10 mm. \u003cstrong\u003eF. \u003c/strong\u003eOccludin endocytosis was inhibited in OTU\u003csup\u003emut\u003c/sup\u003e AGS cells. Endocytic degradation of occludin was assessed by biotin-labeling endocytosis assay and membrane/cytosol fractionation. Data are presented as mean ± SEM (n = 3 independent experiments). Significant differences are indicated (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"FIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/b5131a596ff624997448845f.png"},{"id":92440169,"identity":"830abdd8-1bf0-4796-808d-a8b5315eb159","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3020912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRhoA/ROCK2 signaling is involved in A20-induced migration and occludin degradation in GC cells.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eKnockdown of A20 reduced RhoA expression in MKN45 cells. \u003cstrong\u003eB. \u003c/strong\u003eIncreased RhoA expression was detected in AO/WT1 AGS cells. \u003cstrong\u003eC.\u003c/strong\u003e IF staining showed co-localization of A20 (red) with RhoA (green) in AGS cells. \u003cstrong\u003eD. \u003c/strong\u003eRhoA knockdown upregulated occludin expression in both AO/WT1 and vector control AGS cells. \u003cstrong\u003eE. \u003c/strong\u003eKnockdown of RhoA or treatment with the Rho inhibitor Rho i suppressed cell migration in both AO/WT1 and vector control AGS cells. \u003cstrong\u003eF. \u003c/strong\u003eOTU\u003csup\u003emut\u003c/sup\u003e decreased ROCK2 phosphorylation without affecting ERK phosphorylation in AGS cells. Data are presented as mean ± SEM (n = 3 independent experiments). Statistically significant differences are indicated (*\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":"FIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/5c4f0d044ddfa15a0efd6035.png"},{"id":92439643,"identity":"24c072e7-d95a-443e-a27d-89c732ba9627","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5314270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA20 interacts with and stabilizes RhoA through its OTU domain.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eCo-IP confirmed the interaction between A20 and RhoA in AO/WT1 and vector control AGS cells. \u003cstrong\u003eB. \u003c/strong\u003eRhoA pull-down assays revealed reduced A20-RhoA interaction in OTU\u003csup\u003emut \u003c/sup\u003eAGS cells. \u003cstrong\u003eC. \u003c/strong\u003eIn situ IF staining showed that OTU\u003csup\u003emut\u003c/sup\u003e blocked A20-RhoA binding affinity in AGS cells. \u003cstrong\u003eD. \u003c/strong\u003eTriple IF staining showed that co-localization of A20, RhoA, and endocytic occludin was disrupted in OTU\u003csup\u003emut\u003c/sup\u003e AGS cells.\u003cstrong\u003e E. \u003c/strong\u003ePull-down of GTP‐bound RhoA (GTP‐RhoA) from vector control, AO/WT1, and OTU\u003csup\u003emut\u003c/sup\u003e AGS lysates showed that OTU\u003csup\u003emut\u003c/sup\u003e impaired RhoA activation and occludin binding. \u003cstrong\u003eF.\u003c/strong\u003e Time-lapse imaging showed that AO/WT1 AGS cells had greater dynamic co-localization and motility of cytosolic occludin-GFP and RhoA-OFP than vector control and OTU\u003csup\u003emut\u003c/sup\u003e cells did. \u003cstrong\u003eG.\u003c/strong\u003e OTU\u003csup\u003emut\u003c/sup\u003e increased RhoA degradation in AGS cells. Data are presented as mean ± SEM (n = 3 independent experiments). Statistically significant differences are indicated (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"FIG7.png","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/dc39b0a96fd67f04b1592708.png"},{"id":108668299,"identity":"58cf1b3b-915b-44a4-8b45-ab457ee83b4f","added_by":"auto","created_at":"2026-05-07 07:08:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":35588014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/7c8ac904-9189-4a70-8cf1-6ff208c9dce4.pdf"},{"id":92440162,"identity":"b43b554f-7a6f-4cd5-9fb5-524f523ea5f1","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15904,"visible":true,"origin":"","legend":"Supplementary Table S1.","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/71ffbd7f9190342f63aab6ad.docx"},{"id":92440165,"identity":"d2cbcf9f-cfb4-4119-ba2e-720f2dfc5a25","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17770,"visible":true,"origin":"","legend":"Supplementary Table S2.","description":"","filename":"SupplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/636953310db0294558e31b1a.docx"},{"id":92440168,"identity":"06a5adfa-1729-4a73-8711-b2833f8f59d2","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19208,"visible":true,"origin":"","legend":"Supplementary Table S3.","description":"","filename":"SupplementaryTableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/0bd25e94acc3e0b489a35a99.docx"},{"id":92439629,"identity":"b1d1372a-7386-4d24-b39c-b0b7c86a0139","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":22882,"visible":true,"origin":"","legend":"Supplementary Table S4.","description":"","filename":"SupplementaryTableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/90d04a06107b7b1b4704c7ed.docx"},{"id":92441059,"identity":"af2902da-6927-44a8-8d84-86a350373fb6","added_by":"auto","created_at":"2025-09-29 18:28:09","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":615846,"visible":true,"origin":"","legend":"Supplemental Figure S1","description":"","filename":"SupplementalFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/348cd1bd2f3881feeeb22e4b.docx"},{"id":92440173,"identity":"7f0e1639-4dbd-4d6c-96e8-3a1cc9136d55","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":538411,"visible":true,"origin":"","legend":"Supplemental Figure S2","description":"","filename":"SupplementalFigure2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/806069fc542f7105bd293333.docx"},{"id":92441276,"identity":"5c1b9e47-420d-494e-8503-b858944b393d","added_by":"auto","created_at":"2025-09-29 18:36:09","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":380389,"visible":true,"origin":"","legend":"Supplemental Figure S3","description":"","filename":"SupplementalFigure3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/26c15abc29ca7c29854cbcf1.docx"},{"id":92439647,"identity":"0b1b2143-85a4-464f-83d7-be7d1d5848bb","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":825906,"visible":true,"origin":"","legend":"Supplemental Figure S4","description":"","filename":"SupplementalFigure4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/a6ac2926a859f1860c4ae2f6.docx"},{"id":92440170,"identity":"910752b1-fbd4-465c-a0d2-fb04dec97879","added_by":"auto","created_at":"2025-09-29 18:12:09","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":431464,"visible":true,"origin":"","legend":"Supplemental Figure S5","description":"","filename":"SupplementalFigure5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/1618c4974a9eb325f5f4c62e.docx"},{"id":92440441,"identity":"ae808b61-43c4-4b54-8429-96340e0b3665","added_by":"auto","created_at":"2025-09-29 18:20:09","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":381395,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure S6\u003c/p\u003e","description":"","filename":"SupplementalFigure6.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/dd991ac725d366ba6f4a7780.docx"},{"id":92439646,"identity":"59d14c29-31c1-4aa6-8775-483cff7d6be5","added_by":"auto","created_at":"2025-09-29 18:04:09","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":2048030,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure S7\u003c/p\u003e","description":"","filename":"SupplementalFigure7.docx","url":"https://assets-eu.researchsquare.com/files/rs-6780956/v1/0fced81b556f349b97ff5bcb.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"A20 enhances migration and metastasis of gastric cancer cells by promoting occludin degradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGastric cancer (GC) is one of the deadliest malignancies worldwide, ranking 4th in cancer incidence and 5th in cancer-related mortality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While early-stage GC is often associated with a favorable prognosis, more than 62% of patients are diagnosed with metastatic disease, which significantly worsens clinical outcomes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advancements in basic research and targeted therapies that have improved treatment efficacy, the prognosis for late-stage GC remains poor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, unveiling the molecular mechanisms underlying GC progression and metastasis is critical for the development of effective therapeutic strategies.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that protumor inflammation can activate oncogenic signaling pathways that facilitate cancer progression and metastasis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. GC has been recognized as an inflammation-associated cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], but the specific mechanisms by which inflammatory factors drive GC remain to be fully elucidated. One key player in inflammation signaling is TNF-α\u0026ndash;induced protein 3 (TNFAIP3, also known as A20), which is rapidly induced by NF-κB upon stimulation with cytokines or environmental stressors via binding to the \u003cem\u003eTNFAIP3\u003c/em\u003e promoter [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A20 typically acts as a negative regulator of NF-κB signaling by targeting IκBα, thereby terminating the inflammatory response [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, accumulating evidence has shown that high A20 expression may play a pro-oncogenic role in various cancers through NF-κB independent mechanisms [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCancer cell migration is a prerequisite for metastasis, which involves disruption of cell-cell junctions, cytoskeletal rearrangement, extracellular matrix remodeling, and chemotactic responses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, epithelial-mesenchymal transition (EMT) is also regarded as a pivotal biological process in tumor metastasis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The metastatic cascade is orchestrated by a complex network of extracellular and intracellular signaling events [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent studies have indicated that elevated A20 expression promotes cancer progression and metastasis across several tumor types. For instance, in melanoma, A20 fosters tumor growth, metastasis, and resistance to BRAF-targeted therapies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A20 also inhibits the cell surface translocation of the \u0026ldquo;eat-me\u0026rdquo; signal calreticulin via suppression of stanniocalcin 1 degradation to facilitate immune evasion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In breast cancer and GC, A20 has been shown to promote EMT and malignant transformation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the detailed mechanism by which A20 drives GC metastasis remains largely unknown.\u003c/p\u003e \u003cp\u003eIn this study, we identified a novel mechanism by which A20 facilitates gastric cancer cell migration through the degradation of occludin, an essential tight junction component. Our results demonstrate that A20 promotes GC cell migration by inducing endocytic lysosomal degradation of occludin, thereby disrupting cell-cell adhesion. These findings suggest that A20 serves as a key regulator of GC metastasis. Targeting the A20-mediated occludin degradation pathway could offer a promising therapeutic strategy to inhibit GC cell migration and metastatic spread.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell line and culture conditions\u003c/h2\u003e \u003cp\u003eHuman gastric cancer (GC) cell lines AGS, SNU-1, MKN45, NCI-N87, and KATO III were obtained from the Bioresource Collection and Research Center (BCRC, Taiwan), while the HR cell line was acquired from the Cell Bank of the Clinical Medicine Research Center, College of Medicine, National Cheng Kung University Hospital (NCKUH). All cell lines were cultured in PRMI medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) 1% glutamine and 1% antibiotic-antimycotic, and were incubated in a humidified atmosphere of 5% CO2 at 37℃. The short tandem repeat (STR) analysis was used for cell line authentication.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransformation and transfection in GC cell lines\u003c/h3\u003e\n\u003cp\u003eTo establish stable transfected cell lines, GC cell lines were separately infected with the appropriate pLKO.1 lentiviral vectors containing \u003cem\u003eTNFAIP3\u003c/em\u003e (TRCN0000050961 and TRCN0000218517), \u003cem\u003eRHOA\u003c/em\u003e (TRCN0000047710 and TRCN0000047712), and non-target Luciferin short hairpin RNA (shRNA) vectors (TRCN0000072243) that were purchased from the National RNAi Core Facility, Academia Sinica, Taipei, Taiwan. Briefly, GC cells were grown overnight with 0.5 mL of viral supernatant containing 8 \u0026micro;g/mL polybrene in medium. Fresh medium containing 2 \u0026micro;g/mL puromycin was added in the next day. Cells were maintained in the continuous presence of puromycin. Plasmids pCMV-flag (PS100001, Origene), pCMV-A20-Myc-flag (RC221337, Origene), occludin cDNA ORF Clone, Human, C-GFPSpark\u0026reg; tag (HG15134-ACG, SinoBiological), and RhoA cDNA ORF Clone, Human, C-OFPSpark\u0026reg; tag (HG12110-ACR, SinoBiological) were constructed and then were used to transfected AGS cells by lipofectamin 3000. Cells were maintained in the continuous presence of G418 (500 \u0026micro;g/mL, A1720, Sigma-Aldrich) or Hygromycin B (200 \u0026micro;g/mL, 31282-04-9, Sigma-Aldrich). The knockdown and overexpression efficiencies were checked by Western blotting. The mutagenesis of A20 was performed using the QuikChange Lightning- Mutagenesis Kit (Stratagene, USA). The primers for A20 plasmid mutagenesis and Sanger sequencing are shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2.\u003c/p\u003e\n\u003ch3\u003eTranswell migration assay\u003c/h3\u003e\n\u003cp\u003eCell migration assays were performed using 24-well inserts (Falcon cell culture inserts, 8-\u0026micro;m pore size; BD Biosciences). In brief, the lower chamber was filled with 600\u0026micro;L of growth medium, and 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells seeded into the upper chamber containing 100\u0026micro;L serum free medium were incubated at 37\u0026deg;C for 9\u0026ndash;48 hours (depending on cell lines). Then cells attaching to the upper side of the membrane were removed gently with a cotton swab and rinsed. Cells that migrated through the membrane and attached to the bottom membrane were fixed in methanol for 10 minutes at room temperature and were stained with hematoxylin (MERCK). The number of migrating cells was quantified by counting 5 independent symmetrical visual fields under the microscope.\u003c/p\u003e\n\u003ch3\u003eWound healing assay\u003c/h3\u003e\n\u003cp\u003eCulture-Inserts 2 Well (80209, ibidi) was placed on a 6 well plate, providing two cell culture reservoirs divided by a 500 \u0026micro;m wall. A total of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells in 100 \u0026micro;L of serum-free medium were seeded in each well of the insert. Once the cells reached confluence, the cell density was monitored under the microscope until a 100% optically confluent cell layer was confirmed. The Culture-Insert was then gently removed using sterile tweezers. The cell layer was washed with growth medium to remove cell debris and non-adherent cells. Subsequently, 2 mL of growth medium was added to each well, and the cell migration process was monitored by capturing images at multiple time points over the following hours. The images were quantified and analyzed to determine the area covered by migrated cells.\u003c/p\u003e\n\u003ch3\u003eProtein degradation analysis\u003c/h3\u003e\n\u003cp\u003eAfter GC cells were collected and the supernatant was removed, the cell pellet was resuspended in serum-free medium. A total of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e GC cells were then plated and incubated overnight at 37\u0026deg;C in serum-free medium. For occludin dynamics analysis, the serum-free medium was replaced with medium containing 10% FBS and incubated for 0, 2, 4, 6 or 8 hours. In RhoA degradation analysis, the serum-free medium was replaced with 10% FBS medium supplemented with cycloheximide (CHX, 20 \u0026micro;g/mL, 239763-M, Sigma-Aldrich) and incubated for 0, 15, 30, 60 or 90 minutes. All cell lysates were collected and stored at -20\u0026deg;C until further analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal model\u003c/h2\u003e \u003cp\u003eAn orthotopic mouse model of human GC was used in this study. MKN45 cells (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were suspended in 20 uL of Matrigel and were orthotopically injected into the stomach wall of NOD-SCID mice. After tumor development for 4 weeks, the body weight and survival rate of the mice were monitored. At week 12, the mice were sacrificed for tumor collection. Primary tumors, organs, and metastatic lesions were harvested from each mouse and weighed for subsequent analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReal-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from GC cell lines using TRIzol Reagent (Invitrogen, USA) and chloroform (Sigma, USA), and was reverse transcribed into cDNA. Expression of mRNA was measured using the ABI 7500 Fast Real-Time PCR System (ABI, USA). Results were normalized to GADPH using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primers used for mRNA expression analysis are listed in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eWestern blotting analysis\u003c/h3\u003e\n\u003cp\u003eWhole cell lysates were collected using RIPA lysis buffer, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher). A mixture of 25 \u0026micro;g protein and 6\u0026times; loading dye was heated at 100\u0026deg;C for 5 minutes and then placed on ice for an additional 5 minutes. Protein samples were loaded onto a 12% SDS-polyacrylamide gel for electrophoresis and subsequently transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk diluted in 0.05% TBST for 1 hour at room temperature. Following blocking, the membrane was incubated with primary antibodies overnight at 4\u0026deg;C and then with secondary antibodies for 1 hour at room temperature. Protein signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) according to manufacturer\u0026rsquo;s instruction and were visualized using the iBright FL1000 Imaging System. The antibodies used to detect protein levels are listed in Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF) staining\u003c/h2\u003e \u003cp\u003eGC cells were seeded onto chamber slides at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well. After fixation with paraformaldehyde and permeabilization with Triton X-100, the samples were incubated with primary antibodies overnight at 4℃, followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour. F-actin was stained with Rhodamine Phalloidin (PHDR1, Cytoskeleton), and nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI). IF images were acquired using a Confocal Laser Scanning Microscope FV3000 (Olympus, Tokyo, Japan). Fluorescence signals were quantified using ImageJ software (NIH, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePull-down immunoprecipitation (IP) and IP-mass spectrometry (IP-MS)\u003c/h2\u003e \u003cp\u003eIP was performed using the Millipore Protein G Plus/Protein A Magnetic Beads (16\u0026ndash;663, Millipore). Briefly, cells lysates from 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells were incubated with anti-DDK (1\u0026micro;g, 14793S, cell signaling) and anti-RhoA (1\u0026micro;g, sc‑418, Santa Cruz) antibodies. The immunoprecipitated proteins were either analyzed by western blotting or submitted to LC-MS/MS protein identification (Biotools). GTP-bound RhoA was pulled down using Rhotekin-RBD agarose beads (BK036-5, Cytoskeleton) and detected by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was performed independently at least three times under identical conditions. Data are expressed as the mean \u0026plusmn; standard error of the mean (SEM). Statistical differences were analyzed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test and one-way ANOVA, as appropriate. All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA) and SigmaPlot 12.0 (Systat Software Inc., CA, USA). Statistical significance was defined as follows: *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eA20 enhances the migration and metastasis of GC cells\u003c/h2\u003e \u003cp\u003eWe first performed Western blotting to screen A20 expression across various GC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To study the role of A20 in GC, we established A20 overexpressing AGS cell lines (AO/WT1 and AO/WT2) and A20 knockdown cell lines in NCIN87 and MKN45 cells (shA20-1 and shA20-2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). We found that expression levels of A20 were not associated with the proliferation of GC cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). However, wound healing assays showed that A20 overexpression increased the migratory ability of AGS cells, while A20 knockdown reduced the migration of NCIN87 and MKN45 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Similar results were seen in transwell migration assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Additionally, compared with control cells, A20 overexpressing AGS cells exhibited a mesenchymal-like phenotype, whereas A20 knockdown led to a shrunken, less spread morphology in MKN45 cells (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Collectively, these results indicate that A20 is able to increase GC cell migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate whether A20 contributes to GC metastasis, GC cells were orthotopically inoculated into the stomachs of mice. A20 knockdown in MKN45 cells could increase survival compared to controls (p\u0026thinsp;=\u0026thinsp;0.029) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although the difference in stomach tumor weight between control and A20 knockdown groups was marginal (p\u0026thinsp;=\u0026thinsp;0.061), oA20 knockdown significantly reduced metastasis to the peritoneal cavity and distant organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggest that A20 exerts its oncogenic effects by promoting GC cell migration and metastasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eA20 downregulates the tight junction protein occludin in GC cells\u003c/h2\u003e \u003cp\u003ePrevious studies have reported the involvement of A20 in EMT [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Accordingly, we sought to investigated the mechanism by which A20 regulates EMT in GC cells. In E-cadherin negative AGS cells, A20 overexpression reduced the expression of occludin (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Conversely, A20 knockdown in NCIN87 cells increased occludin expression and decreased the levels of vimentin, snail, and twist1/2. Similarly, A20 knockdown in MKN45 cells also upregulated occludin and reduced N-cadherin, vimentin, snail, slug, and twist1/2 expression (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). These results were further confirmed by qPCR analysis in AGS and MKN45 cells (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eNotably, occludin expression was inversely correlated with A20 expression across GC cell lines. As the disruption of cell-cell junction is a critical early step in cell migration during cancer metastasis, and loss of occludin contributes to increased aggressiveness in various cancers [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], we further examined this relationship in clinical specimens. IF staining revealed an inverse correlation between A20 and occludin in GC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A20 overexpression (AO/WT1) induced occludin translocation to the cytoplasm and co-localization with A20 in AGS cells, whereas knockdown of A20 (shA20-1) maintained occludin localization at the cell membrane of NCIN87 and MKN45 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the functional role of occludin in GC cell migration, we silenced occludin in AGS and MKN45 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Depletion of occludin significantly enhanced the migratory capacity of both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), suggesting that A20 promotes GC cell migration, at least in part, by downregulating occludin. Taken together, our findings demonstrate that A20 promotes EMT and facilitates the loss of occludin, consequently enhancing the migratory and metastatic potential of GC cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eA20 induces occludin degradation via endocytosis and lysosomal processing\u003c/h2\u003e \u003cp\u003eIn response to environmental stimuli, occludin can translocate from the cell membrane to the cytoplasm, followed by endocytosis and subsequent proteasomal or lysosomal degradation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, we sought to study whether A20 is involved in this process to regulate occludin degradation. After stimulation with FBS to induce EMT, occludin expression in AGS cells was decreased in a time-dependent manner, and this effect was further potentiated by A20 overexpression. Similarly, in MKN45 cells, occludin expression was also decreased over time, but knockdown of A20 attenuated this reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the proteolytic pathway in A20-mediated occludin degradation, we employed the proteasome inhibitor MG-132 in the presence of cycloheximide (CHX) that inhibits de novo protein synthesis. MG-132 restored occludin expression in vector control AGS cells and A20 knockdown MKN45 cells but had no effect in A20 overexpressing AGS cells and shLuc MKN45 cells., suggesting that A20 promotes occludin degradation through a proteasome independent mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eGiven that lysosomal degradation contributes to occludin loss during tight junction disruption [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and that the endocytosis inhibitor wortmannin can prevent tight-junction disassembly in epithelial cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], we investigated the role of endocytic and lysosomal pathways in A20-mediated occludin degradation. Treatment with the lysosome inhibitors chloroquine (CQ) as well as wortmannin in the presence of cycloheximide (CHX) could significantly inhibit occludin degradation in AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Under the stimulation of FBS, treatment with CQ restored occludin expression in both AGS and MKN45 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Moreover, CQ or wortmannin strongly counteracted A20 overexpression-induced cell migration in AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), supporting the conclusion that A20 promotes GC cell migration by facilitating occludin degradation via enhanced endocytosis and lysosomal activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe ovarian tumor (OTU) domain of A20 is critical to occludin endocytosis and lysosomal degradation\u003c/h2\u003e \u003cp\u003ePrevious research has demonstrated that the A20 OTU domain possesses deubiquitinating activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and the ZnF4 domain and ZnF7 domain have catalytic activity as a E3 ligase [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Mutations such as C103A in the OUT domain, C624A/C627A in the ZnF4 domain, and F770A/G771A in the ZnF7 domain can impair their enzymatic activity, indicating that these amino acid residues are critical for A20 biological functions. To determine which domain is essential for occludin degradation and cell migration, we generated A20 expression constructs harboring the C103A mutation in the OTU domain (OTU\u003csup\u003emut\u003c/sup\u003e), C624A/C627A in the ZnF4 domain (ZnF4\u003csup\u003emut\u003c/sup\u003e), and F770A/G771A in the ZnF7 domain (ZnF7\u003csup\u003emut\u003c/sup\u003e) (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWound healing assays revealed that, in AGS cells, the OTU\u003csup\u003emut\u003c/sup\u003e construct blocked A20-induced cell migration, but Zn4\u003csup\u003emut\u003c/sup\u003e and Zn7\u003csup\u003emut\u003c/sup\u003e did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This inhibitory effect of OTU\u003csup\u003emut\u003c/sup\u003e on cell migration was further confirmed by transwell migration assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To study the impact of OTU\u003csup\u003emut\u003c/sup\u003e on occludin degradation, we performed Western blotting following FBS stimulation. Occludin degradation was enhanced in A20 overexpressing AGS cells (AO/WT1), but this effect was abolished in OTU\u003csup\u003emut\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause occludin degradation involves endocytosis and cytosolic trafficking, we examined the co-localization of occludin with early endosomes using IF staining for EEA1. The co-localization signal in AO/WT1 AGS cells was stronger than in OTU\u003csup\u003emut\u003c/sup\u003e or vector control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Given our results that lysosomal degradation contributed to occludin loss, we performed dual IF staining for the lysosomal marker LAMP1 and occludin. The co-localization of occludin with LAMP1 was reduced in OTUmut cells compared to AO/WT1 AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Additionally, both biotin-based endocytosis assays and membrane/cytosol fractionation showed that occludin internalization was promoted in AO/WT1 AGS cells but was decreased in OTU\u003csup\u003emut\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Taken together, these results demonstrate that the OTU domain of A20 is essential to promote occludin endocytosis and subsequent lysosomal degradation, thereby facilitating GC cell migration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eA20-induced occludin degradation involves activation of the RhoA/ROCK2 signaling pathway\u003c/h2\u003e \u003cp\u003eTo date, no studies have described how A20 regulates occludin degradation. To elucidate this mechanism, we conducted immunoprecipitation followed by mass spectrometry (IP-MS) to identify A20-interacting proteins potentially involved in occludin degradation and cancer metastasis. KEGG pathway analysis of proteins pulled down with Flag-tagged A20 revealed RhoA as a binding partner, known to be associated with tight junction assembly, actin cytoskeleton regulation, and endocytosis (Supplementary Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In A20 knockdown MKN45 cells, RhoA expression was decreased and negatively correlated to occludin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Conversely, A20 overexpression enhanced RhoA expression 24 hours after FBS stimulation and thus promoted AGS cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). IF staining further demonstrated increased expression and co-localization of A20 and RhoA in AO/WT1 AGS cells compared to vector control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). RhoA knockdown increased occludin expression in both AO/WT1 and vector control AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE); similar effects were observed with a RhoA inhibitor (Rho i). These results suggest that RhoA acts downstream of A20 to drive GC cell migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt has been reported that RhoA promotes occludin endocytosis by activating ERK or ROCK2 signaling [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study, we observed that only ROCK2 phosphorylation was prolonged in AO/WT1 AGS cells, and this phosphorylation was diminished in OTU\u003csup\u003emut\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Activation of RhoA/ROCK2 signaling promotes downstream F-actin assembly, a key indicator of cell migration. Phalloidin-Rhodamine staining revealed that AO/WT1 AGS cells exhibited increased expression of F-actin (red), which co-localized with endocytic occludin (green) (Supplementary Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA). To further assess the involvement of ROCK2 phosphorylation in occludin degradation, AGS and MKN45 cells were treated with an endocytosis inhibitor dynole and a ROCK2 inhibitor Y-27632. The inhibition of either endocytosis or ROCK2 effectively reduced occludin degradation in AGS and MKN45 cells (Supplementary Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB). Taken together, these results suggest that A20 promotes occludin degradation in GC cells through activation of RhoA/ROCK2 signaling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe OTU domain of A20 interacts with and stabilizes RhoA for occludin degradation\u003c/h2\u003e \u003cp\u003eTo confirm the interaction between A20 and RhoA, we performed co-IP, which showed reciprocal binding between the two proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Their interaction was significantly weakened in OTU\u003csup\u003emut\u003c/sup\u003e AGS cells after FBS stimulation, indicating that the C103A mutation in A20 OTU domain disrupts the binding of A20 with RhoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The interaction was further validated in HEK293T cells co-transfected with A20 and RhoA (Supplementary Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Proximity ligation assay (PLA) also showed a strong interaction between A20 and RhoA in AO/WT1 AGS cells but a marked reduction in OTU\u003csup\u003emut\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), accompanied by increased F-actin expression, suggesting enhanced cell motility. Triple IF staining demonstrated cytoplasmic co-localization of A20, RhoA, and occludin in AO/WT1 AGS and shLuc MKN45 cells, whereas occludin remained membrane-bound in vector, OTU\u003csup\u003emut,\u003c/sup\u003e and shA20 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and Supplementary Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA). Rhotekin-RBD pull-down assays showed that A20 overexpression increased RhoA-GTP levels, while the OTU mutant impaired RhoA activity and disrupted its association with occludin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Live-cell imaging using occludin-GFP and RhoA-OFP confirmed enhanced cytosolic mobility only in AO/WT1 AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and Supplementary Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eB). Collectively, these results demonstrate that A20 OTU domain facilitates occludin endocytosis and degradation by stabilizing and activating RhoA in GC cells. To determine whether the OTU domain of A20 is required for RhoA protein stabilization, GC cells were treated with CHX. After 90 minutes of CHX treatment, the degradation of RhoA was slower in AO/WT1 AGS cells than in vector control and OTU\u003csup\u003emut\u003c/sup\u003e AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Overall, these results suggest that the OTU domain of A20 protein can stabilize RhoA, thereby promoting occludin degradation and enhancing the migratory capacity of GC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identify a novel mechanism in which A20 facilitates GC cell migration through its OTU domain by inducing occludin internalization and subsequent lysosomal degradation. While A20 has been previously implicated in epithelial-mesenchymal transition (EMT) and tumor progression, our findings clarify how A20 contributes to tight junction disassembly, a key step in cancer cell dissemination.\u003c/p\u003e \u003cp\u003ePrevious studies have associated A20 with the EMT in GC cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], aligns with our findings that expression of EMT markers was affected by A20 knockdown. Among the EMT markers, occludin, a critical component of tight junctions, plays a central role in maintaining epithelial barrier integrity. Its depletion contributes to increased cellular permeability and is commonly observed during cancer metastasis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Occludin has been also linked to gastric epithelial hyperplasia and inflammation in animal models [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and to cytosolic redistribution in GC cells following H. pylori infection [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our study confirms occludin as a tumor suppressor, as its silencing increased GC cell migration, and identifies A20 as a promoter of occludin degradation.\u003c/p\u003e \u003cp\u003eAlthough tight junction loss has been known as a facilitator of tumor invasion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the role of A20 in this process remains vague. In contrast to our findings, Kolodziej et al. reported that A20 maintained tight junctions in intestinal epithelial cells by preventing LPS-induced occludin loss via its N-terminal activity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This discrepancy may be due to cell type specificity or different stimuli, suggesting the context-dependent regulatory roles of A20 in inflammation and cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Despite earlier evidence has associated A20 with metastasis in inflammation-associated cancers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], this is the first detailed demonstration of A20 driving GC cell migration by promoting lysosomal occludin degradation.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that A20 is localized to endocytic compartments and interacts with lysosome-associated proteins LAMP1 and LAPTM5 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, biotin-labeling endocytosis assays revealed that A20 enhances occludin internalization, an important early event of GC cell migration. Pharmacological inhibition of lysosomes or endocytosis suppressed both occludin degradation and GC cell motility, indicating that targeting this pathway to prevent occludin degradation, for example, using CQ, might be a promising anti-metastatic strategy. Further investigation is needed to define the trafficking route and machinery that mediate the endocytic pathway of occludin.\u003c/p\u003e \u003cp\u003eRhoA is a small GTPase protein belonging to the Rho family, which plays a well-established role in cancer progression, including tight-junction breakdown, EMT induction, and increased migration [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Tight junction components like occludin, ZO-1, and claudins form a barrier limiting cell dissemination, which can be disrupted by RhoA signaling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Blockade of the RhoA signaling pathway effectively suppresses cancer aggressiveness [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A meta-analysis by Nam et al. linked RhoA expression to advanced tumor stage and poor differentiation in GC [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Despite its relevance, little is known about the regulation of RhoA protein expression. Our results show that A20 can stabilize RhoA and consequently facilitate occludin degradation, indicating that A20 is a critical upstream regulator of RhoA in GC metastasis. Notably, RhoA silencing or inhibition could significantly restore occludin levels in A20 overexpressing cells.\u003c/p\u003e \u003cp\u003eGiven the ubiquitin-editing enzymatic activity of A20, we explored the functional domains responsible for RhoA regulation. Prior studies have highlighted the oncogenic functions of its zinc finger domain in promoting EMT by mediating multiple mono-ubiquitin modification of Snail [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], but our study found that the OTU deubiquitinase domain is also crucial for GC cell migration. We identified that A20 binds and stabilizes RhoA through its OTU domain. Mutation of the catalytic cysteine residue (C103A) in the OTU domain abrogated ROCK2 phosphorylation, suggesting that A20 acts as a regulator of the RhoA/ROCK2 pathway. However, the precise mechanism by which the OTU domain modulates RhoA stabilization and the downstream signaling pathways remains to be elucidated.\u003c/p\u003e \u003cp\u003eRhoA signaling has been reported to be essential for cytoskeletal reorganization, a prerequisite for cancer cell motility [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Activated RhoA drives F-actin polymerization and reassembly of myosin and tubulin filaments, facilitating cell adhesion and migration [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Live-cell imaging in this study showed co-localization and coordinated movement of A20, RhoA, and occludin, suggesting that A20 not only destabilizes tight junctions but also modulates cytoskeletal architecture to promote GC migration. Thus, A20, via its OTU domain, serves as a central modulator of both tight-junction integrity and cellular polarity.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provided strong evidence that A20 stabilizes RhoA through its OTU domain to induce occludin internalization and lysosomal degradation, thus promoting GC cell migration and metastasis. This newly identified A20/RhoA/occludin signaling axis offers mechanistic insight into GC progression and highlights the A20 OTU domain as a promising therapeutic target against metastasis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egastric cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEMT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eepithelial-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factorα\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTNFAIP3\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor α-induced protein 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOTU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eovarian tumor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eZnF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eZinc finger\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHRP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunofluorescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePLA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eproximity ligation assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCQ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echloroquine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCHX\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecycloheximide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within this article (and its supplementary information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Taiwan Ministry of Science and Technology (NSTC 109-2314-B-006-028-MY2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYTK: Methodology, investigation, writing-original draft. HCW: Conceptualization, writing-review and editing. YSS: Conceptualization, resources, visualization, supervision, funding acquisition, project administration, writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving human tissues and data in this study were approved by the Institutional Review Board (IRB) of NCKUH (IRB No. B-ER-107-432). The requirement for informed consent was waived by the IRB. All methods were conducted in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the manuscript and given consent for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A: \u003cstrong\u003eCancer Statistics, 2021\u003c/strong\u003e. \u003cem\u003eCA: a cancer journal for clinicians\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e71\u003c/strong\u003e(1):7-33.\u003c/li\u003e\n \u003cli\u003eKinami S, Saito H, Takamura H: \u003cstrong\u003eSignificance of Lymph Node Metastasis in the Treatment of Gastric Cancer and Current Challenges in Determining the Extent of Metastasis\u003c/strong\u003e. \u003cem\u003eFrontiers in oncology\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e11\u003c/strong\u003e:806162.\u003c/li\u003e\n \u003cli\u003eGuan WL, He Y, Xu RH: \u003cstrong\u003eGastric cancer treatment: recent progress and future perspectives\u003c/strong\u003e. \u003cem\u003eJournal of hematology \u0026amp; oncology\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e16\u003c/strong\u003e(1):57.\u003c/li\u003e\n \u003cli\u003eHibino S, Kawazoe T, Kasahara H, Itoh S, Ishimoto T, Sakata-Yanagimoto M, Taniguchi K: \u003cstrong\u003eInflammation-Induced Tumorigenesis and Metastasis\u003c/strong\u003e. \u003cem\u003eInternational journal of molecular sciences\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e22\u003c/strong\u003e(11).\u003c/li\u003e\n \u003cli\u003eO\u0026apos;Brien VP, Kang Y, Shenoy MK, Finak G, Young WC, Dubrulle J, Koch L, Rodriguez Martinez AE, Williams J, Donato E\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eSingle-cell Profiling Uncovers a Muc4-Expressing Metaplastic Gastric Cell Type Sustained by Helicobacter pylori-driven Inflammation\u003c/strong\u003e. \u003cem\u003eCancer research communications\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e3\u003c/strong\u003e(9):1756-1769.\u003c/li\u003e\n \u003cli\u003eRihawi K, Ricci AD, Rizzo A, Brocchi S, Marasco G, Pastore LV, Llimpe FLR, Golfieri R, Renzulli M: \u003cstrong\u003eTumor-Associated Macrophages and Inflammatory Microenvironment in Gastric Cancer: Novel Translational Implications\u003c/strong\u003e. \u003cem\u003eInternational journal of molecular sciences\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e22\u003c/strong\u003e(8).\u003c/li\u003e\n \u003cli\u003eWaldum H, Fossmark R: \u003cstrong\u003eInflammation and Digestive Cancer\u003c/strong\u003e. \u003cem\u003eInternational journal of molecular sciences\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e24\u003c/strong\u003e(17).\u003c/li\u003e\n \u003cli\u003eKrikos A, Laherty CD, Dixit VM: \u003cstrong\u003eTranscriptional activation of the tumor necrosis factor alpha-inducible zinc finger protein, A20, is mediated by kappa B elements\u003c/strong\u003e. \u003cem\u003eThe Journal of biological chemistry\u0026nbsp;\u003c/em\u003e1992, \u003cstrong\u003e267\u003c/strong\u003e(25):17971-17976.\u003c/li\u003e\n \u003cli\u003eMothes J, Ipenberg I, Arslan S, Benary U, Scheidereit C, Wolf J: \u003cstrong\u003eA Quantitative Modular Modeling Approach Reveals the Effects of Different A20 Feedback Implementations for the NF-kB Signaling Dynamics\u003c/strong\u003e. \u003cem\u003eFrontiers in physiology\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e:896.\u003c/li\u003e\n \u003cli\u003ePeng X, Zhang C, Zhou ZM, Wang K, Gao JW, Qian ZY, Bao JP, Ji HY, Cabral VLF, Wu XT: \u003cstrong\u003eA20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics\u003c/strong\u003e. \u003cem\u003eInflammation research : official journal of the European Histamine Research Society [et al]\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e71\u003c/strong\u003e(5-6):695-710.\u003c/li\u003e\n \u003cli\u003eLuo M, Wang X, Wu S, Yang C, Su Q, Huang L, Fu K, An S, Xie F, To KKW\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eA20 promotes colorectal cancer immune evasion by upregulating STC1 expression to block \u0026quot;eat-me\u0026quot; signal\u003c/strong\u003e. \u003cem\u003eSignal transduction and targeted therapy\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e8\u003c/strong\u003e(1):312.\u003c/li\u003e\n \u003cli\u003ePaul S, Bhardwaj M, Kang SC: \u003cstrong\u003eGSTO1 confers drug resistance in HCT‑116 colon cancer cells through an interaction with TNFalphaIP3/A20\u003c/strong\u003e. \u003cem\u003eInternational journal of oncology\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e61\u003c/strong\u003e(5).\u003c/li\u003e\n \u003cli\u003eLiu J, Yang S, Wang Z, Chen X, Zhang Z: \u003cstrong\u003eUbiquitin ligase A20 regulates p53 protein in human colon epithelial cells\u003c/strong\u003e. \u003cem\u003eJournal of biomedical science\u0026nbsp;\u003c/em\u003e2013, \u003cstrong\u003e20\u003c/strong\u003e(1):74.\u003c/li\u003e\n \u003cli\u003eNehme Z, Roehlen N, Dhawan P, Baumert TF: \u003cstrong\u003eTight Junction Protein Signaling and Cancer Biology\u003c/strong\u003e. \u003cem\u003eCells\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e12\u003c/strong\u003e(2).\u003c/li\u003e\n \u003cli\u003eBergers G, Fendt SM: \u003cstrong\u003eThe metabolism of cancer cells during metastasis\u003c/strong\u003e. \u003cem\u003eNature reviews Cancer\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e21\u003c/strong\u003e(3):162-180.\u003c/li\u003e\n \u003cli\u003eHuang Y, Hong W, Wei X: \u003cstrong\u003eThe molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis\u003c/strong\u003e. \u003cem\u003eJournal of hematology \u0026amp; oncology\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e15\u003c/strong\u003e(1):129.\u003c/li\u003e\n \u003cli\u003eAttiq A, Afzal S: \u003cstrong\u003eTrinity of inflammation, innate immune cells and cross-talk of signalling pathways in tumour microenvironment\u003c/strong\u003e. \u003cem\u003eFrontiers in pharmacology\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e14\u003c/strong\u003e:1255727.\u003c/li\u003e\n \u003cli\u003eMa J, Wang H, Guo S, Yi X, Zhao T, Liu Y, Shi Q, Gao T, Li C, Guo W: \u003cstrong\u003eA20 promotes melanoma progression via the activation of Akt pathway\u003c/strong\u003e. \u003cem\u003eCell death \u0026amp; disease\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e(9):794.\u003c/li\u003e\n \u003cli\u003eLee JH, Jung SM, Yang KM, Bae E, Ahn SG, Park JS, Seo D, Kim M, Ha J, Lee J\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eA20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1\u003c/strong\u003e. \u003cem\u003eNature cell biology\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e19\u003c/strong\u003e(10):1260-1273.\u003c/li\u003e\n \u003cli\u003eDu B, Liu M, Li C, Geng X, Zhang X, Ning D, Liu M: \u003cstrong\u003eThe potential role of TNFAIP3 in malignant transformation of gastric carcinoma\u003c/strong\u003e. \u003cem\u003ePathology, research and practice\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e215\u003c/strong\u003e(8):152471.\u003c/li\u003e\n \u003cli\u003eOsanai M, Murata M, Nishikiori N, Chiba H, Kojima T, Sawada N: \u003cstrong\u003eEpigenetic silencing of occludin promotes tumorigenic and metastatic properties of cancer cells via modulations of unique sets of apoptosis-associated genes\u003c/strong\u003e. \u003cem\u003eCancer research\u0026nbsp;\u003c/em\u003e2006, \u003cstrong\u003e66\u003c/strong\u003e(18):9125-9133.\u003c/li\u003e\n \u003cli\u003eWang M, Liu Y, Qian X, Wei N, Tang Y, Yang J: \u003cstrong\u003eDownregulation of occludin affects the proliferation, apoptosis and metastatic properties of human lung carcinoma\u003c/strong\u003e. \u003cem\u003eOncology reports\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e40\u003c/strong\u003e(1):454-462.\u003c/li\u003e\n \u003cli\u003eLi Y, Yuan T, Zhang H, Liu S, Lun J, Guo J, Wang Y, Zhang Y, Fang J: \u003cstrong\u003ePHD3 inhibits colon cancer cell metastasis through the occludin-p38 pathway\u003c/strong\u003e. \u003cem\u003eActa biochimica et biophysica Sinica\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e55\u003c/strong\u003e(11):1749-1757.\u003c/li\u003e\n \u003cli\u003eMortell KH, Marmorstein AD, Cramer EB: \u003cstrong\u003eFetal bovine serum and other sera used in tissue culture increase epithelial permeability\u003c/strong\u003e. \u003cem\u003eIn vitro cellular \u0026amp; developmental biology : journal of the Tissue Culture Association\u0026nbsp;\u003c/em\u003e1993, \u003cstrong\u003e29A\u003c/strong\u003e(3 Pt 1):235-238.\u003c/li\u003e\n \u003cli\u003eKim KA, Kim D, Kim JH, Shin YJ, Kim ES, Akram M, Kim EH, Majid A, Baek SH, Bae ON: \u003cstrong\u003eAutophagy-mediated occludin degradation contributes to blood-brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models\u003c/strong\u003e. \u003cem\u003eFluids and barriers of the CNS\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e17\u003c/strong\u003e(1):21.\u003c/li\u003e\n \u003cli\u003eUtech M, Mennigen R, Bruewer M: \u003cstrong\u003eEndocytosis and recycling of tight junction proteins in inflammation\u003c/strong\u003e. \u003cem\u003eJournal of biomedicine \u0026amp; biotechnology\u0026nbsp;\u003c/em\u003e2010, \u003cstrong\u003e2010\u003c/strong\u003e:484987.\u003c/li\u003e\n \u003cli\u003eSoni D, Wang DM, Regmi SC, Mittal M, Vogel SM, Schluter D, Tiruppathi C: \u003cstrong\u003eDeubiquitinase function of A20 maintains and repairs endothelial barrier after lung vascular injury\u003c/strong\u003e. \u003cem\u003eCell death discovery\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e4\u003c/strong\u003e(1):60.\u003c/li\u003e\n \u003cli\u003eYin H, Karayel O, Chao YY, Seeholzer T, Hamp I, Plettenburg O, Gehring T, Zielinski C, Mann M, Krappmann D: \u003cstrong\u003eA20 and ABIN-1 cooperate in balancing CBM complex-triggered NF-kappaB signaling in activated T cells\u003c/strong\u003e. \u003cem\u003eCellular and molecular life sciences : CMLS\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e79\u003c/strong\u003e(2):112.\u003c/li\u003e\n \u003cli\u003eWang Y, Zhang J, Yi XJ, Yu FS: \u003cstrong\u003eActivation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells\u003c/strong\u003e. \u003cem\u003eExperimental eye research\u0026nbsp;\u003c/em\u003e2004, \u003cstrong\u003e78\u003c/strong\u003e(1):125-136.\u003c/li\u003e\n \u003cli\u003eFeng S, Zou L, Wang H, He R, Liu K, Zhu H: \u003cstrong\u003eRhoA/ROCK-2 Pathway Inhibition and Tight Junction Protein Upregulation by Catalpol Suppresses Lipopolysaccaride-Induced Disruption of Blood-Brain Barrier Permeability\u003c/strong\u003e. \u003cem\u003eMolecules\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e23\u003c/strong\u003e(9).\u003c/li\u003e\n \u003cli\u003eMartin TA, Jiang WG: \u003cstrong\u003eLoss of tight junction barrier function and its role in cancer metastasis\u003c/strong\u003e. \u003cem\u003eBiochimica et biophysica acta\u0026nbsp;\u003c/em\u003e2009, \u003cstrong\u003e1788\u003c/strong\u003e(4):872-891.\u003c/li\u003e\n \u003cli\u003eSaitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T, Tsukita S: \u003cstrong\u003eComplex phenotype of mice lacking occludin, a component of tight junction strands\u003c/strong\u003e. \u003cem\u003eMolecular biology of the cell\u0026nbsp;\u003c/em\u003e2000, \u003cstrong\u003e11\u003c/strong\u003e(12):4131-4142.\u003c/li\u003e\n \u003cli\u003eWroblewski LE, Shen L, Ogden S, Romero-Gallo J, Lapierre LA, Israel DA, Turner JR, Peek RM, Jr.: \u003cstrong\u003eHelicobacter pylori dysregulation of gastric epithelial tight junctions by urease-mediated myosin II activation\u003c/strong\u003e. \u003cem\u003eGastroenterology\u0026nbsp;\u003c/em\u003e2009, \u003cstrong\u003e136\u003c/strong\u003e(1):236-246.\u003c/li\u003e\n \u003cli\u003eHuang S, Zhang J, Li Y, Xu Y, Jia H, An L, Wang X, Yang Y: \u003cstrong\u003eDownregulation of Claudin5 promotes malignant progression and radioresistance through Beclin1-mediated autophagy in esophageal squamous cell carcinoma\u003c/strong\u003e. \u003cem\u003eJournal of translational medicine\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e21\u003c/strong\u003e(1):379.\u003c/li\u003e\n \u003cli\u003eKolodziej LE, Lodolce JP, Chang JE, Schneider JR, Grimm WA, Bartulis SJ, Zhu X, Messer JS, Murphy SF, Reddy N\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eTNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions\u003c/strong\u003e. \u003cem\u003ePloS one\u0026nbsp;\u003c/em\u003e2011, \u003cstrong\u003e6\u003c/strong\u003e(10):e26352.\u003c/li\u003e\n \u003cli\u003eYokoyama Y, Tamachi T, Iwata A, Maezawa Y, Meguro K, Yokota M, Takatori H, Suto A, Suzuki K, Hirose K\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eA20 (Tnfaip3) expressed in CD4(+) T cells suppresses Th2 cell-mediated allergic airway inflammation in mice\u003c/strong\u003e. \u003cem\u003eBiochemical and biophysical research communications\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e629\u003c/strong\u003e:47-53.\u003c/li\u003e\n \u003cli\u003eLi L, Hailey DW, Soetandyo N, Li W, Lippincott-Schwartz J, Shu HB, Ye Y: \u003cstrong\u003eLocalization of A20 to a lysosome-associated compartment and its role in NFkappaB signaling\u003c/strong\u003e. \u003cem\u003eBiochimica et biophysica acta\u0026nbsp;\u003c/em\u003e2008, \u003cstrong\u003e1783\u003c/strong\u003e(6):1140-1149.\u003c/li\u003e\n \u003cli\u003eGlowacka WK, Alberts P, Ouchida R, Wang JY, Rotin D: \u003cstrong\u003eLAPTM5 protein is a positive regulator of proinflammatory signaling pathways in macrophages\u003c/strong\u003e. \u003cem\u003eThe Journal of biological chemistry\u0026nbsp;\u003c/em\u003e2012, \u003cstrong\u003e287\u003c/strong\u003e(33):27691-27702.\u003c/li\u003e\n \u003cli\u003eSharif M, Baek YB, Naveed A, Stalin N, Kang MI, Park SI, Soliman M, Cho KO: \u003cstrong\u003ePorcine Sapovirus-Induced Tight Junction Dissociation via Activation of RhoA/ROCK/MLC Signaling Pathway\u003c/strong\u003e. \u003cem\u003eJournal of virology\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e95\u003c/strong\u003e(11).\u003c/li\u003e\n \u003cli\u003eHasan MK, Widhopf GF, 2nd, Zhang S, Lam SM, Shen Z, Briggs SP, Parker BA, Kipps TJ: \u003cstrong\u003eWnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis\u003c/strong\u003e. \u003cem\u003eNPJ breast cancer\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e5\u003c/strong\u003e:35.\u003c/li\u003e\n \u003cli\u003eXu Z, Gu C, Yao X, Guo W, Wang H, Lin T, Li F, Chen D, Wu J, Ye G\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eCD73 promotes tumor metastasis by modulating RICS/RhoA signaling and EMT in gastric cancer\u003c/strong\u003e. \u003cem\u003eCell death \u0026amp; disease\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e(3):202.\u003c/li\u003e\n \u003cli\u003eTerry S, Nie M, Matter K, Balda MS: \u003cstrong\u003eRho signaling and tight junction functions\u003c/strong\u003e. \u003cem\u003ePhysiology\u0026nbsp;\u003c/em\u003e2010, \u003cstrong\u003e25\u003c/strong\u003e(1):16-26.\u003c/li\u003e\n \u003cli\u003eKim JH, Park S, Lim SM, Eom HJ, Balch C, Lee J, Kim GJ, Jeong JH, Nam S, Kim YH: \u003cstrong\u003eRational design of small molecule RHOA inhibitors for gastric cancer\u003c/strong\u003e. \u003cem\u003eThe pharmacogenomics journal\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e20\u003c/strong\u003e(4):601-612.\u003c/li\u003e\n \u003cli\u003eNam S, Lee Y, Kim JH: \u003cstrong\u003eRHOA protein expression correlates with clinical features in gastric cancer: a systematic review and meta-analysis\u003c/strong\u003e. \u003cem\u003eBMC cancer\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e22\u003c/strong\u003e(1):798.\u003c/li\u003e\n \u003cli\u003eChen Z, Liu S, Xia Y, Wu K: \u003cstrong\u003eMiR-31 Regulates Rho-Associated Kinase-Myosin Light Chain (ROCK-MLC) Pathway and Inhibits Gastric Cancer Invasion: Roles of RhoA\u003c/strong\u003e. \u003cem\u003eMedical science monitor : international medical journal of experimental and clinical research\u0026nbsp;\u003c/em\u003e2016, \u003cstrong\u003e22\u003c/strong\u003e:4679-4691.\u003c/li\u003e\n \u003cli\u003eOh M, Batty S, Banerjee N, Kim TH: \u003cstrong\u003eHigh extracellular glucose promotes cell motility by modulating cell deformability and contractility via the cAMP-RhoA-ROCK axis in human breast cancer cells\u003c/strong\u003e. \u003cem\u003eMolecular biology of the cell\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e34\u003c/strong\u003e(8):ar79.\u003c/li\u003e\n \u003cli\u003eXie L, Huang H, Zheng Z, Yang Q, Wang S, Chen Y, Yu J, Cui C: \u003cstrong\u003eMYO1B enhances colorectal cancer metastasis by promoting the F-actin rearrangement and focal adhesion assembly via RhoA/ROCK/FAK signaling\u003c/strong\u003e. \u003cem\u003eAnnals of translational medicine\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e9\u003c/strong\u003e(20):1543.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\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":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"gastric cancer, tumor necrosis factor α induced protein 3, A20, occludin, RhoA, metastasis","lastPublishedDoi":"10.21203/rs.3.rs-6780956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6780956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic inflammation is a well-established risk factor in the development of gastric cancer (GC). Tumor necrosis factor α-induced protein 3 (TNFAIP3, also known as A20) is an inflammation-associated protein that functions as an oncogene in various cancers, but the role of A20 in GC progression remains unclear. In this study, we found that overexpression of A20 significantly enhanced GC cell migration, whereas A20 knockdown suppressed this effect. A20 promoted epithelial-mesenchymal transition and led to the loss of the tight junction protein occludin. Mechanistically, A20 induced occludin endocytosis and lysosomal degradation via its ovarian tumor (OTU) domain. Pull-down assays revealed that A20 interacts with the migration-related protein RhoA, increasing its stability, and thereby sustaining ROCK2 phosphorylation, which contributes to occludin degradation. PLA further showed that mutation of the OTU domain disrupted the interaction between A20 and RhoA in AGS cells, indicating the necessity of the OTU domain for this interaction. In conclusion, our findings demonstrate that A20 promotes GC cell migration by stabilizing RhoA and facilitating occludin degradation, underscoring A20 as a potential therapeutic target to inhibit GC metastasis.\u003c/p\u003e","manuscriptTitle":"A20 enhances migration and metastasis of gastric cancer cells by promoting occludin degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 18:04:04","doi":"10.21203/rs.3.rs-6780956/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"34481633-c333-4972-b123-1f462884b320","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":49375452,"name":"Biological sciences/Cancer/Tumour biomarkers"},{"id":49375453,"name":"Health sciences/Diseases/Cancer/Cancer microenvironment"}],"tags":[],"updatedAt":"2026-05-07T07:07:44+00:00","versionOfRecord":{"articleIdentity":"rs-6780956","link":"https://doi.org/10.1038/s41420-026-03082-2","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2026-03-28 04:00:00","publishedOnDateReadable":"March 28th, 2026"},"versionCreatedAt":"2025-09-29 18:04:04","video":"","vorDoi":"10.1038/s41420-026-03082-2","vorDoiUrl":"https://doi.org/10.1038/s41420-026-03082-2","workflowStages":[]},"version":"v1","identity":"rs-6780956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6780956","identity":"rs-6780956","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}