Abstract Gastric cancer remains a leading cause of cancer-related mortality worldwide, characterized by poor prognosis due to its aggressive nature and high metastatic potential. While the E2-conjugating enzyme UBE2I (UBC9), essential for SUMOylation, has been implicated in various cancers, its precise role in gastric cancer remains poorly understood. In the study, we demonstrate significant UBC9 overexpression in gastric cancer tissues, which correlates with poor clinical outcomes. Functional analyses revealed that UBC9 knockdown significantly suppressed gastric cancer cell proliferation, migration, and invasion in vitro and in vivo, whereas UBC9 overexpression enhanced these malignant phenotypes. Through integrated transcriptomic and proteomic analyses, we identified ATF2 (Activating Transcription Factor 2) as a crucial downstream effector of UBC9-mediated oncogenic signaling. The mechanistic relationship between these factors was confirmed as ATF2 knockdown substantially attenuated the oncogenic effects of UBC9 overexpression. This newly identified UBC9-ATF2 regulatory axis promotes gastric cancer progression by enhancing cellular proliferation and metastatic potential. Our findings establish UBC9 and ATF2 as promising prognostic biomarkers and potential therapeutic targets, suggesting that intervention in the UBC9-ATF2 axis may provide novel therapeutic strategies for inhibiting gastric cancer progression and improving patient outcomes. Supplementary Information The online version contains supplementary material available at 10.1186/s12957-025-03922-y. Keywords: Gastric cancer, UBC9, SUMOylation, ATF2, Prognosis Introduction Gastric cancer remains a significant global health burden, ranking as the fifth most common cancer and the fifth leading cause of cancer-related mortality worldwide [[60]1]. Despite advancements in diagnostic and therapeutic strategies, the prognosis for gastric cancer patients remains poor, primarily due to late-stage diagnosis, and high rates of metastasis [[61]2–[62]4]. Understanding the molecular mechanisms underlying gastric cancer progression is crucial for the development of effective diagnostic markers and therapeutic targets. Post-translational modifications (PTMs) play pivotal roles in regulating various cellular processes, including protein stability, localization, and activity [[63]5]. Among these PTMs, Small Ubiquitin-like Modifier (SUMO)ylation has emerged as a critical regulator in cancer biology. SUMOylation involves the attachment of SUMO proteins to target substrates, mediated by a cascade of enzymatic activities involving E1 activating enzymes, E2 conjugating enzymes, and E3 ligases [[64]6–[65]8]. UBE2I (also known as UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation, making it a central player in this modification process. UBC9 has been implicated in numerous cellular functions and its dysregulation has been associated with various malignancies [[66]9–[67]12]. In melanoma, high levels of UBC9 are associated with sensitivity to the cytotoxic effects of chemotherapy drugs, suggesting its potential role in tumor resistance [[68]13]. In breast cancer, UBC9 expression is closely related to tumor grade and prognosis. Research has found that SUMOylation of UBC9 in breast cancer cells can influence the migration and invasion capabilities of tumor cells [[69]14]. While previous studies have demonstrated elevated UBC9 expression in gastric cancer tissues [[70]15], its functional implications in gastric cancer cells remain unexplored. Activating Transcription Factor 2 (ATF2), a member of the ATF/CREB family, is a transcription factor involved in various cellular processes, including cell cycle progression, apoptosis, and stress responses [[71]16]. ATF2 has been shown to be elevated in gastric cancer and correlates with worse clinical outcomes and increased tumor aggressiveness [[72]17]. It has also been reported to interact with UBC9 [[73]18], suggesting a potential regulatory relationship. This study explores UBC9’s role in gastric cancer by analyzing expression patterns in clinical samples and assessing prognostic significance across cohorts. Functional assays examined the effects of UBC9 knockdown and overexpression on cancer cell proliferation, migration, and invasion both in vitro and in vivo. Transcriptomic and proteomic analyses, focusing on its interaction with ATF2, identified UBC9-mediated pathways. These findings suggest UBC9 as a potential prognostic biomarker and therapeutic target, aiming to improve clinical outcomes. Methods Patients and specimens A total of 100 gastric cancer tissue samples and corresponding normal gastric tissue samples were collected from patients at the Fujian Provincial Hospital between June 2018 and October 2019. The study protocol was approved by the Research Ethics Committee of Fujian Provincial Hospital, and all participating patients provided written informed consent. Survival analysis of UBC9 and ATF2 Survival analysis of UBC9 and ATF2 was primarily conducted using the BEST platform ([74]https://rookieutopia.hiplot.com.cn/app_direct/BEST/) [[75]19]. We obtained survival analysis results for UBC9 and ATF2 from the TCGA, [76]GSE28541, [77]GSE14208 and [78]GSE13861 datasets. The [79]GSE28541 dataset included 40 patients with advanced gastric cancer from the University of Texas MD Anderson Cancer Center. The [80]GSE13861 dataset comprised 65 patients diagnosed with primary gastric adenocarcinoma, sourced from Yonsei Cancer Center in Seoul, Korea. The [81]GSE14208 dataset involved 122 patients with metastatic gastric cancer from the National Cancer Center Hospital in Goyang, Korea. The TCGA dataset encompassed 348 patients with primary gastric cancer, complete with prognostic follow-up data, representing a diverse ethnic background that includes Black, White, and Asian individuals. Immunohistochemistry (IHC) staining analysis Immunoperoxidase staining was used to assess UBC9 protein expression in gastric cancer tissues. Tissue sections were incubated with an anti-UBC9 antibody (Santa Cruz Biotechnology, 1:50). Two pathologists independently assessed the staining scores. Positive cells were grouped by percentage (0–25%, 26–50%, 51–75%, 76–100%) and staining intensity was scored from 0 (no staining) to 3 (strong staining). The final score was calculated by multiplying both values. Scores below 4 were classified as low expression, and scores of 4 or higher as high expression. Western blotting Cells were lysed in RIPA buffer with protease inhibitors, and protein concentrations were measured using the Micro BCA Protein Assay Kit. Proteins were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated with primary antibodies against UBC9 (1:1000 dilution), ATF2 (1:500 dilution), and GAPDH (1:5000 dilution). The primary antibodies were purchased from Proteintech. Detection was performed using IRDye^® secondary antibodies and the Odyssey^® CLx Imaging System. Immunofluorescence experiment Cells were seeded on coverslips in 24-well plates at 1 × 10^5 cells/well. At 40–50% confluence, cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and blocked with 10% BSA. After overnight incubation with primary antibodies at 4 °C, secondary antibodies were applied for 1 h in the dark. Cover slips were mounted with DAPI and imaged by confocal microscopy. Differential expression analysis of genes and proteins Gene and protein expression levels were analyzed using the “limma” package in R, with differential expression defined as|log2FC| ≥ 0.585 and p-value < 0.05. Functional enrichment analysis KEGG and GO Biological Process enrichment analyses were performed to identify pathways and processes associated with differentially expressed genes and proteins, using an adjusted p-value < 0.05 for significance. Cell culture Human gastric cancer cell lines (AGS, SNU-1, HGC-27) and the normal gastric epithelial line (GES-1) were obtained from ATCC. Cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO[2] humidified incubator. Cell proliferation assay Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) method. Cells were suspended and adjusted to a concentration of 1 × 10^4 cells/ml. Aliquots of 2000 cells were then seeded into each well of a 96-well plate. At 24, 48, and 72 h, 10 µl of CCK-8 reagent was added to each well. After incubating for an additional hour, the absorbance was measured at 450 nm using a microplate spectrophotometer. Cell migration assay Cells were plated in 6-well plates at 1 × 10^6 cells/well and grown to near confluence. A linear wound was created in the cell monolayer. Detached cells were rinsed away. Wound closure was evaluated at 0 and 48 h using microscopy, and the migration area quantified with ImageJ. Zebrafish xenograft model Zebrafish were obtained from Fuzhou Bio-Service Biotechnology Co. Ltd. (Fuzhou, China). AGS cells were labeled with fluorescence using a 5 µM solution of CellTracker™ CM-DiI (C700, Invitrogen, CA, USA), a red-fluorescent lipophilic dye. These labeled cells were subsequently injected into zebrafish larvae through microinjection, with approximately 200 cells introduced per larva, and ten larvae forming an experimental group. To monitor tumor cell proliferation within the zebrafish host, fluorescence imaging was conducted at two time points: 2 h and 48 h after the xenotransplantation. Additionally, to evaluate the presence of distant metastasis, images of the tail regions were captured at 2 h and 24 h post-injection. All zebrafish experiments were conducted in strict accordance with the ethical guidelines and regulations established by the Institutional Animal Care and Use Committee (IACUC) of Fujian Provincial Hospital. The experimental procedures involving zebrafish larvae (less than 5 days post fertilization) do not require prior ethics approval for such developmental stages. All protocols also adhered to the ARRIVE guidelines for reporting animal research, ensuring humane care and minimizing animal use and suffering throughout the study. Statistical analysis Data were analyzed using GraphPad Prism 6.0. Results are shown as mean ± standard deviation. Statistical tests included one-way ANOVA or Student’s t-test for group comparisons. Survival analyses were performed using the Kaplan-Meier method, and differences between groups were assessed using the log-rank test. To account for multiple comparisons, a Bonferroni correction was applied, and p-values less than 0.05 was considered statistically significant. Results Significant upregulation of UBC9 in gastric cancer tissues linked to adverse prognosis To understand the role of UBC9 in gastric cancer, we first examined its expression pattern in the TCGA_STAD cohort. Analysis revealed that UBC9 expression was significantly upregulated in gastric cancer tissues compared to normal tissues (Fig. [82]1A). We further investigated the relationship between UBC9 expression and various clinical characteristics. No significant differences in UBC9 expression were observed when patients were stratified by age, gender, Lauren classification, or race (Fig. [83]1B-E). Similarly, UBC9 expression showed no significant variation across different TNM stages (Fig. [84]1F). Fig. 1. [85]Fig. 1 [86]Open in a new tab UBC9 expression patterns and prognostic significance in gastric cancer. (A) Comparison of UBC9 levels in normal vs. tumor tissues (TCGA_STAD). (B-F) UBC9 expression stratified by clinical characteristics: (B) age, (C) gender, (D) Lauren classification, (E) race, and (F) TNM stages. (G-I) Kaplan-Meier survival analyses of gastric cancer patients stratified by UBC9 expression levels: (G) overall survival in [87]GSE28541 cohort, (H) disease-specific survival in TCGA_STAD cohort, and (I) relapse-free survival in [88]GSE13861 cohort. The classification into high and low expression groups was primarily based on the optimal cutoff value To evaluate the prognostic significance of UBC9 in gastric cancer, we performed survival analyses across multiple independent cohorts. In the [89]GSE28541 cohort, patients with high UBC9 expression demonstrated significantly worse overall survival compared to those with low expression (Fig. [90]1G). This finding was further validated in the TCGA_STAD cohort, where high UBC9 expression was significantly associated with poor disease-specific survival (Fig. [91]1H). Consistent with these results, analysis of the [92]GSE13861 cohort also revealed that high UBC9 expression correlated with worse relapse-free survival (Fig. [93]1I). These findings collectively suggest that UBC9 overexpression is a common feature in gastric cancer and serves as a potential prognostic biomarker associated with poor clinical outcomes. UBC9 knockdown suppresses gastric cancer cell proliferation and metastatic potential in vitro To investigate the biological function of UBC9 in gastric cancer, we established stable UBC9 knockdown cell lines using two different shRNAs in AGS, SNU-1, and HGC-27 cells. Western blot analysis confirmed the effective downregulation of UBC9 expression in all three cell lines (Fig. [94]2A-C). CCK-8 assays revealed that UBC9 knockdown significantly suppressed cell proliferation in all three gastric cancer cell lines (Fig. [95]2D-F). Fig. 2. [96]Fig. 2 [97]Open in a new tab Effects of UBC9 knockdown on gastric cancer cells. (A-C) Western blot for UBC9 knockdown using shRNAs in (A) AGS, (B) SNU-1, and (C) HGC-27 cells. (D-F) CCK-8 proliferation assays for cell proliferation in (D) AGS, (E) SNU-1, and (F) HGC-27 cells. (G-I) Wound healing assays for migration in (G) AGS, (H) SNU-1, and (I) HGC-27 cells. (J-L) Transwell assays for invasion for (J) AGS, (K) SNU-1, and (L) HGC-27 cells. *, p < 0.05, **, p < 0.01, ***, p < 0.001 We next examined the impact of UBC9 knockdown on cell migration using wound healing assays. After 48 h, UBC9 knockdown cells showed significantly reduced migration capacity in all three cell lines (Fig. [98]2G-I). Furthermore, transwell invasion assays demonstrated that UBC9 knockdown markedly decreased the invasive capability of all three gastric cancer cell lines (Fig. [99]2J-L). These results collectively indicate that UBC9 plays a crucial role in promoting gastric cancer cell proliferation, migration, and invasion. UBC9 knockdown demonstrates significant in vivo effects on proliferation and metastatic behavior of gastric cancer cells To evaluate the impact of UBC9 knockdown on the proliferation and metastasis of gastric cancer AGS cells in vivo, we used a zebrafish model. Fluorescence areas for yolk (indicating proliferation) and tail (indicating metastasis) were analyzed at different time points. At the 2-hour time point, there were no significant differences in the yolk fluorescence area among the UBC9 knockdown groups and the negative control. However, at the 48-hour mark, both UBC9 knockdown groups exhibited a significant reduction in yolk fluorescence area compared to the control group, suggesting impaired cell proliferation (Fig. [100]3A). Fig. 3. [101]Fig. 3 [102]Open in a new tab In vivo effects of UBC9 knockdown on proliferation and metastasis of gastric cancer cells in a zebrafish model. (A) Representative images and quantitative analysis of yolk fluorescence area at 2- and 48-hours post-injection in AGS cells. (B) Representative images and quantitative analysis of tail fluorescence area at 2- and 24-hours post-injection. ns, p > 0.05; ***, p < 0.001 At the 2-hour observation, fluorescence in the tail area showed no significant differences among the knockdown and control groups. By the 24-hour mark, a significant decrease in tail fluorescence was observed in the UBC9 knockdown groups compared to the NC group, indicating reduced metastatic potential (Fig. [103]3B). These results highlight the crucial role of UBC9 in promoting both proliferation and metastasis of gastric cancer cells in vivo. Prognostic implications and nuclear localization of UBC9 protein in gastric cancer Next, we investigated UBC9 expression in 100 gastric cancer patients and its localization in gastric cell lines. IHC staining of tumor and adjacent normal tissues revealed higher UBC9 staining in tumor tissues (Fig. [104]4A). This observation is quantified in Fig. [105]4B, demonstrating a significantly higher IHC score for UBC9 in cancer tissues compared to normal tissues. Furthermore, Kaplan-Meier survival analysis indicates that elevated UBC9 expression correlates with poorer overall survival (Fig. [106]4C). To determine whether UBC9 expression is an independent prognostic factor for gastric cancer, we performed multivariate Cox regression analysis adjusting for clinicopathological variables including sex, age, pStage, pT Stage, pN Stage, and pM Stage (Table [107]1). The univariate analysis showed that pStage, pT Stage, pN Stage, pM Stage and UBC9 expression were significantly associated with overall survival. Multivariate analysis revealed that pN Stage and UBC9 expression remained independent prognostic factors for overall survival in gastric cancer patients. Finally, immunofluorescence analysis of UBC9-GFP in normal gastric epithelial cells (GES-1) and gastric cancer cells (AGS) shows predominant nuclear localization in both cell types (Fig. [108]4D). Fig. 4. [109]Fig. 4 [110]Open in a new tab Expression and localization of UBC9 protein in gastric cancer and its prognostic significance. (A) Immunohistochemical staining of UBC9 in tumor and adjacent normal tissues from two representative gastric cancer patients. (B) Quantitative IHC analysis. (C) Kaplan-Meier survival analysis. (D) Immunofluorescence of UBC9-GFP localization in cells. The classification into high and low expression groups was primarily based on the optimal cutoff value. ***, p < 0.001 Table 1. Univariate and multivariate analysis Characteristics Univariate Multivariate HR (95% CI) P value HR (95% CI) P value Sex  Female 0.96(0.54–1.74) > 0.05 N/A  Male Reference Age  ≤ 60 0.87(0.47–1.59) > 0.05 N/A  >60 Reference p Stage  I&II 0.30(0.17–0.534) < 0.001 0.97(0.30–3.12) > 0.05  III&IV Reference Reference pT Stage  2 0.39(0.23–0.68) < 0.001 0.46(0.2018-1.11) > 0.05  3&4 Reference Reference pN Stage  0&1 0.26(0.15–0.45) < 0.001 0.30(0.11–0.72) < 0.01  2&3 Reference Reference pM Stage  0 0.26(0.1–0.67) < 0.01 0.75(0.39–1.45) < 0.05  1 Reference Reference UBC9  Low expression 0.40(0.18–0.80) < 0.001 0.31(0.10–0.89) < 0.05  High expression Reference Reference [111]Open in a new tab Multi-Omics identification of ATF2 as a downstream effector of UBC9 in gastric cancer In this study, we performed a comprehensive transcriptomic analysis to investigate the effects of UBC9 knockdown on gene expression in AGS cells. Our findings revealed that a total of 1452 genes were significantly downregulated, while 1842 genes showed significant upregulation after UBC9 knockdown (Fig. [112]5A, Table [113]S1). The heatmap further illustrated the distinct expression patterns between UBC9 knockdown cells and control cells (Fig. [114]5B). Fig. 5. [115]Fig. 5 [116]Open in a new tab Multi-omics analysis identifies ATF2 as a downstream effector of UBC9 in gastric cancer. (A) Volcano plot of DEGs upon UBC9 knockdown. (B) Heatmap of DEGs. (C-F) KEGG and GO analysis of DEGs. (G) Volcano plot of DEPs. (H) Heatmap of DEPs. (I-L) KEGG and GO analysis of DEPs. (M) Venn diagram of DEGs and DEPs. (N) ATF2 expression in TCGA. (O-T) Kaplan-Meier survival curves for ATF2 expression. (U) Western blot of ATF2 after UBC9 modulation. The classification into high and low expression groups was primarily based on the optimal cutoff value To elucidate the biological significance of these transcriptional changes, we conducted KEGG and GO enrichment analyses on the upregulated and downregulated genes. The KEGG analysis indicated that the upregulated genes were primarily enriched in pathways related to herpes simplex virus type 1 infection, human papillomavirus infection, p53 signaling, and apoptotic pathways across multiple species (Fig. [117]5C). Additionally, GO analysis revealed that these genes were involved in processes such as protein localization, cellular macromolecule localization, intracellular transport, and post-Golgi vesicle-mediated transport (Fig. [118]5D). For downregulated genes, KEGG analysis showed enrichment in pathways related to the ribosome function, Huntington’s disease, oxidative phosphorylation, and DNA replication (Fig. [119]5E). GO analysis indicated that these downregulated genes were involved in biological processes such as protein complex subunit organization, mRNA metabolism, and RNA catabolism (Fig. [120]5F). In addition to transcriptomic analysis, we also investigated the impact of UBC9 knockdown on protein expression through proteomic analysis. Results showed that 46 proteins were significantly downregulated, while 74 proteins were significantly upregulated following UBC9 knockdown (Fig. [121]5G&H, Table [122]S2). KEGG pathway enrichment analysis on the upregulated proteins demonstrated significant involvement in metabolic pathways, arachidonic acid metabolism, IL-17 signaling pathway, α-linolenic acid metabolism, metabolite pathways of linoleic acid, porphyrin and chlorophyll metabolism, and ovarian steroidogenesis pathways (Fig. [123]5I). GO analysis for the upregulated proteins indicated participation in biological processes such as homeostasis, negative regulation of molecular function, small molecule biosynthesis, myeloid leukocyte activation, and cellular responses to cisplatin (Fig. [124]5J). Conversely, KEGG analysis showed that the downregulated proteins were enriched in pathways like NOD-like receptor signaling, phosphoinositide metabolism, FcγR-mediated phagocytosis, choline metabolism in cancer, and fatty acid biosynthesis (Fig. [125]5K). GO analysis for downregulated proteins revealed enrichment in biological processes related to cytokine responses, tRNA processing, regulation of cell shape, lysosomal assembly, and phagosome maturation (Fig. [126]5L). We then performed an integrated analysis of transcriptomic and proteomic data, revealing that 43 genes exhibited significant changes at both the mRNA and protein levels following UBC9 knockdown. A total of 3251 genes displayed significant changes exclusively at the mRNA level, while 77 genes showed significant changes solely at the protein level (Fig. [127]5M). Given the role of UBC9 in regulating SUMOylation and influencing protein localization and stability, we explored the correlation of these 77 proteins with UBC9. Among these, the transcription factor ATF2 has been reported to interact with UBC9 [[128]20], promoting proliferation and metastasis in gastric cancer cells [[129]17]. Our results indicated that ATF2 protein levels were significantly reduced following UBC9 knockdown, suggesting that UBC9 may regulate downstream target genes through ATF2, thereby fostering malignant progression in gastric cancer. Consequently, we examined ATF2 expression and its prognostic implications in gastric cancer. The expression of ATF2 in gastric cancer was analyzed using the TCGA_STAD cohort, revealing that ATF2 is significantly overexpressed in tumor tissues compared to normal tissues (Fig. [130]5N). Further survival analysis based on TCGA_STAD data indicated that high ATF2 expression is associated with poor overall survival in gastric cancer patients (Fig. [131]5O). This observation was corroborated by additional cohorts, [132]GSE28541 and [133]GSE14208, where elevated ATF2 levels also predicted unfavorable outcomes (Fig. [134]5P&Q). In TCGA_STAD cohorts, patients with higher ATF2 expression showed decreased disease-specific survival and disease-free survival rates (Fig. [135]5R&S). Consistent with these results, analysis of the [136]GSE14208 cohort also revealed that high UBC9 expression correlated with worse progress-specific survival (Fig. [137]5T). We further investigated the relationship between ATF2 expression and various clinical characteristics. No significant differences in ATF2 expression were observed when patients were stratified by age, gender, Lauren classification, or race (Supplementary Fig. [138]1A-D). Similarly, ATF2 expression showed no significant variation across different TNM stages (Supplementary Fig. [139]1E). Next, we further investigated the influence of UBC9 on the expression of ATF2. The results demonstrated that knockdown of UBC9 in AGS cells significantly suppressed ATF2 expression. Conversely, overexpression of UBC9 led to a marked enhancement of ATF2 expression. These findings suggest that UBC9 plays a crucial regulatory role in modulating ATF2 levels, thereby potentially impacting various cellular processes in gastric cancer cells (Fig. [140]5U). UBC9 overexpression enhances the proliferation and metastatic behavior of gastric cancer cell through ATF2 dependency Finally, we explored the effects of UBC9 overexpression on AGS cell functionality and its dependency on ATF2. The proliferation assay results in Fig. [141]6A show that cells with UBC9 overexpression (UBC9-OS) exhibited a significant increase in relative OD values over 72 h compared to the negative control (NC), indicating enhanced cell proliferation. However, this increased proliferation was notably reduced when ATF2 was knocked down (UBC9-OS + ATF2-shRNA), suggesting that the proliferation effects of UBC9 are partially dependent on ATF2. Figure [142]6B presents the migration assay results. AGS cells with UBC9 overexpression displayed significant enhancement in migration rates at 48 h post-wounding compared to NC. The migration capacity was diminished in the group where ATF2 was knocked down, confirming ATF2’s role in mediating UBC9-induced migration enhancement. In Fig. [143]6C, the invasion assay demonstrated that UBC9 overexpression significantly increased the invasion capabilities of AGS cells compared to NC. Similar to the migration assay, ATF2 knockdown resulted in a marked decrease in invasive capacity, highlighting the necessity of ATF2 for UBC9-mediated invasion enhancement. Fig. 6. [144]Fig. 6 [145]Open in a new tab UBC9 promotes proliferation, migration, and invasion of gastric cancer cells via ATF2. (A) CCK-8 assay for proliferation. (B) Wound healing assay for migration. (C) Transwell invasion assay. NS, p > 0.05; **, p < 0.01, ***, p < 0.001 Discussion Gastric cancer remains a significant global health challenge, being one of the leading causes of cancer-related mortality worldwide [[146]21–[147]23]. The molecular mechanisms underlying its progression and metastasis are complex and not yet fully understood. In this study, we investigated the role of UBC9 in gastric cancer. Our results demonstrate that UBC9 is significantly upregulated in gastric cancer tissues and is associated with poor clinical outcomes. Furthermore, we identified ATF2 as a crucial downstream effector of UBC9, mediating its effects on cell proliferation and metastasis. This study provides new insights into the molecular pathways involved in gastric cancer progression and highlights potential therapeutic targets. Our comprehensive analysis of the TCGA cohort revealed that both UBC9 and ATF2 are significantly overexpressed in gastric cancer tissues compared to normal controls. Notably, neither UBC9 nor ATF2 expression showed significant correlation with clinicopathological parameters such as patient age, gender, Lauren classification, race, or TNM stage. While gastric cancer exhibits heterogeneity, including Lauren classification-based intestinal and diffuse subtypes, our data showed no significant UBC9 or ATF2 expression differences between these subtypes. We further investigated whether UBC9 and ATF2 were part of the gene signatures identified by Russi et al. that distinguish between diffuse and intestinal gastric cancer subtypes [[148]24]. Russi et al. defined a diffuse signature comprising 1659 genes upregulated in both GC vs. normal mucosa and diffuse vs. intestinal samples, and an intestinal signature of 1839 genes upregulated in both GC vs. healthy mucosa and in intestinal vs. diffuse samples. Our analysis indicated that UBC9 and ATF2 were not present within either the diffuse or intestinal signature gene sets identified in the Russi et al. study, suggesting that their role may be independent of these broad subtype classifications. These findings indicate that the upregulation of UBC9 and ATF2 represents a common molecular alteration in gastric cancer, largely independent of these major clinical variables. This robustness across different clinical subgroups highlights the potential utility of UBC9 and ATF2 as generalizable biomarkers for gastric cancer diagnosis and prognosis. It is pertinent to address the heterogeneity inherent in the publicly sourced cohorts utilized in this study. The TCGA dataset, for instance, comprised 348 patients with primary gastric cancer from diverse ethnic backgrounds, including Black, White, and Asian individuals, offering broad representation1. In contrast, [149]GSE28541 focused on 40 patients with advanced gastric cancer from a single US center (University of Texas MD Anderson Cancer Center), [150]GSE13861 included 65 patients with primary gastric adenocarcinoma from Yonsei Cancer Center in Seoul, Korea, and [151]GSE14208 involved 122 patients with metastatic gastric cancer from the National Cancer Center Hospital in Goyang, Korea1. These cohorts thus varied in terms of disease stage (primary vs. advanced vs. metastatic), geographical origin, and ethnic composition. Despite these potential sources of molecular and clinical variability, the consistent observation of UBC9 and ATF2 overexpression and their association with poor prognosis across these distinct cohorts strongly supports the broad applicability of our findings regarding the UBC9-ATF2 axis. UBC9’s role in various cancers highlights its significance not only in gastric cancer but also as a common feature in multiple tumor types. High expression of UBC9 has been associated with aggressive tumor behavior and poor prognosis across various cancers, including breast, colon, lung, and liver cancer. For instance, in breast cancer, elevated UBC9 levels have been linked to increased cell proliferation and resistance to apoptosis, facilitating tumor progression [[152]14, [153]25]. In colorectal cancer, studies have shown that UBC9 regulates several oncogenic pathways that contribute to cancer cell proliferation and survival. Its involvement in the EMT process has also been noted, promoting migration and invasion capabilities [[154]26, [155]27]. The ability of UBC9 to modulate the stability of key oncoproteins through SUMOylation illustrates its potential as a therapeutic target. Recent studies have explored small molecule inhibitors of UBC9, underscoring its relevance in cancer therapy. In hepatocellular carcinoma, UBC9 has been implicated in modulating the tumor microenvironment. By facilitating the SUMOylation of proteins involved in metastasis, UBC9 promotes aggressive tumor phenotypes. Targeting UBC9 in HCC models showed promising results, reducing tumor growth and dissemination [[156]13]. Functional assays further elucidated the role of UBC9 in gastric cancer cell proliferation and metastasis. UBC9 knockdown in AGS, SNU-1, and HGC-27 cell lines resulted in significant suppression of cell proliferation, migration, and invasion. These in vitro findings were corroborated by in vivo experiments using a zebrafish model. UBC9 knockdown led to reduced proliferation and metastatic potential of gastric cancer cells in vivo, as evidenced by decreased fluorescence areas in the yolk and tail regions of zebrafish embryos. These results indicate that UBC9 is essential for the proliferation and metastatic behavior of gastric cancer cells. At the molecular level, UBC9 functions as the sole E2-conjugating enzyme in the SUMOylation pathway, a post-translational modification process that influences various cellular functions, including protein stability, localization, and interactions [[157]28–[158]31]. The overexpression of UBC9 could lead to aberrant SUMOylation of target proteins, thereby promoting oncogenic processes. The nuclear localization of UBC9 observed in both normal and cancerous gastric cells suggest its involvement in regulating nuclear proteins critical for cell proliferation and survival. Our multi-omics analysis identified ATF2 as a downstream effector of UBC9 in gastric cancer. ATF2 is known to regulate genes involved in cell cycle progression, apoptosis, and stress responses [[159]17, [160]32]. The oncogenic role of ATF2 has been documented in multiple cancers. For instance, in gastric cancer, ATF2 is elevated and correlates with worse clinical outcomes and increased tumor aggressiveness. In melanoma, ATF2 drives cell proliferation and survival [[161]33]. It has been reported that ATF2 activates transcription of pro-survival genes while repressing apoptotic pathways, promoting tumorigenesis in various contexts. In pancreatic cancer, ATF2 facilitates the maintenance of stem-like properties in cancer cells, further enhancing tumor recurrence and metastasis [[162]34]. In gastric cancer, ATF2 promotes gastric cancer cell progression [[163]35]. The findings of our study indicate that UBC9 plays a significant role in regulating ATF2 in gastric cancer. While UBC9 has been shown to interact with ATF2, our data suggest that UBC9 primarily enhances ATF2 expression without affecting its mRNA levels, implying a post-transcriptional regulatory mechanism rather than direct transcriptional control. This raises an important question regarding the exact nature of their interaction. It is plausible that UBC9 directly SUMOylates ATF2, thereby stabilizing the protein and promoting its oncogenic activities. Alternatively, UBC9 may exert its influence on ATF2 indirectly through the modification of other regulatory proteins that impact ATF2 stability or activity. For instance, UBC9 could SUMOylate proteins that affect ATF2 degradation or activation pathways. Future studies, including co-immunoprecipitation and in vitro SUMOylation assays, are necessary to clarify whether UBC9 directly SUMOylates ATF2 or regulates its function through intermediaries. The interplay between UBC9 and ATF2 suggests a regulatory axis. ATF2 has been reported to interact with UBC9. UBC9 may enhance the expression of genes that facilitate cell proliferation, migration, and invasion by increasing the expression of ATF2 protein. Further supporting this notion, functional assays demonstrated that the pro-tumorigenic effects of UBC9 overexpression are, at least in part, dependent on ATF2. Overexpression of UBC9 in AGS cells enhanced proliferation, migration, and invasion. However, silencing ATF2 in these cells significantly attenuated these effects, indicating that ATF2 is a critical mediator of UBC9’s oncogenic functions. The identification of UBC9 and ATF2 as key players in gastric cancer progression has important therapeutic implications. Targeting UBC9 could decrease the expression of ATF2, thereby inhibiting tumor growth and metastasis. Several small molecule inhibitors of UBC9 have been developed and shown to exhibit anti-tumor activity in preclinical models [[164]11]. Of particular interest is TAK-981 (subasumstat), a first-in-class inhibitor of the SUMO-activating enzyme SAE, which blocks the initial step of the SUMOylation cascade. Preclinical studies have demonstrated that TAK-981 can effectively suppress tumor growth and enhance anti-tumor immunity in several cancer types by disrupting SUMOylation-dependent oncogenic pathways [[165]36]. While specific studies focused on gastric cancer are still limited, the well-established importance of the UBC9-ATF2 axis in gastric cancer suggests that SUMOylation inhibitors like TAK-981 may hold therapeutic promise in this context as well. Further research is needed to evaluate the efficacy and potential clinical benefit of such inhibitors for gastric cancer patients. In addition to UBC9, ATF2 itself represents a promising pharmacological target in gastric cancer. ATF2 is a transcription factor that governs genes involved in cell cycle control, apoptosis, and metastasis. Pharmacological inhibition of ATF2-such as through small molecules that disrupt its phosphorylation, dimerization, or DNA binding-has shown efficacy in other tumor models. Combining UBC9 blockade with strategies that directly inhibit ATF2 may provide synergistic anti-cancer effects by shutting down both the upstream SUMOylation machinery and its major oncogenic effector. Future work should explore combined inhibition approaches and evaluate their potential to more effectively suppress gastric cancer progression while minimizing toxicity to normal tissues. Another intriguing strategy is the application of synthetic lethality in gastric cancer therapy. Inhibition of UBC9 could increase cancer cell susceptibility to established treatments, such as chemotherapy, by disrupting multiple cellular stress response pathways that are SUMOylation-dependent. For instance, UBC9 inhibition has been shown to sensitize cancer cells to DNA-damaging agents and impair the repair of chemotherapy-induced DNA damage. Exploiting these vulnerabilities-potentially by combining UBC9 or SUMO pathway inhibition with conventional cytotoxic agents-may enhance therapeutic efficacy and overcome resistance mechanisms in gastric cancer. One of the notable limitations of this study is the absence of mammalian in vivo models, such as mice, which are traditionally used in cancer research to evaluate tumor biology and treatment responses. Although zebrafish models provide unique advantages, including transparent embryos and rapid development, they may not fully mirror the complex physiological and pathological processes that occur in mammalian systems. The reliance on zebrafish for our investigations into UBC9 and ATF2 dynamics in gastric cancer limits the translatability of our findings to human disease. Specifically, differences in the immune response, metabolic processes, and tumor microenvironment between zebrafish and mammals could influence the interactions and therapeutic efficacy we observe in our model. Future studies incorporating mammalian models will be essential for validating our results and understanding the functional implications of UBC9 and ATF2 in a more clinically relevant context. By addressing these gaps, we can enhance our understanding of their roles in gastric cancer and further explore potential therapeutic avenues. In conclusion, our study demonstrates that UBC9 is significantly upregulated in gastric cancer tissues and is closely associated with poor clinical outcomes. Functional experiments revealed that UBC9 enhances gastric cancer cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, UBC9 regulates the expression of ATF2, a crucial downstream effector, thereby promoting malignant progression. These findings position UBC9 and ATF2 as potential prognostic biomarkers and therapeutic targets in gastric cancer (Fig. [166]7). Targeting the UBC9-ATF2 axis may offer novel strategies for inhibiting tumor growth and metastasis, although the essential roles of SUMOylation in normal cellular functions necessitate careful consideration to minimize potential side effects. Fig. 7. [167]Fig. 7 [168]Open in a new tab Proposed summary mechanism. UBC9 promotes the proliferation and metastasis of gastric cancer cells by increasing the expression of the ATF2 protein Electronic supplementary material Below is the link to the electronic supplementary material. [169]12957_2025_3922_MOESM1_ESM.xls^ (466KB, xls) Supplementary Table S1: Differentially expressed genes after UBC9 knockdown [170]12957_2025_3922_MOESM2_ESM.xls^ (36.5KB, xls) Supplementary Table S2: Differentially expressed proteins after UBC9 knockdown [171]Supplementary Material 3^ (606.6KB, pdf) [172]Supplementary Material 4^ (71.1KB, docx) Acknowledgements