Supplementary Figure 9. Fluorescence Co-localization Quantitative Analysis

Supplementary Figure 8. Transcriptional regulation of UBE2B and BIRC2 by NF-κB.

ABSTRACT Radiation‐induced enteritis (RIE) is a severe, dose‐limiting toxicity of cancer radiotherapy lacking mechanism‐based therapies. While the gut microbiome regulates radiation injury, harnessing it therapeutically remains challenging. Here, we show that the natural product β‐elemene protects against RIE through a synergistic mechanism coordinating host and microbial responses. β‐elemene directly rescues the radiation‐disrupted interaction between the lactate transporter MCT1 and its chaperone CD147 in intestinal epithelial cells, priming them for enhanced lactate uptake. Concurrently, β‐elemene selectively enriches for Lactobacillus gasseri , increasing intestinal lactate production. The convergence of host priming and elevated lactate availability triggers a metabo‐epigenetic cascade. Specifically, lactate drives the lactylation of the chromatin‐associated protein RBBP4, which in turn recruits EP300 to activate the transcription of essential DNA damage repair genes. We further identify EP300 as a lactyl‐transferase, establishing a self‐amplifying positive feedback loop that robustly enhances the repair signal. Our findings delineate a complete drug‐microbe‐metabolite‐epigenome axis, establishing a ‘prime‐and‐fuel’ therapeutic strategy where a single agent orchestrates inter‐kingdom communication to promote tissue regeneration.

Supplementary Figure 2. UBE2B promotes GC cell proliferation

Supplementary Table 3. GSEA Results

Supplementary Figure 1. Identification of UBE2B as a key prognostic marker in GC

Supplementary Figure 6. TRAF1 is a substrate of the ubiquitin ligase BIRC2

Ubiquitin (Ub)-conjugating enzymes (E2 enzyme) are essential mediators of Ub-dependent signaling cascades, governing diverse cellular processes such as proteolysis and transcriptional regulation. Despite increasing evidence linking E2 enzymes to tumorigenesis, their precise roles in gastric cancer remain incompletely defined. In this study, we identified UBE2B as a key oncogenic E2 enzyme significantly upregulated in gastric cancer tissues through integrative bioinformatics analysis and clinical validation. High UBE2B expression was associated with poor patient prognosis and aggressive clinicopathologic features. Functional assays demonstrated that UBE2B promotes gastric cancer cell proliferation both in vitro and in vivo. Mechanistically, UBE2B interacts with the E3 ligase baculoviral IAP repeat-containing 2 (BIRC2) to catalyze K63-linked ubiquitination of TNF receptor-associated factor 1 (TRAF1), thereby amplifying NF-κB signaling. Furthermore, chromatin immunoprecipitation and luciferase reporter assays revealed that the NF-κB subunit P65 directly binds to the UBE2B promoter, enhancing its transcription and forming a feedforward regulatory loop. This UBE2B-BIRC2-TRAF1 axis, coupled with the UBE2B-TRAF1-P65 feedback circuitry, establishes a self-sustaining mechanism that drives NF-κB hyperactivation and tumor cell proliferation. Collectively, our findings highlight UBE2B as a critical modulator of gastric cancer progression and a potential target for therapeutic intervention. IMPLICATIONS:This study characterizes the UBE2B-BIRC2-TRAF1 axis as a driver of NF-κB hyperactivation, identifying UBE2B as a prognostic biomarker and a potential therapeutic target for disrupting this oncogenic feedback loop in gastric cancer.

Supplementary Figure 7. P65 promotes the transcription of UBE2B

Supplementary Figure 3. UBE2B promotes gastric cancer cell proliferation through activation of the NF-kB signaling pathway

Supplementary Figure 11. UBE2B knockdown suppresses gastric cancer cell growth in vivo.

Supplementary Figure 5. Validation of UBE2B-mediated K63-linked polyubiquitination of TRAF1.

RASSF1A is a well-established tumor suppressor implicated in various human malignancies; however, its specific role in the metastasis of gastric cancer (GC) remains poorly understood. In this study, we observed that RASSF1A expression is frequently silenced in GC tissues compared to adjacent normal tissues, with its loss significantly correlating with metastatic progression. Immunohistochemical analysis further revealed a negative correlation between RASSF1A levels and the infiltration of neutrophils, as well as the formation of neutrophil extracellular traps (NETs). Mechanistically, knockdown of RASSF1A triggers the JNK-JUN signaling pathway, which subsequently accelerates ribosome biogenesis. This metabolic shift enhances the translation of pro-inflammatory chemokines, thereby promoting neutrophil recruitment and NETs formation. Furthermore, our findings demonstrate that RASSF1A interacts with LTBR, effectively inhibiting the LIGHT-induced recruitment of TRAF2 and suppressing downstream JNK-JUN activation. Collectively, our results suggest that RASSF1A functions as a critical inhibitor of GC metastasis by modulating the chemokine-neutrophil axis, offering novel insights into its role as a therapeutic target in GC.

Supplementary Table 1. Correlation between UBE2B expression in GC tissue and clinicopathological Features of GC patients

Supplementary Figure 10. Clinical significance and expression profile of BIRC2 in gastric cancer.

Supplementary Figure 4. Validation of UBE2B Downstream Target Points

Radiotherapy is important for advanced and metastatic gastric cancer (GC), but radioresistance limits its benefit. Pyroptosis has emerged as a potential strategy to overcome radioresistance, yet its regulatory mechanisms remain unclear. Using LC-MS/MS-based proteomic profiling of tumor tissues from patients with GC treated with neoadjuvant chemoradiotherapy, we identified tumor-intrinsic lymphotoxin beta receptor (LTβR), previously considered mainly an immune cell membrane protein, as a candidate determinant of poor radiotherapy response. Functional studies in GC cell lines, xenografts, and patient-derived organoids (PDOs) showed that LTβR depletion enhanced radiosensitivity, whereas LTβR overexpression promoted radioresistance. Integrated RNA-seq, Ribo-seq, and polysome profiling showed that LTβR loss reduced translational efficiency of SARM1 under irradiation. Mechanistically, irradiation increased LTβR stability in a glycosylation-dependent manner and promoted nuclear translocation. In the nucleus, LTβR enhanced TRIM28-mediated SUMOylation of PCBP2, promoting cytoplasmic redistribution of PCBP2 and increased translational efficiency of SARM1. Consistently, LTβR suppressed irradiation-induced pyroptosis through the NLRP3/caspase-1/GSDMD pathway. We further identified EMD638683 as an LTβR-binding compound through structure-based virtual screening, and showed that cRGD-modified liposomes improved its tumor-targeted delivery and enhanced LTβR reduction, radiosensitization, and tumor suppression in PDO and xenograft models. Together, these findings highlight LTβR as a promising therapeutic target to improve radiotherapy efficacy in GC.

ABSTRACTBackgroundLymphatic metastasis in gastric cancer (GC) profoundly influences its prognosis, but the precise mechanism remains elusive. In this study, we identified the long noncoding RNA MIR181A2HG as being upregulated in GC and associated with LNs metastasis and prognosis.MethodsThe expression of MIR181A2HG in GC was identified through bioinformatics screening analysis and qRT‐PCR validation. Both in vitro and in vivo functional studies revealed that MIR181A2HG facilitates lymphangiogenesis and lymphatic metastasis. Techniques such as immunofluorescence, immunohistochemistry, qRT‐PCR, ELISA, CHIP, RNA‐pulldown, luciferase reporter assay, and Co‐IP were employed to investigate the mechanism of MIR181A2HG in LNs metastasis of GC.ResultsMIR181A2HG overexpressed in GC signifies an unfavorable prognosis and drives M2 polarization of TAMs enhancing lymphangiogenesis. Mechanistically, MIR181A2HG/miR‐5680 axis as a novel ceRNA regulatory axis to upregulate versican (VCAN). On one hand, VCAN interacts with CD44 receptors on the surface of TAMs through paracrine secretion, promoting M2 macrophage polarization and subsequently enhancing the secretion of VEGF‐C, ultimately facilitating lymphangiogenesis. On the other hand, VCAN binds to CD44 receptors on the surface of GC cells through autocrine secretion, activating the Hippo pathway and upregulating SP1, thereby promoting the transcription of MIR181A2HG and establishing a feedback loop driving lymphatic metastasis.ConclusionThis study highlights the pivotal role of MIR181A2HG in GC progression and LNs metastasis. MIR181A2HG‐based targeted therapy would represent a novel strategy for GC.

BACKGROUND:Aberrant ribosome biogenesis promotes gastric cancer (GC) progression and contributes to chemoresistance by sustaining protein synthesis, upon which GC cell survival depends. However, the regulatory role of cancer-testis-associated long noncoding RNAs (CT-lncRNAs) in modulating ribosome biogenesis in GC remains largely unexplored. METHODS:First, we performed a screening of lncRNAs and identified CT-lncRNA LINC01940 on the basis of integrated expression and survival analyses using The Cancer Genome Atlas (TCGA) data. Subsequently, the impact of LINC01940 on GC progression and chemosensitivity was evaluated using in vitro cell functional assays, patient-derived organoid models, and in vivo subcutaneous tumor xenograft experiments. To further elucidate the underlying mechanisms, we employed a comprehensive approach combining bioinformatics analyses, RNA sequencing, fluorescence in situ hybridization, translation assays, ribosomal DNA (rDNA) transcription assays, methylated RNA immunoprecipitation, co-immunoprecipitation mass spectrometry, fluorescence multiplex immunohistochemistry, and RNA pull-down mass spectrometry. RESULTS:Normally, testis-specific LINC01940 is aberrantly upregulated in GC and associated with poor prognosis. Functional assays demonstrated that LINC01940 promotes GC cell proliferation and invasion and confers resistance to cisplatin. Mechanistically, LINC01940 is stabilized by methyltransferase 16 (METTL16)/ insulin-like growth factor 2 messenger RNA binding protein 3 (IGF2BP3)-mediated N6-methyladenosine (m6A) modification, which enhances its ability to act as a scaffold promoting the interaction between the small ubiquitin-like modifier 2 (SUMO2) E3 ligase TATA-box binding protein associated factor 15 (TAF15) and Nucleolar protein 11 (NOL11), promoting the SUMOylation of NOL11 and enhancing its protein stability. This, in turn, increases ribosomal DNA transcription and ribosome biogenesis, thereby promoting GC progression and chemoresistance. CONCLUSIONS:LINC01940 is a cancer-testis lncRNA that promotes GC progression and cisplatin resistance by enhancing ribosome biogenesis via the METTL16/IGF2BP3-TAF15-NOL11 axis. These findings suggest its potential as a prognostic biomarker and therapeutic target in GC.

Metastasis is a major determinant of prognosis in gastric cancer (GC), and microRNAs (miRNAs) play crucial roles in driving the metastatic process. This study aimed to identify key miRNAs involved in GC metastasis and elucidate their underlying mechanisms. GC tissues from patients with and without metastasis were subjected to miRNA sequencing to identify differentially expressed miRNAs. Expression differences between GC and normal tissues, as well as their correlation with patient survival, were analyzed using data from The Cancer Genome Atlas and an internal cohort. miR-378d expression was measured by RT-qPCR in the internal cohort, and its association with clinicopathological features and prognosis was analyzed. Gene Set Enrichment Analysis (GSEA) was performed to investigate the potential mechanisms by which miR-378d influences GC metastasis. The findings were validated through in vitro wound healing, transwell assays, western blotting, and immunofluorescence, as well as in vivo models. MiRNA sequencing identified miR-378d as significantly downregulated in GC tissues and associated with poor prognosis. GSEA showed that miR-378d was negatively correlated with epithelial-mesenchymal transition (EMT). In vitro and in vivo experiments demonstrated that upregulation of miR-378d inhibited GC cell migration and invasion. Mechanistically, miR-378d suppressed EMT by downregulating METTL4 expression. miR-378d inhibits GC metastasis by suppressing EMT through the downregulation of METTL4, offering novel insights into the role of miRNAs in GC progression and highlighting potential therapeutic targets for intervention.