MAGEA4 promotes gastric cancer progression by inducing chromosomal instability through the STAU1/c-Myc axis

MAGEA4 promotes gastric cancer progression by inducing chromosomal instability through the STAU1/c-Myc axis | 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 Research Article MAGEA4 promotes gastric cancer progression by inducing chromosomal instability through the STAU1/c-Myc axis HONG DENG, Ziwen Long, Cuiping Ren, Changyu Chen, Bingbing Zou, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9348456/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to investigate the clinical significance, biological function, and molecular mechanisms of MAGEA4 in the progression of gastric cancer (GC). Analysis of the TCGA database and 40 pairs of GC tissues and matched normal mucosa samples revealed that MAGEA4 expression was significantly upregulated in GC. Immunohistochemical analysis using two tissue microarrays containing 195 GC samples demonstrated that high MAGEA4 expression is significantly associated with advanced tumor stage, lymph node metastasis and unfavorable patient prognosis. Both in vitro and in vivo experiments confirmed that MAGEA4 significantly promoted the proliferation and metastasis of GC cells. Investigations of the underlying mechanisms revealed that MAGEA4 promotes GC progression by inducing chromosomal instability (CIN). This was evidenced by increased γH2AX (a sensitive DNA damage marker for double-strand breaks), abnormal chromosome numbers, abnormal mitosis, and other CIN features. At the molecular level, MAGEA4 enhances the translation efficiency of c-Myc through the RNA-binding protein STAU1, thereby inducing CIN. Knockdown of STAU1 effectively reversed the CIN phenotype and malignant phenotypes induced by MAGEA4 overexpression in GC cells. In summary, this research demonstrated the crucial role of MAGEA4 in promoting GC progression by inducing CIN via the STAU1/c-Myc axis. Consequently, MAGEA4 represents a highly promising novel therapeutic target for GC. Chromosomal instability Gastric cancer c-Myc MAGEA4 STAU1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Gastric cancer (GC) is a major global health challenge, being one of the most frequently diagnosed cancers and a leading cause of cancer-related mortality (1). According to GLOBOCAN 2020 data, GC accounts for over one million new cases and more than 700,000 deaths annually worldwide (2). Notably, while its global incidence is declining, GC rates are rising in specific demographics, particularly among adults under 50 years old (3). Additionally, the prognosis of GC is generally poor, particularly when diagnosed at advanced stages, with a five-year survival rate of less than 10% (4). Given this clinical reality, continued investigation into the molecular mechanisms driving GC progression is essential to identify novel therapeutic targets. For a long time, the pathological classification of GC has primarily relied on histological features. According to the Lauren classification, GC can be divided into two main types: intestinal type and diffuse type (5). However, traditional pathological typing cannot accurately reflect the molecular characteristics of tumors. In recent years, the emergence of The Cancer Genome Atlas (TCGA) molecular classification of GC has provided significant guidance for personalized treatment. Among these, the chromosomal instability (CIN) subtype accounts for approximately half of all GC cases (6). CIN refers to the missegregation of chromosomes during mitosis, leading to changes in chromosome number or structure, and is the main driver of tumor progression (7, 8). The causes of CIN include abnormalities in DNA damage repair, centrosome amplification, defects in kinetochore-microtubule attachment, and impaired spindle assembly checkpoint function (9-11). CIN is not only closely associated with the progression of various cancers but also plays a critical role in tumor metastasis and therapy resistance (12). CIN continuously generates genomic diversity, which can accelerate the emergence of subclones with specific genotypes that confer therapeutic resistance (7, 13). CIN can also enhance cellular adaptability and tolerance by triggering epigenetic reprogramming and reinforcing cancer stem cell properties (14, 15). Additionally, CIN activates the cGAS–STING pathway through the rupture of micronuclei and the release of dsDNA/dsRNA. The chronic activation of cGAS–STING can remodel the immune microenvironment, ultimately facilitating tumor metastasis and treatment resistance (16-18). As such, it is of great significance to study the molecular mechanisms underlying CIN. MAGEA4 (Melanoma Antigen Gene A4) is a cancer/testis antigen (CTA) characterized by its aberrant overexpression in a wide range of malignancies yet negligible expression in normal tissues (19, 20). This restricted expression profile positions MAGEA4 as a promising target for tumor therapy. Evidence indicates that MAGEA4 promotes tumorigenesis in non-small cell lung cancer (NSCLC) through recruitment and retention of IgA-positive plasma cells in the lungs (21). Furthermore, MAGEA4 can support cancer cell survival and accelerate tumor progression by increasing DNA damage tolerance through inducing RAD18 expression, a ubiquitin ligase associated with translesion synthesis (22). Beyond driving tumorigenesis and tumor progression, MAGEA4 is also implicated in modulating immune responses within the tumor microenvironment (23). Although MAGEA4 expression in GC is well documented, its precise biological functions and the underlying molecular mechanisms have not been fully elucidated (24). Here, we investigated the role and mechanism of MAGEA4 in GC pathogenesis. Our results revealed a significant upregulation of MAGEA4 in GC tissues, which was closely correlated with adverse clinical outcomes. Through in vitro and in vivo experiments, we demonstrated that MAGEA4 substantially enhanced the proliferation, migration, and invasion of GC cells. Mechanistically, we identified that MAGEA4 drives CIN and facilitates tumor progression by activating the STAU1/c-Myc signaling axis. Materials and Methods Patients and tissue samples A total of 40 pairs of GC tissues and matched adjacent normal mucosal tissues were included from the First Affiliated Hospital of Anhui Medical University. None of the patients had received any local or systemic antitumor therapy prior to surgery. Tumor staging was determined according to the National Comprehensive Cancer Network (NCCN) 2010 guidelines. In addition, two tissue microarrays containing 195 GC samples and 71 normal gastric mucosa samples were constructed for subsequent analysis. All studies on human specimens were approved by the Clinical Research Ethics Committee of Anhui Medical University, and written informed consent was obtained from all participants. Cell lines and cell culture Cell lines used in this study, including human GC cell lines (HGC-27, MKN-45, NCI-N87, AGS, and KATO III), the normal gastric epithelial cell line GES-1, and the kidney epithelial-like 293T cell line, were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured at 37°C in a humidified atmosphere with 5% CO₂ in their specified culture medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Western blotting Cells were lysed on ice for 30 minutes in radioimmunoprecipitation assay (RIPA) buffer containing a protease and phosphatase inhibitor cocktail. After lysis, samples were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was then mixed with loading buffer and boiled for 10 minutes at 100°C. The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with Tris-buffered saline containing 5% skim milk for 2.5 hours, then incubated with the corresponding primary antibodies overnight. Subsequently, the membranes were incubated with alkaline phosphatase-conjugated secondary antibodies at room temperature for 1 hour. Finally, immunoreactive bands were detected using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (Sangon Biotech, Shanghai, China). Details regarding the antibodies utilized are provided in Supplementary Table S1. Quantitative real-time PCR analysis (qRT-PCR) Total RNA was extracted from cells using Trizol reagent (Accurate Biotechnolog, China), and equal amounts were reverse-transcribed to complementary DNA (cDNA) using the 5× Evo M-MLV RT Premix (Accurate Biotechnolog, China). Gene expression levels were detected by quantitative real-time PCR (qPCR) using the 2× SYBR Green Pro Taq HS Premix (Accurate Biotechnolog, China). The expression levels of all target genes were normalized to those of the housekeeping gene Gapdh. Details regarding the primer sequences used are provided in Supplementary Table S2. Immunohistochemistry (IHC) analysis Immunohistochemical analysis was run on two tissue microarrays with 71 pairs of GC and matched normal gastric mucosa samples and 124 primary GC samples. A semi-quantitative scoring system was used based on two parameters: staining intensity and the percentage of positive areas. The intensity of staining was categorized into four distinct levels: negative, weakly positive, positive, and strongly positive, which correspond to scores of 0, 1, 2, and 3, respectively. The percentage of positive area was categorized into 0%-25%, 25%-50%, 50%-75%, and above 75%, corresponding to scores of 1, 2, 3, and 4, respectively. The scores of the two parameters were multiplied, and a total score of 3 or higher was used to indicate significant protein overexpression. Gene knockdown and overexpression experiments To transiently knockdown MAGEA4 and STAU1 expression, 1×10 5 cells were transfected using Lipofectamine 2000 (Invitrogen). Specific small interfering RNAs (siRNAs) targeting MAGEA4 and STAU1 were used in the transfection. The siRNA sequences for the MAGEA4 gene were as follows: siRNA #1: 5’-CAA GAU UGG GUG CAG GAA ATT-3’, siRNA#2: 5’-GCG UUG AGG CCC AAG AAG ATT-3’ (General Bio, Anhui, China), and the siRNA sequence for STAU1 gene was siRNA: 5’-CUG UGG GAG GAC AGC AAU UUA TT-3’ (General Bio, Anhui, China). For MAGEA4 overexpression, the MAGEA4 sequence was PCR-amplified and subcloned into the pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro plasmid (General Bio, Anhui, China). Plasmid sequences were confirmed by DNA sequencing before use. The stable plasmid and viral packaging plasmids were then co-transfected with Lipo fectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) into 293T cells to generate lentiviruses. The gastric cancer cells were transduced with the lentiviruses to establish a stable cell line. Cell proliferation assay Cells were seeded in 96-well plates at a density of 5×10³ cells per well and cultured for 5 days. The absorbance was measured and recorded every 24 hours using CCK-8 reagent (Dojondo Laboratories, Kumamoto, Japan). Each experiment was repeated 3 times with 4 technical repeats for each time point per group. Colony formation assay Cells were seeded in 6-well plates at a density of 5×10² cells per well. They were cultured in complete medium at 37°C with 5% CO₂ for 13 days; the medium was refreshed every 4 days. Following the incubation period, cells were incubated with 4% paraformaldehyde for 25 minutes and then stained with Crystal Violet solution for 12 minutes. The stained plates were photographed under a microscope. Colonies consisting of 50 or more cells were counted. Wound‐healing assay Cells were seeded in 6-well plates with complete medium and cultured until they reached full confluence. A linear wound scratch was created in the cell monolayer using a standard 200 μL pipette tip. The scratch width was approximately 300-500 μm. The wounded area was washed twice with 1×PBS to remove detached cells. Wound closure was monitored and imaged at five randomly selected microscopic fields at 0 and 48 hours. Transwell migration and invasion assay Approximately 5×10⁴ cells were seeded into the serum-free medium upper chamber of a Transwell. For the invasion assay, the membranes were pre-coated with Matrigel (BD Bioscience, USA), while no coating was used for the migration assay. The lower chamber was filled with complete medium containing 10% fetal bovine serum (FBS) as a chemoattractant. After 48 hours of incubation at 37°C, the chambers were collected, fixed with 4% paraformaldehyde, and stained with crystal violet. Cells that migrated or invaded to the lower surface of the membrane were counted under a microscope in five randomly selected fields. In vivo tumour growth assay Transduced cells (2×10⁶) were subcutaneously injected into the inguinal region of 4-6 week-old male nude mice. Tumor dimensions were measured every 4 days, and tumor volume was calculated as V = 4π/3 × (width/2) ² × (length/2). All mice were euthanized 30 days after inoculation, marking the experimental endpoint. Subsequently, xenograft tumors were harvested, photographed, and weighed. Six biological replicates per group were used. Lung metastasis model Transduced cells (6×10⁶ per mouse) were injected into nude mice via the tail vein. All mice were euthanized six weeks after injection and lung tissues were harvested for pathological examination, hematoxylin and eosin (H&E) staining, and quantification of metastatic nodules. Each group included five biological replicates. Transcriptome Sequencing and Analysis After passing quality control, total RNA was subjected to RNA-seq using the Hiseq3000 (Illumina, USA). The raw sequencing data were converted into a gene expression matrix following additional quality control and filtering. Subsequently, normalized differential expression analysis was performed on the expression matrix, and significantly differentially expressed genes were further analyzed for functional enrichment using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Chromosome spreading assay Cells at the logarithmic growth phase were treated with colchicine. The drug was added to the culture medium at a final concentration of 0.4 μg/mL and the cells were incubated at 37 °C for 3 hours. The cells were then gently detached, transferred into 2 mL microcentrifuge tubes, and centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, the cell pellet was resuspended in 1 mL of 0.075 mol/L KCl hypotonic solution warmed to 37 °C and incubated at 37 °C for 30 minutes. Next, 1 mL of freshly prepared fixative (methanol: glacial acetic acid = 3:1) was added, mixed thoroughly, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded. The fixation step was repeated twice more, each time by adding 1 mL of fixative, gently mixing, incubating at 37 °C for 30 minutes, and centrifuging under the same conditions. Finally, the cells were resuspended in 50 μL of fixative. The entire suspension was then dropped onto a pre-chilled clean glass slide and gently spread to ensure even coverage. The slide was air-dried at room temperature, stained with 10% Giemsa solution for 10 minutes, rinsed gently under running water, and air-dried at room temperature. The prepared slides were observed and cells counted under an oil immersion microscope. Cell cycle assay A total of 100,000 cells were fixed by resuspension in 80% ethanol and incubated for 12 hours at 4°C. The fixed cells were then washed twice to remove ethanol: first with phosphate-buffered saline (PBS), and then with staining buffer (BD Biosciences, Cat. #554656). The cells were then resuspended in 0.5 mL of PI/RNase Staining Buffer (BD Biosciences, Cat. #550825) and incubated for 15 minutes at room temperature in the dark before flow cytometric analysis. The data were analyzed using FlowJo software, and the proportion of cells with DNA content ＞4N was calculated. Immunofluorescence imaging Cells were seeded in confocal dishes at a density of 2.4-3×10⁵ cells per dish and cultured for 24 hours. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.5% Triton X-100 for 20 min. After blocking cells for 30 min, the cells were incubated with primary antibodies at 4 °C overnight followed by incubation with the corresponding secondary antibodies for 1 h at room temperature. Three 5-minute washes with PBS were performed after each antibody incubation step. Nuclei were stained with 1μg/mL DAPI for 5 min, and samples were mounted with ProLong Gold Antifade Reagent (P10144, Thermo Fisher). Images were acquired using a ZEISS LSM 888 confocal microscope. For mitosis quantitative analysis, at least 100 cells per condition were manually examined to count lagging chromosomes, anaphase bridges, multipolar mitotic events, and micronuclei. Details regarding the antibodies utilized are provided in Supplementary Table S1. H&E staining Paraffin-embedded tissue sections were heated at 60°C for 30 minutes prior to hematoxylin and eosin staining. The staining procedure was performed as follows: sections were deparaffinized in xylene I and II for 15 minutes each, rehydrated through a graded ethanol series (100%, 95%, 85%, and 75%) for 5 minutes each, and rinsed for 5 minutes in distilled water. Subsequently, sections were stained with Harris hematoxylin for 5–8 minutes, rinsed under running tap water to promote bluing, differentiated in 1% acid alcohol for several seconds, and then washed again under running water for 15 minutes to complete the differentiation and bluing steps. Counterstaining was performed with 0.5% eosin Y in ethanol for 1-3 minutes, followed by rapid dehydration through graded ethanol concentrations (85%, 95%, and 100%; about 30 seconds each) and clearing in xylene (two changes, 2 minutes each). Finally, sections were mounted with neutral balsam and examined under a light microscope for histopathological analysis. Statistical analyses Statistical comparisons between groups were performed using Student's t-test. Associations between protein expression levels and clinicopathological parameters were analyzed with a Chi-square test. Survival analysis was performed using the Kaplan-Meier method, and differences between groups were assessed by the log-rank test. All statistical analyses were performed using GraphPad Prism software (version 10.1.2), using a two-tailed P value < 0.05 as the threshold for statistical significance. Results MAGEA4 is up‐regulated in GC patients and associated with poor prognosis The TCGA and Genotype-Tissue Expression (GTEx) databases revealed that MAGEA4 was upregulated in various cancers, including esophageal carcinoma, lung adenocarcinoma, and stomach adenocarcinoma (Fig. 1A). Analysis of the Kaplan-Meier plot database found that increased expression of MAGEA4 was correlated with poorer overall survival (OS) rates in GC (Fig. 1B). Differential expression analysis comparing normal gastric mucosae and GC tissues from the TCGA database identified 1,672 differentially expressed genes comprising 922 upregulated and 750 downregulated genes (Fig. 1C). Among the most differentially expressed genes, MAGEA4 ranked third (Fig. 1D). To assess the expression of MAGEA4 in GC, we first using quantitative analysis of 40 matched tissue pairs to detect the expression level of MAGEA4 mRNA levels in GC and normal gastric mucosa tissue. The results indicated that MAGEA4 mRNA was markedly upregulated in GC (Fig. 1E). Consistent with this, the upregulation of MAGEA4 protein in GC tissues and GC cell lines was also confirmed (Fig. 1F and G). Scoring of immunohistochemical analysis on tissue microarrays containing 71 pairs of GC samples and their matched normal gastric mucosae, and 124 primary GC samples demonstrated that MAGEA4 expression was significantly higher in GC compared to normal gastric mucosae (Fig. 1H-J). An immunohistochemical score was calculated by integrating staining intensity and the proportion of positive cells, and all patients were divided into a MAGEA4 high-expression group and a low-expression group for subsequent analysis. Correlation analysis revealed that high MAGEA4 expression was significantly associated with higher AJCC stage and lymph node metastasis of GC (Supplementary Table S3). Furthermore, Kaplan-Meier survival analysis demonstrated that GC patients with high MAGEA4 expression had significantly shorter overall survival (OS) and disease-free survival (DFS) (Fig. 1K). This finding aligned with the trend of GC observed in the Kaplan-Meier Plotter database. Subsequent multivariate Cox regression analysis further confirmed that high MAGEA4 expression was an independent predictive factor for poor OS and DFS in GC patients (Table 1). In summary, MAGEA4 expression is significantly up-regulated in GC, and its high expression is closely associated with poor prognosis. MAGEA4 stimulates the proliferation, migration, and invasion of GC in vitro To evaluate the role of MAGEA4 in GC progression, we knocked down MAGEA4 in HGC-27 cells which exhibit high MAGEA4 endogenous expression. Conversely, we stably overexpressed MAGEA4 in MKN-45 cells which show low basal MAGEA4 expression (Fig. 2A). The CCK8 proliferation assay and colony formation assay demonstrated that MAGEA4 knockdown significantly suppressed HGC-27 cell proliferation, whereas MAGEA4 overexpression markedly enhanced the proliferation of MKN-45 cells (Fig. 2B and C). Furthermore, wound healing and Transwell migration assays revealed that MAGEA4 silencing markedly impaired the migratory ability of GC cells, while its overexpression promoted cell migration (Fig. 2D and E). Similarly, the invasive capacity of GC cells was significantly reduced upon MAGEA4 knockdown and enhanced by MAGEA4 overexpression (Fig. 2F). In summary, these results indicate that MAGEA4 stimulates the proliferation, migration, and invasion of GC cells in vitro . MAGEA4 promotes the proliferation and metastasis of GC cells in vivo To assess the effect of MAGEA4 on GC growth in vivo , we performed a tumor burden assay using MKN-45 cells with stable MAGEA4 overexpression. Tumors derived from MAGEA4-overexpressing cells displayed enhanced proliferative activity compared with controls (n = 6 per group; Fig. 3A), and at the experimental endpoint, both tumor size and weight were significantly greater in the MAGEA4-overexpressed group (Fig. 3 B and C). These results indicate that MAGEA4 promotes tumor growth of GC cells in vivo . In the lung metastasis model, MAGEA4-overexpressing or control MKN-45 cells were injected into the tail veins of nude mice (n = 5 per group). The incidence of lung metastasis was 100% (5/5) in the MAGEA4-overexpression group compared to 40% (2/5) in the control group (Fig. 3D). In addition, the number of pulmonary metastatic nodules was significantly higher in MAGEA4-overexpressed groups than in controls (Fig. 3E). These findings demonstrate that MAGEA4 plays a critical role in promoting metastasis of GC cells. MAGEA4 induces chromosomal instability (CIN) in GC cells To elucidate how MAGEA4 contributes to GC progression, we performed RNA sequencing on transcriptomes of HGC-27 cells with MAGEA4 knockdown and control cells. Gene ontology analysis of results from RNA-seq analysis revealed significant alterations in genes involved in biological processes such as DNA replication, chromosome segregation, and mitotic G2 DNA damage checkpoint signaling (Fig. 4A). This finding prompted us to investigate the role of MAGEA4 in cell cycle progression and DNA damage repair. Western blotting analysis and immunofluorescence imaging showed that γH2AX, a biomarker for DNA double-strand breaks (25), was significantly reduced in MAGEA4-knockdown HGC-27 cells, but was increased in MAGEA4-overexpressing MKN-45 cells (Fig. 4B and C). Representative images are provided in supplementary (Fig. S1). Chromosome spreading assays showed that MAGEA4 overexpression induced a shift from diploidy to aneuploidy in MKN-45 cells (Fig. 4D). Cell cycle analysis further confirmed that MAGEA4 overexpression increased the proportion of aneuploidy cells (Fig. 4E and G). Given that DNA damage and aneuploidy are recognized as consequences of CIN in cancer cells (26), we sought to determine whether MAGEA4 induces characteristic CIN features such as misaligned chromosomes, anaphase bridges, asymmetric anaphase, multipolar mitosis, or micronuclei (27, 28). Results demonstrated that the number of abnormal mitotic cells, such as those displaying misaligned chromosomes, anaphase bridges, and asymmetric anaphase, was significantly higher in MAGEA4-overexpressing MKN-45 cells (Fig. 4F and H). Additionally, the incidences of micronuclei and multipolar mitosis were significantly elevated in MAGEA4-overexpressing MKN-45 cells (Fig. 4I-L). Consistent with these in vitro results, H&E-stained sections from clinical samples with high MAGEA4 expression also showed more CIN-associated features (Fig. 4 M and N). In summary, our study demonstrates that elevated MAGEA4 expression induces chromosomal instability, resulting in chromosomal aberrations and DNA damage in GC cells. MAGEA4 enhances c-Myc expression at the translational level via a STAU1-dependent pathway To elucidate the mechanism by which MAGEA4 induces CIN, we determined the expression of CIN-related proteins, p53 and c-Myc (29-33) in GC cells with modified MAGEA4 expression as indicated by Western blotting analysis. While p53 protein levels remained largely unchanged with both MAGEA4 knockdown and overexpression (Fig. 5A), c-Myc protein expression was significantly upregulated in MAGEA4-overexpressed cells and downregulated in MAGEA4-depleted cells (Fig. 5B). The RNA-seq data on transcriptomes of HGC-27 cells with MAGEA4 knockdown and control cells revealed 291 genes upregulated and 534 genes downregulated (Fig. 5C), however, no significant changes in c-Myc mRNA levels were observed (Fig. 5D), leading us to hypothesize that a post-transcriptional mechanism enhances c-Myc expression. Previous studies have reported that the RNA-binding protein STAU1 enhances the translation efficiency of c-Myc by binding to the 5' untranslated region (5'UTR) of its transcript(34, 35). Notably, our RNA-seq analysis identified STAU1 among the top 20 most significantly altered genes (Fig. 5E) with STAU1 protein levels decreasing significantly after MAGEA4 silencing and increasing after MAGEA4 overexpression (Fig. 5F). RT-qPCR analysis confirmed that STAU1 mRNA expression mirrored these changes while c-Myc mRNA levels remained stable (Fig. 5G). To determine whether MAGEA4 enhances c-Myc expression via STAU1, we performed STAU1 knockdown in both MAGEA4-overexpressed and control MKN-45 cells. Depletion of STAU1 eliminated the increase in c-Myc protein induced by MAGEA4 overexpression (Fig. 5H). In fact, c-Myc expression was also inhibited by STAU1 knockdown. These findings demonstrate that MAGEA4 upregulates c-Myc expression at the translational level via a STAU1-dependent pathway. MAGEA4 promotes malignant phenotypes of GC cells by inducing CIN via the STAU1/c-Myc axis Previous studies reported c-Myc as driver of genomic instability (36) , and our results have established that MAGEA4 upregulates c-Myc expression at the translational level via STAU1. We further investigated the impact of the STAU1/c-Myc axis on CIN phenotypes and malignant behaviors in GC cells. Colony formation assays revealed that STAU1 knockdown not only reversed the enhanced proliferation induced by MAGEA4 overexpression in MKN-45 cells, but also suppressed the basal colony-forming ability in control cells (Fig. 6A). Similarly, in the Transwell migration and invasion assays, STAU1 depletion abolished the pro-migratory and pro-invasive effects induced by MAGEA4 overexpression, while impairing these capacities in wild-type MKN-45 cells (Fig. 6B and C). Thus, MAGEA4 promotes the malignant phenotypes of GC cells in a STAU1-dependent manner. Chromosome spread assays and DNA content analysis indicated that STAU1 knockdown markedly attenuated the increase in chromosomal numerical abnormalities resulting from MAGEA4 overexpression (Fig. 6D, F and H). Western blotting analysis and immunofluorescence imaging showed that STAU1 silencing reduced γH2AX expression in both MAGEA4-overexpressed and control cells (Fig. 5H and Fig. 6E, G). The frequencies of abnormal mitosis, multipolar mitosis, and micronuclei formation were similarly reduced (Fig. 6I-K). Representative images are provided in supplementary (fig. S2). These results indicated that MAGEA4 promotes the proliferation, metastasis and invasion of GC cells by inducing CIN through the STAU1/c-Myc signaling axis. Discussion In recent years, there has been growing interest in the role of MAGEA4 in promoting tumor progression. However, its specific mechanism in GC remains unclear. This study aimed to investigate the clinical significance, biological function, and molecular mechanism of MAGEA4 in the evolution of GC. Mechanistically, we found that MAGEA4 upregulates c-Myc expression at the translational level via STAU1, thereby inducing CIN and promoting GC development (Fig. 7). As a cancer/testis antigen, MAGEA4 is highly expressed in tumor tissues such as esophageal squamous cell carcinoma, intrahepatic cholangiocarcinoma, and non-small cell lung cancer (NSCLC), where its elevated expression is associated with adverse clinical outcomes (37-39). Evidence indicates that MAGEA4 can increase the DNA damage tolerance of cancer cells, thus supporting their survival (22). The Cancer Genome Atlas (TCGA) database revealed that MAGEA4 is highly expressed in GC tissues but barely detectable in normal tissues, suggesting its potential as an ideal therapeutic target. To further explore its role in GC, we analyzed 40 pairs of matched clinical samples and observed significant upregulation of MAGEA4 in tumor tissues compared to adjacent normal tissues. Moreover, two tissue microarrays containing 195 GC samples and 71 normal gastric mucosa samples supported a strong correlation between high MAGEA4 expression and poor prognosis. Through gain‐ and loss‐of‐function experiments, we showed that MAGEA4 enhances the proliferation, migration, and invasion abilities of GC cells both in vitro and in vivo . These findings indicated that MAGEA4 plays a critical role in promoting GC progression. To elucidate how MAGEA4 contributes to GC progression we performed RNA sequencing on transcriptomes of HGC-27 cells with MAGEA4 knockdown and control cells. Gene Ontology analysis revealed significant alterations in biological processes related to chromosome segregation, DNA replication, and mitotic G2 DNA damage checkpoint signaling, all of which are closely associated with CIN. CIN is commonly observed in cancer patients and correlates with tumor aggressiveness and metastasis, serving as a key driver of tumor progression (7, 8, 40, 41). According to TCGA, GC is classified into four molecular subtypes: Epstein-Barr virus-positive (EBV+), microsatellite instability (MSI), genomically stable (GS), and chromosomal instability (CIN)(6). Among these, the CIN subtype is the most prevalent (42) and typically exhibits a higher mutation burden and more complex genomic features than the other subtypes (43, 44). We then conducted a series of experiments to examine whether MAGEA4 affects CIN in GC cells. The results showed that MAGEA4-overexpressed cells exhibited markedly increased CIN features, including γH2AX foci accumulation, aneuploidy, abnormal mitosis, multipolar division, and micronucleus formation. Furthermore, in GC tissue sections, the frequency of these CIN-related markers was significantly higher in MAGEA4-high samples compared to MAGEA4-low samples. These results collectively support that MAGEA4 induces CIN in GC cells. Building upon the established role of CIN in GC progression (14), our work focused on deciphering the molecular mechanism by which MAGEA4 induces this pathogenic state. The development of CIN involves a complex network of proteins, among which P53 and c-Myc are particularly prominent. Deficiencies in P53 function are well-documented to increase cellular susceptibility to chromosome misalignment and unequal segregation during mitosis (45, 46). Furthermore, the critical role of P53 in orchestrating DNA damage repair means its loss severely compromises this protective capacity, thereby amplifying CIN manifestations (47-49). In contrast, c-Myc drives CIN through two principal avenues: first, by accelerating cell proliferation via regulation of cell cycle-related genes, which increases the frequency of mitotic errors; and second, by impairing the normal function of the DNA repair system, thereby undermining genomic stability (31, 50-53). Our investigation into the expression of these key regulators in MAGEA4-modulated cell models revealed a distinct pattern: Western blotting showed that P53 protein levels remained largely unaltered in both MAGEA4 knockdown and overexpression models, whereas c-Myc protein expression exhibited a striking dependence on MAGEA4 status despite the absence of corresponding changes in c-Myc mRNA levels pointing toward post-transcriptional regulation. This hypothesis was subsequently validated through confirmation that MAGEA4 enhances c-Myc protein synthesis at the translational level via the RNA-binding protein STAU1. To establish the functional centrality of STAU1 within this pathway, we performed STAU1 knockdown experiments in both MAGEA4-overexpressed and control MKN-45 cell lines. The results showed that depleting STAU1 effectively reversed the CIN phenotype and other malignant characteristics induced by MAGEA4 overexpression. Notably, STAU1 knockdown in native MKN-45 cells was sufficient to significantly reduce baseline CIN and suppress the malignant phenotype. Together, these findings delineate a clear mechanistic pathway wherein MAGEA4 induces CIN via the STAU1/c-Myc signaling axis, thereby promoting the progression of GC. Conclusion In summary, our findings establish MAGEA4 as a key driver of GC progression and a potential predictor of poor patient prognosis. By inducing chromosomal instability through the STAU1/c-Myc axis, MAGEA4 fuels tumor aggressiveness. Its highly restricted expression in tumor tissue positions MAGEA4 as a promising and specific therapeutic target. Future efforts should focus on elucidating the upstream mechanisms that govern MAGEA4's control over STAU1. Abbreviations GC: gastric cancer CIN: chromosomal instability MAGEA4: Melanoma Antigen Gene A4 Declarations Ethics approval and consent to participate Experiments involving human participants in this study obtained written informed consent from all relevant individuals and were approved by the Clinical Research Ethics Committee of Anhui Medical University (Approval No.: PJ 2024-13-63). Experiments involving mice in this study were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (Approval No.: LLSC20200141). Consent for publication All Authors agreed to the manuscript. Availability of data and materials All the data supporting the findings of this study are available within the article and its supplemental files. Competing interests The authors declare that they have no known competing financial interests or personal relationships. Funding This work was supported by National Natural Science Foundation of China (82002545, 82573137, 81902451) and Shanghai Sailing Program (20YF1409200) and the Natural Science Research Project of Anhui Higher Education Institution (2025AHGXZK31473) and Clinical and Translational Research Project of Anhui Province (202427b10020121). Authors' contributions H.D and ZW.L wrote the manuscript and participated in the entire experiment. CP.R analyzed and processed the data. CY.C and BB.Z collected the specimens. YS.J participated in data processing. CC.L and CY.F provided financial support and designed the experiment, and made substantial contributions to the writing of the manuscript. 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Supplementary Files Supplementary.docx UncroppedGelsandBlotsimage.docx Table1.docx Cite Share Download PDF Status: Posted 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-9348456","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621739935,"identity":"f9c0bdeb-2066-4554-ba52-d808fb56f0b1","order_by":0,"name":"HONG DENG","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"HONG","middleName":"","lastName":"DENG","suffix":""},{"id":621739936,"identity":"4d0a55f6-dc68-4085-be80-41924966bf91","order_by":1,"name":"Ziwen Long","email":"","orcid":"","institution":"Fudan University Shanghai Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Ziwen","middleName":"","lastName":"Long","suffix":""},{"id":621739937,"identity":"bcc7769a-f21a-4a15-a438-251bbc4ecab2","order_by":2,"name":"Cuiping Ren","email":"","orcid":"","institution":"School of Basic Medical Sciences ,Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cuiping","middleName":"","lastName":"Ren","suffix":""},{"id":621739938,"identity":"40425249-3658-43c7-b75e-ca37c794911c","order_by":3,"name":"Changyu Chen","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changyu","middleName":"","lastName":"Chen","suffix":""},{"id":621739939,"identity":"97f18d12-e1f0-410b-a415-edf53c18ed78","order_by":4,"name":"Bingbing Zou","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bingbing","middleName":"","lastName":"Zou","suffix":""},{"id":621739940,"identity":"70fc4d08-f718-4069-a189-036702583096","order_by":5,"name":"Yuanshuang Jin","email":"","orcid":"","institution":"Gansu University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuanshuang","middleName":"","lastName":"Jin","suffix":""},{"id":621739941,"identity":"28d4fa1a-adc2-4248-88f0-20d613e2669a","order_by":6,"name":"Changyi Fang","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changyi","middleName":"","lastName":"Fang","suffix":""},{"id":621739942,"identity":"b72647d6-6d72-4ac5-be91-f4e0818e3972","order_by":7,"name":"Chenchen Liu","email":"data:image/png;base64,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","orcid":"","institution":"Zhongshan Hospital","correspondingAuthor":true,"prefix":"","firstName":"Chenchen","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-04-07 18:10:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9348456/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9348456/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106961732,"identity":"58258282-176d-445b-856a-5240a64720f2","added_by":"auto","created_at":"2026-04-15 09:26:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3506790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAGEA4 is up‐regulated in GC patients and associated with poor prognosis\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e A pan-cancer analysis of MAGEA4 expression in the TCGA and GTEx databases. Note: red and blue represent tumor and normal samples, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Survival curves of OS in gastric cancer (GC). \u003cstrong\u003e(C)\u003c/strong\u003e The differential expression analysis on GC samples and normal gastric mucosae from the TCGA database. \u003cstrong\u003e(D)\u003c/strong\u003e The rank of GC samples and normal gastric mucosae differentially expressed genes from the TCGA database. \u003cstrong\u003e(E)\u003c/strong\u003e The MAGEA4 mRNA levels in 40 pairs of GC tissues and matched normal gastric mucosae; \u003cem\u003e****p \u003c/em\u003e\u0026lt; 0.0001. \u003cstrong\u003e(F)\u003c/strong\u003e The MAGEA4 protein levels in six pairs of GC tissues and matched normal gastric mucosae. β-tubulin was used as an internal control. \u003cstrong\u003e(G) \u003c/strong\u003eThe MAGEA4 protein levels in GES‐1 and five GC cell lines. \u003cstrong\u003e(H)\u003c/strong\u003e Immunohistochemical (IHC) analysis of MAGEA4 expression performed using a tissue microarray containing 71 pairs of primary GC samples and matched normal gastric mucosae. Representative IHC images showed high and low expression of MAGEA4 in tissues; scale bar =50μm. \u003cstrong\u003e(I) \u003c/strong\u003eThe IHC score of MAGEA4 in GC tissues and paired normal gastric mucosae; \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003e(J) \u003c/strong\u003eImmunohistochemical (IHC) analysis of MAGEA4 expression using a tissue microarray containing 124 GC samples. Representative IHC images showed high and low expression of MAGEA4 in GC sample; scale bar =200μm. \u003cstrong\u003e(K) \u003c/strong\u003eInfluence of MAGEA4 expression patterns on overall survival (OS) and disease‐free survival (DFS) by Kaplan‐Meier analyses (Log‐rank test). Low expression of MAGEA4, n = 128; high expression of MAGEA4, n = 67.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/d8910c6325ae2f27362e2d47.png"},{"id":106961343,"identity":"f4794f12-8986-4659-b5eb-ce74417086be","added_by":"auto","created_at":"2026-04-15 09:25:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3677100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAGEA4 promotes the proliferation, migration, and invasion of gastric cancer\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eWestern blotting of the effective knockdown and overexpression of MAGEA4, β-tubulin (internal control). \u003cstrong\u003e(B)\u003c/strong\u003e CCK-8 assays showed that MAGEA4 depletion suppressed the proliferation of GC cell, whereas its overexpression promoted GC cell proliferation; \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003e(C) \u003c/strong\u003eThe effect of MAGEA4 on the colony-forming ability of GC cell; \u003cem\u003e**p \u003c/em\u003e\u0026lt; 0.01. \u003cstrong\u003e(D)\u003c/strong\u003e Migration of indicated cells assessed by wound healing assay. Migration rates were calculated following measurement of wound widths at 0 and 48 hours post scratching. Migration rate (%) = [(0-hour width - 48-hour width) / 0-hour width] × 100%; \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(E, F)\u003c/strong\u003e Transwell assays detected the role of MAGEA4 in GC cell migration \u003cstrong\u003e(E)\u003c/strong\u003e and invasion \u003cstrong\u003e(F)\u003c/strong\u003e;\u003cem\u003e **p\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/57435967bb4c23ac69415f3c.png"},{"id":106903838,"identity":"a6a5c124-a49b-45f2-a91d-ab1ee0e6749e","added_by":"auto","created_at":"2026-04-14 15:13:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4692797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAGEA4 promotes the proliferation and metastasis of GC cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eTumor growth curves of MAGEA4-overexpressing and wild-type MKN-45 cells in nude mice (2×10⁶ cells injected subcutaneously; n=6 per group). Quantification of tumor volume at the indicated time points, with data presented as mean ± SD; \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003e(B, C)\u003c/strong\u003e Images of representative xenograft tumors\u003cstrong\u003e (B)\u003c/strong\u003e, and the average tumor weight \u003cstrong\u003e(C)\u003c/strong\u003e, with data presented as mean ± SD; \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(D)\u003c/strong\u003e Establishment of a lung metastasis model via tail vein injection of wild-type or MAGEA4-overexpressing MKN-45 cells (6×10⁶ cells per mouse; n=5 per group). Representative images showed the H\u0026amp;E sections of lung metastatic nodules. \u003cstrong\u003e(E) \u003c/strong\u003eNumber of lung metastatic nodules per experimental group; \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/812f39dc02131e96ebbadce1.png"},{"id":108006005,"identity":"b374fda2-17db-4258-af50-c60933cc004e","added_by":"auto","created_at":"2026-04-28 12:51:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3480725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAGEA4 induces chromosomal instability (CIN) in GC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eGO biological processes significantly altered by MAGEA4-siRNA transfection. \u003cstrong\u003e(B) \u003c/strong\u003eWestern blotting of the DNA damage marker γH2AX. GAPDH was used as an internal control. \u003cstrong\u003e(C)\u003c/strong\u003e The effect of MAGEA4 knockdown and overexpression on γH2AX was detected by immunofluorescence. The bar graph shows the average number of lesion foci per nucleus,＞40 cells/group; \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(D)\u003c/strong\u003e Chromosome spreading assay assessed the impact of MAGEA4 overexpression on chromosomal numerical changes in MKN-45 cell. One representative experiment out of 100 analyzed metaphase cells is shown; \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001.\u003cstrong\u003e (E, G)\u003c/strong\u003e Cell cycle analysis showing the DNA content in different phases of MKN-45 cell following MAGEA4 overexpression. Cells with a DNA content \u0026gt;4N were quantified as aneuploid. In each experiment, 10,000 cells were collected, and three independent replicates were performed; \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(F, H)\u003c/strong\u003e High-resolution immunofluorescence was used to detect the induction of mitotic defects by MAGEA4 overexpression, using α-tubulin (green) to label spindles, phospho-histone H3 (PH3, red) to label chromosomes, and DAPI (blue) to stain nuclear DNA. \u003cstrong\u003e(F)\u003c/strong\u003e Representative images of chromosome segregation during metaphase and anaphase in MKN-45 cells. Chromosomal segregation abnormalities in MAGEA4-overexpressed MKN-45 cells included non-aligned chromosomes in metaphase, anaphase bridges, and asymmetrical anaphase.\u003cstrong\u003e (H) \u003c/strong\u003eQuantification of the percentage of cells exhibiting these mitotic defects. A total of 100 mitotic cells were counted per replicate in each of three independent experiments; \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003e(I, K) \u003c/strong\u003eRepresentative images\u003cstrong\u003e (I)\u003c/strong\u003e and quantification \u003cstrong\u003e(K)\u003c/strong\u003e of spindle tripolarity/multipolarity formation in MKN-45 cell after MAGEA4 overexpression. Staining: α-tubulin (green), PH3 (red), DAPI (blue). A total of 100 mitotic cells were counted per replicate over three independent experiments; \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(J, L) \u003c/strong\u003eRepresentative images \u003cstrong\u003e(J) \u003c/strong\u003eand quantification\u003cstrong\u003e (L) \u003c/strong\u003eof micronuclei formation in MKN-45 cells following MAGEA4 overexpression. Staining: α-tubulin (green), PH3 (red), DAPI (blue). A total of 100 cells were counted per replicate over three independent experiments; \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(M, N)\u003c/strong\u003e Representative images \u003cstrong\u003e(M)\u003c/strong\u003eand quantification\u003cstrong\u003e (N)\u003c/strong\u003e of aberrant chromosomal segregation and tri-/multi-polar spindles in GC tissue sections with low (n = 128) vs. high (n = 67) MAGEA4 expression by H\u0026amp;E staining. A total of 50 mitotic cells were counted per tissue section; \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/2a5f1abac83d0c6fc48474c5.png"},{"id":106961226,"identity":"0ef457e6-4159-459c-9f7a-19781e11bfdd","added_by":"auto","created_at":"2026-04-15 09:24:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2360666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAGEA4 enhances c-Myc expression at the translational level via STAU1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eWestern blotting was performed to assess the effect of MAGEA4 on P53 protein levels. β-actin was used as an internal control; ns, non-significant. \u003cstrong\u003e(B) \u003c/strong\u003eWestern blotting was performed to assess the effect of MAGEA4 on c-Myc protein. β-actin was used as an internal control; \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01.\u003cstrong\u003e(C, D, E)\u003c/strong\u003e RNA-seq analysis of MAGEA4-siRNA versus Control group. \u003cstrong\u003e(C) \u003c/strong\u003eVolcano plot of differentially expressed genes. \u003cstrong\u003e(D) \u003c/strong\u003ec-Myc expression remained largely unchanged at the mRNA level. \u003cstrong\u003e(E) \u003c/strong\u003eTop 20 most significantly altered genes. \u003cstrong\u003e(F)\u003c/strong\u003e The effect of MAGEA4 on STAU1 protein levels analyzed by Western blotting, using β-actin as a loading control; \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001.\u003cstrong\u003e (G) \u003c/strong\u003eRT-qPCR analysis of the effect of MAGEA4 on STAU1 and c-Myc mRNA levels; ns, non-significant; \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01.\u003cstrong\u003e (H) \u003c/strong\u003eWestern blotting was performed to assess the effect of STAU1 knockdown on c-Myc and γH2AX protein expression in both MAGEA4-overexpressing and control MKN-45 cell, using β-actin as an internal control; ns, non-significant; \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e****p\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/c5923563167af7aa931149a7.png"},{"id":106903842,"identity":"f0fb2aa0-76e2-4c18-9be3-4e684f413ee3","added_by":"auto","created_at":"2026-04-14 15:13:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4111212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMAGEA4 promotes malignant phenotypes of GC cells by inducing CIN via the STAU1/c-Myc axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B, C) \u003c/strong\u003eThe influence of STAU1 expression on the proliferation, migration, and invasion abilities of MAGEA4-overexpressing and wild-type MKN-45 cells. \u003cstrong\u003e(A) \u003c/strong\u003eColony formation assays showed that STAU1 depletion reversed the pro-proliferative effect of MAGEA4 overexpression. STAU1 knockdown alone inhibits basal colony-forming ability of wild-type MKN-45 cells. Transwell migration \u003cstrong\u003e(B) \u003c/strong\u003eand invasion \u003cstrong\u003e(C) \u003c/strong\u003eassays showed that STAU1 depletion abolished the pro-migratory and pro-invasive effect induced by MAGEA4 overexpression. STAU1 knockdown also suppresses cell basal migration and invasion of wild-type MKN-45 cells; \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003e(D) \u003c/strong\u003eEffect of STAU1 knockdown on chromosomal numerical abnormalities in the MAGEA4-overexpressing and wild-type MKN-45 cells. Results indicated that STAU1 knockdown markedly attenuated the increase in chromosomal numerical abnormalities resulting from MAGEA4 overexpression; ns, non-significant; \u003cem\u003e***p\u003c/em\u003e\u0026lt; 0.001. \u003cstrong\u003e(E)\u003c/strong\u003e Representative images of γH2AX foci in MAGEA4-overexpressing and wild-type MKN-45 cells following STAU1 knockdown. Red, γH2AX staining; blue, DAPI staining of nuclei.\u003cstrong\u003e (F) \u003c/strong\u003eThe DNA content in different phases of MAGEA4-overexpressing and wild-type MKN-45 cells following STAU1 knockdown assessed by cell cycle analysis. Cells with a DNA content \u0026gt;4N were quantified as aneuploid.\u003cstrong\u003e (G) \u003c/strong\u003eQuantification of the γH2AX foci showed that STAU1 silencing reduced γH2AX expression in both MAGEA4-overexpressed and wild-type MKN-45 cells. The average number of lesion foci per nucleus,＞50 cells/group, is shown; \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001.\u003cstrong\u003e (H)\u003c/strong\u003eThe results of cell cycle analysis show that STAU1 knockdown not only prevented the increase in aneuploid cells induced by MAGEA4 overexpression but also reduced the aneuploid cells in wild-type MKN-45 cells. In each experiment, 10,000 cells were collected, and three independent replicates were performed; \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003e(I, J, K)\u003c/strong\u003e Quantification of the effect of STAU1 knockdown on the frequency of abnormal mitosis \u003cstrong\u003e(I)\u003c/strong\u003e, spindle tripolarity/multipolarity\u003cstrong\u003e (J)\u003c/strong\u003e, and micronuclei \u003cstrong\u003e(K) \u003c/strong\u003ein the MAGEA4-overexpressing and wild-type MKN-45 cells; \u003cem\u003e*p\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**p\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003e***p\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/7ae8c6d4ddda9ecd64bcb712.png"},{"id":106903840,"identity":"34ace9cd-e6c1-4df1-aa10-77e139dc425d","added_by":"auto","created_at":"2026-04-14 15:13:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1126734,"visible":true,"origin":"","legend":"\u003cp\u003eMAGEA4 upregulates the translation of c-Myc via STAU1, thereby inducing chromosomal instability (CIN) and ultimately promoting the progression of GC.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/b28d9402e4c43ce13376444c.png"},{"id":108008340,"identity":"ba8b5441-6138-4c4d-b847-3c908d3afefd","added_by":"auto","created_at":"2026-04-28 13:06:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22728845,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/53d37855-4550-4eb4-ace4-76e8861f5b6e.pdf"},{"id":106903833,"identity":"f2aa86ad-e44b-4b42-90c0-863c0bf1da60","added_by":"auto","created_at":"2026-04-14 15:13:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1116463,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/c9763738eb79262a1e34314c.docx"},{"id":106960210,"identity":"ef5677fa-f8cc-4d23-949e-c4b279c3e90e","added_by":"auto","created_at":"2026-04-15 09:19:23","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3877882,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedGelsandBlotsimage.docx","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/98df1473fbf5964ba65a91f6.docx"},{"id":106903837,"identity":"2b380738-af47-4881-b45f-525c438a09e3","added_by":"auto","created_at":"2026-04-14 15:13:57","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":136822,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9348456/v1/12c8d345954e7221d55616e0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MAGEA4 promotes gastric cancer progression by inducing chromosomal instability through the STAU1/c-Myc axis","fulltext":[{"header":"Background","content":"\u003cp\u003eGastric cancer (GC) is a major global health challenge, being one of the most frequently diagnosed cancers and a leading cause of cancer-related mortality (1). According to GLOBOCAN 2020 data, GC accounts for over one million new cases and more than 700,000 deaths annually worldwide (2). Notably, while its global incidence is declining, GC rates are rising in specific demographics, particularly among adults under 50 years old (3).\u0026nbsp;Additionally, the prognosis of GC is generally poor, particularly when diagnosed at advanced stages, with a five-year survival rate of less than 10%\u0026nbsp;(4). Given this clinical reality, continued investigation into the molecular mechanisms driving GC progression is essential to identify novel therapeutic targets.\u003c/p\u003e\n\u003cp\u003eFor a long time, the pathological classification of GC has primarily relied on histological features. According to the Lauren classification, GC can be divided into two main types: intestinal type and diffuse type (5). However, traditional pathological typing cannot accurately reflect the molecular characteristics of tumors. In recent years, the emergence of The Cancer Genome Atlas (TCGA) molecular classification of GC has provided significant guidance for personalized treatment. Among these, the chromosomal instability (CIN) subtype accounts for approximately half of all GC cases (6).\u0026nbsp;CIN refers to the missegregation of chromosomes during mitosis, leading to changes in chromosome number or structure, and is the main driver of tumor progression\u0026nbsp;(7, 8). The causes of CIN include abnormalities in DNA damage repair, centrosome amplification, defects in kinetochore-microtubule attachment, and impaired spindle assembly checkpoint function\u0026nbsp;(9-11). CIN is not only closely associated with the progression of various cancers but also plays a critical role in tumor metastasis and therapy resistance\u0026nbsp;(12).\u0026nbsp;CIN continuously generates genomic diversity, which can accelerate the emergence of subclones with specific genotypes that confer therapeutic resistance\u0026nbsp;(7, 13). CIN can also enhance cellular adaptability and tolerance by triggering epigenetic reprogramming and reinforcing cancer stem cell properties\u0026nbsp;(14, 15). Additionally, CIN activates the cGAS\u0026ndash;STING pathway through the rupture of micronuclei and the release of dsDNA/dsRNA. The chronic activation of cGAS\u0026ndash;STING can remodel the immune microenvironment, ultimately facilitating tumor metastasis and treatment resistance\u0026nbsp;(16-18). As such, it is of great significance to study the molecular mechanisms underlying CIN.\u003c/p\u003e\n\u003cp\u003eMAGEA4 (Melanoma Antigen Gene A4) is a cancer/testis antigen (CTA) characterized by its aberrant overexpression in a wide range of malignancies yet negligible expression in normal tissues (19, 20). This restricted expression profile positions MAGEA4 as a promising target for tumor therapy. Evidence indicates that MAGEA4 promotes tumorigenesis in non-small cell lung cancer (NSCLC) through recruitment and retention of IgA-positive plasma cells in the lungs\u0026nbsp;(21). Furthermore, MAGEA4 can support cancer cell survival and accelerate tumor progression by increasing DNA damage tolerance through inducing RAD18 expression, a ubiquitin ligase associated with translesion synthesis (22). Beyond driving tumorigenesis and tumor progression, MAGEA4 is also implicated in modulating immune responses within the tumor microenvironment\u0026nbsp;(23). Although MAGEA4 expression in GC is well documented, its precise biological functions and the underlying molecular mechanisms have not been fully elucidated\u0026nbsp;(24).\u003c/p\u003e\n\u003cp\u003eHere, we investigated the role and mechanism of MAGEA4 in GC pathogenesis. Our results revealed a significant upregulation of MAGEA4 in GC tissues, which was closely correlated with adverse clinical outcomes. Through in vitro and in vivo experiments, we demonstrated that MAGEA4 substantially enhanced the proliferation, migration, and invasion of GC cells. Mechanistically, we identified that MAGEA4 drives CIN and facilitates tumor progression by activating the STAU1/c-Myc signaling axis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and tissue samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 40 pairs of GC tissues and matched adjacent normal mucosal tissues were included from the First Affiliated Hospital of Anhui Medical University. None of the patients had received any local or systemic antitumor therapy prior to surgery. Tumor staging was determined according to the National Comprehensive Cancer Network (NCCN) 2010 guidelines. In addition, two tissue microarrays containing 195 GC samples and 71 normal gastric mucosa samples were constructed for subsequent analysis. All studies on human specimens were approved by the Clinical Research Ethics Committee of Anhui Medical University, and written informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lines used in this study, including human GC cell lines (HGC-27, MKN-45, NCI-N87, AGS, and KATO III), the normal gastric epithelial cell line GES-1, and the kidney epithelial-like 293T cell line, were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China).\u0026nbsp;All cell lines were cultured at 37\u0026deg;C in a humidified atmosphere with 5% CO₂ in their specified culture medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed on ice for 30 minutes in radioimmunoprecipitation assay (RIPA) buffer containing a protease and phosphatase inhibitor cocktail. After lysis, samples were centrifuged at 12,000 rpm for 15 minutes at 4\u0026deg;C. The supernatant was then mixed with loading buffer and boiled for 10 minutes at 100\u0026deg;C. The proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes.\u0026nbsp;The membranes were blocked with Tris-buffered saline containing 5% skim milk for 2.5 hours, then incubated with the corresponding primary antibodies overnight. Subsequently, the membranes were incubated with alkaline phosphatase-conjugated secondary antibodies at room temperature for 1 hour. Finally, immunoreactive bands were detected using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (Sangon Biotech, Shanghai, China). Details regarding the antibodies utilized are provided in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR analysis (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using Trizol reagent (Accurate Biotechnolog, China), and equal amounts were reverse-transcribed to complementary DNA (cDNA) using the 5\u0026times; Evo M-MLV RT Premix (Accurate Biotechnolog, China). Gene expression levels were detected by quantitative real-time PCR (qPCR) using the 2\u0026times; SYBR Green Pro Taq HS Premix (Accurate Biotechnolog, China). The expression levels of all target genes were normalized to those of the housekeeping gene Gapdh.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDetails regarding the primer sequences used are provided in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis was run on two tissue microarrays with 71 pairs of GC and matched normal gastric mucosa samples and 124 primary GC samples. A semi-quantitative scoring system was used based on two parameters: staining intensity and the percentage of positive areas. The intensity of staining was categorized into four distinct levels: negative, weakly positive, positive, and strongly positive, which correspond to scores of 0, 1, 2, and 3, respectively. The percentage of positive area was categorized into 0%-25%, 25%-50%, 50%-75%, and above 75%, corresponding to scores of 1, 2, 3, and 4, respectively. The scores of the two parameters were multiplied, and a total score of 3 or higher was used to indicate significant protein overexpression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene knockdown and overexpression experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo transiently knockdown MAGEA4 and STAU1 expression, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were transfected using Lipofectamine 2000 (Invitrogen). Specific small interfering RNAs (siRNAs) targeting MAGEA4 and STAU1 were used in the transfection. The siRNA sequences for the MAGEA4 gene were as follows: siRNA #1: 5\u0026rsquo;-CAA GAU UGG GUG CAG GAA ATT-3\u0026rsquo;, siRNA#2: 5\u0026rsquo;-GCG UUG AGG CCC AAG AAG ATT-3\u0026rsquo; (General Bio, Anhui, China), and the siRNA sequence for STAU1 gene was siRNA: 5\u0026rsquo;-CUG UGG GAG GAC AGC AAU UUA TT-3\u0026rsquo; (General Bio, Anhui, China).\u0026nbsp;For MAGEA4 overexpression, the MAGEA4 sequence was PCR-amplified and subcloned into the pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro plasmid (General Bio, Anhui, China). Plasmid sequences were confirmed by DNA sequencing before use. The stable plasmid and viral packaging plasmids were then co-transfected with Lipo fectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) into 293T cells to generate lentiviruses. The gastric cancer cells were transduced with the lentiviruses to establish a stable cell line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 96-well plates at a density of 5\u0026times;10\u0026sup3; cells per well and cultured for 5 days. The absorbance was measured and recorded every 24 hours using CCK-8 reagent\u0026nbsp;(Dojondo Laboratories, Kumamoto, Japan). Each experiment was repeated 3 times with 4 technical repeats for each time point per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 6-well plates at a density of 5\u0026times;10\u0026sup2; cells per well. They were cultured in complete medium at 37\u0026deg;C with 5% CO₂ for 13 days; the medium was refreshed every 4 days. Following the incubation period, cells were\u0026nbsp;incubated with 4% paraformaldehyde for 25 minutes and then stained with Crystal Violet solution for 12 minutes. The stained plates were photographed under a microscope. Colonies consisting of 50 or more cells were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound‐healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 6-well plates with complete medium and cultured until they reached full confluence. A linear wound scratch was created in the cell monolayer using a standard 200 \u0026mu;L pipette tip. The scratch width was approximately 300-500 \u0026mu;m. The wounded area was washed twice with 1\u0026times;PBS to remove detached cells.\u0026nbsp;Wound closure was monitored and imaged at five randomly selected microscopic fields at 0 and 48 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell migration and invasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 5\u0026times;10⁴ cells were\u0026nbsp;seeded into the serum-free medium upper chamber of a Transwell. For the invasion assay, the membranes were pre-coated with Matrigel (BD Bioscience, USA), while no coating was used for the migration assay. The lower chamber was filled with complete medium containing 10% fetal bovine serum (FBS) as a chemoattractant. After 48 hours of incubation at 37\u0026deg;C, the chambers were collected, fixed with 4% paraformaldehyde, and stained with crystal violet. Cells that migrated or invaded to the lower surface of the membrane were counted under a microscope in five randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo tumour growth assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransduced cells (2\u0026times;10⁶) were subcutaneously injected into the inguinal region of 4-6 week-old male nude mice.\u0026nbsp;Tumor dimensions were measured every 4 days, and tumor volume was calculated as V = 4\u0026pi;/3 \u0026times; (width/2) \u0026sup2; \u0026times; (length/2).\u0026nbsp; All mice were euthanized 30 days after inoculation, marking the experimental endpoint. Subsequently, xenograft tumors were harvested, photographed, and weighed. Six biological replicates per group were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLung metastasis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransduced cells (6\u0026times;10⁶ per mouse) were injected into nude mice via the tail vein. All mice were euthanized six weeks after injection and lung tissues were harvested for pathological examination, hematoxylin and eosin (H\u0026amp;E) staining, and quantification of metastatic nodules. Each group included five biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome Sequencing and Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter passing quality control, total RNA was subjected to RNA-seq using the Hiseq3000 (Illumina, USA). The raw sequencing data were converted into a gene expression matrix following additional quality control and filtering. Subsequently, normalized differential expression analysis was performed on the expression matrix, and significantly differentially expressed genes were further analyzed for functional enrichment using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosome spreading assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells at the logarithmic growth phase were treated with colchicine. The drug was added to the culture medium at a final concentration of 0.4 \u0026mu;g/mL and the cells were incubated at 37 \u0026deg;C for 3 hours. The cells were then gently detached, transferred into 2 mL microcentrifuge tubes, and centrifuged at 1000 rpm for 5 minutes. After removing the supernatant, the cell pellet was resuspended in 1 mL of 0.075 mol/L KCl hypotonic solution warmed to 37 \u0026deg;C and incubated at 37 \u0026deg;C for 30 minutes. Next, 1 mL of freshly prepared fixative (methanol: glacial acetic acid = 3:1) was added, mixed thoroughly, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded. The fixation step was repeated twice more, each time by adding 1 mL of fixative, gently mixing, incubating at 37 \u0026deg;C for 30 minutes, and centrifuging under the same conditions. Finally, the cells were resuspended in 50 \u0026mu;L of fixative. The entire suspension was then dropped onto a pre-chilled clean glass slide and gently spread to ensure even coverage. The slide was air-dried at room temperature, stained with 10% Giemsa solution for 10 minutes, rinsed gently under running water, and air-dried at room temperature. The prepared slides were observed and cells counted under an oil immersion microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 100,000 cells were fixed by resuspension in 80% ethanol and incubated for 12 hours at 4\u0026deg;C. The fixed cells were then washed twice to remove ethanol: first with phosphate-buffered saline (PBS), and then with staining buffer (BD Biosciences, Cat. #554656). The cells were then resuspended in 0.5 mL of PI/RNase Staining Buffer (BD Biosciences, Cat. #550825) and incubated for 15 minutes at room temperature in the dark before flow cytometric analysis. The data were analyzed using FlowJo software, and the proportion of cells with DNA content\u0026nbsp;＞4N was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in confocal dishes at a density of 2.4-3\u0026times;10⁵ cells per dish and cultured for 24 hours. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.5% Triton X-100 for 20 min. After blocking cells for 30 min, the cells were incubated with primary antibodies at 4 \u0026deg;C overnight followed by incubation with the corresponding secondary antibodies for 1 h at room temperature. Three 5-minute washes with PBS were performed after each antibody incubation step. Nuclei were stained with 1\u0026mu;g/mL DAPI for 5 min, and samples were mounted with ProLong Gold Antifade Reagent (P10144, Thermo Fisher). Images were acquired using a ZEISS LSM 888 confocal microscope. For mitosis quantitative analysis, at least 100 cells per condition were manually examined to count lagging chromosomes, anaphase bridges, multipolar mitotic events, and micronuclei. Details regarding the antibodies utilized are provided in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded tissue sections were heated at 60\u0026deg;C for 30 minutes prior to hematoxylin and eosin staining. The staining procedure was performed as follows: sections were deparaffinized in xylene I and II for 15 minutes each, rehydrated through a graded ethanol series (100%, 95%, 85%, and 75%) for 5 minutes each, and rinsed for 5 minutes in distilled water. Subsequently, sections were stained with Harris hematoxylin for 5\u0026ndash;8 minutes, rinsed under running tap water to promote bluing, differentiated in 1% acid alcohol for several seconds, and then washed again under running water for 15 minutes to complete the differentiation and bluing steps. Counterstaining was performed with 0.5% eosin Y in ethanol for 1-3 minutes, followed by rapid dehydration through graded ethanol concentrations (85%, 95%, and 100%; about 30 seconds each) and clearing in xylene (two changes, 2 minutes each). Finally, sections were mounted with neutral balsam and examined under a light microscope for histopathological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical comparisons between groups were performed using Student\u0026apos;s t-test. Associations between protein expression levels and clinicopathological parameters were analyzed with a Chi-square test. Survival analysis was performed using the Kaplan-Meier method, and differences between groups were assessed by the log-rank test. All statistical analyses were performed using GraphPad Prism software (version 10.1.2), using a two-tailed P value \u0026lt; 0.05 as the threshold for statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMAGEA4 is up‐regulated in GC patients and associated with poor prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TCGA and Genotype-Tissue Expression (GTEx) databases revealed that MAGEA4 was upregulated in various cancers, including esophageal carcinoma, lung adenocarcinoma, and stomach adenocarcinoma\u0026nbsp;(Fig. 1A). Analysis of the Kaplan-Meier plot database found that increased expression of MAGEA4 was correlated with poorer overall survival (OS) rates in GC (Fig. 1B). Differential expression analysis comparing normal gastric mucosae and GC tissues from the TCGA database identified 1,672 differentially expressed genes comprising 922 upregulated and 750 downregulated genes (Fig. 1C). Among the most differentially expressed genes, MAGEA4 ranked third (Fig. 1D).\u003c/p\u003e\n\u003cp\u003eTo assess the expression of MAGEA4 in GC, we first using quantitative analysis of 40 matched tissue pairs to detect the expression level of MAGEA4 mRNA levels in GC and normal gastric mucosa tissue. The results indicated that MAGEA4 mRNA was markedly upregulated in GC (Fig. 1E). Consistent with this, the upregulation of\u0026nbsp;MAGEA4\u0026nbsp;protein in GC tissues and GC cell lines was also confirmed (Fig. 1F and G). Scoring of immunohistochemical analysis on tissue microarrays containing 71 pairs of GC samples and their matched normal gastric mucosae, and 124 primary GC samples demonstrated that MAGEA4 expression was significantly higher in GC compared to normal gastric mucosae (Fig. 1H-J). An immunohistochemical score was calculated by integrating staining intensity and the proportion of positive cells, and all patients were divided into a MAGEA4 high-expression group and a low-expression group for subsequent analysis. Correlation analysis revealed that high MAGEA4 expression was significantly associated with higher AJCC stage and lymph node metastasis of GC (Supplementary Table S3). Furthermore, Kaplan-Meier survival analysis demonstrated that GC patients with high MAGEA4 expression had significantly shorter overall survival (OS) and disease-free survival (DFS) (Fig. 1K). This finding aligned with the trend of GC observed in the Kaplan-Meier Plotter database. Subsequent multivariate Cox regression analysis further confirmed that high MAGEA4 expression was an independent predictive factor for poor OS and DFS in GC patients (Table 1).\u003c/p\u003e\n\u003cp\u003eIn summary, MAGEA4 expression is significantly up-regulated in GC, and its high expression is closely associated with poor prognosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAGEA4 stimulates the proliferation, migration, and invasion of GC \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the role of MAGEA4 in GC progression, we knocked down MAGEA4 in HGC-27 cells which exhibit high MAGEA4 endogenous expression. Conversely, we stably overexpressed MAGEA4 in MKN-45 cells which show low basal MAGEA4 expression (Fig. 2A). The CCK8 proliferation assay and colony formation assay demonstrated that MAGEA4 knockdown significantly suppressed HGC-27 cell proliferation, whereas MAGEA4 overexpression markedly enhanced the proliferation of MKN-45 cells (Fig. 2B and C). Furthermore, wound healing and Transwell migration assays revealed that MAGEA4 silencing markedly impaired the migratory ability of GC cells, while its overexpression promoted cell migration (Fig. 2D and E). Similarly, the invasive capacity of GC cells was significantly reduced upon MAGEA4 knockdown and enhanced by MAGEA4 overexpression (Fig. 2F). In summary, these results indicate that MAGEA4 stimulates the proliferation, migration, and invasion of GC cells \u003cem\u003ein\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAGEA4 promotes the proliferation and metastasis of GC cells \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the effect of MAGEA4 on GC growth \u003cem\u003ein vivo\u003c/em\u003e, we performed a tumor burden assay using MKN-45 cells with stable MAGEA4 overexpression. Tumors derived from MAGEA4-overexpressing cells displayed enhanced proliferative activity compared with controls (n = 6 per group; Fig. 3A), and at the experimental endpoint, both tumor size and weight were significantly greater in the MAGEA4-overexpressed group (Fig. 3 B and C). These results indicate that MAGEA4 promotes tumor growth of GC cells \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn the lung metastasis model, MAGEA4-overexpressing or control MKN-45 cells were injected into the tail veins of nude mice (n = 5 per group). The incidence of lung metastasis was 100% (5/5) in the MAGEA4-overexpression group compared to 40% (2/5) in the control group (Fig. 3D). In addition, the number of pulmonary metastatic nodules was significantly higher in MAGEA4-overexpressed groups than in controls (Fig. 3E). These findings demonstrate that MAGEA4 plays a critical role in promoting metastasis of GC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAGEA4 induces chromosomal instability (CIN) in GC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate how MAGEA4 contributes to GC progression, we performed RNA sequencing on transcriptomes of HGC-27 cells with MAGEA4 knockdown and control cells. Gene ontology analysis of results from RNA-seq analysis revealed significant alterations in genes involved in biological processes such as DNA replication, chromosome segregation, and\u0026nbsp;mitotic G2 DNA damage checkpoint signaling (Fig. 4A). This finding prompted us to investigate the role of MAGEA4 in cell cycle progression and DNA damage repair. Western blotting analysis and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eimmunofluorescence imaging showed that \u0026gamma;H2AX,\u0026nbsp;a biomarker for DNA double-strand breaks\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(25), was significantly reduced in MAGEA4-knockdown HGC-27 cells, but was increased in MAGEA4-overexpressing MKN-45 cells (Fig. 4B and C). Representative images are provided in supplementary (Fig. S1).\u003c/p\u003e\n\u003cp\u003eChromosome spreading assays showed that MAGEA4 overexpression induced a shift from diploidy to aneuploidy in MKN-45 cells\u0026nbsp;(Fig. 4D). Cell cycle analysis further confirmed that MAGEA4 overexpression increased the proportion of aneuploidy cells (Fig. 4E and G). Given that DNA damage and aneuploidy are recognized as consequences of CIN in cancer cells (26), we sought to determine whether MAGEA4 induces characteristic CIN features such as misaligned chromosomes, anaphase bridges, asymmetric anaphase, multipolar mitosis, or micronuclei (27, 28). Results demonstrated that the number of abnormal mitotic cells, such as those displaying misaligned chromosomes, anaphase bridges, and asymmetric anaphase, was significantly higher in MAGEA4-overexpressing MKN-45 cells (Fig. 4F and H). Additionally, the incidences of micronuclei and multipolar mitosis were significantly elevated in MAGEA4-overexpressing MKN-45 cells (Fig. 4I-L). Consistent with these \u003cem\u003ein vitro\u003c/em\u003e results, H\u0026amp;E-stained sections from clinical samples with high MAGEA4 expression also showed more CIN-associated features (Fig. 4 M and N). In summary, our study demonstrates that elevated MAGEA4 expression induces chromosomal instability, resulting in chromosomal aberrations and DNA damage in GC cells.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAGEA4 enhances c-Myc expression at the translational level via a STAU1-dependent pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism by which MAGEA4 induces CIN, we determined the expression of CIN-related proteins, p53 and c-Myc (29-33) in GC cells with modified MAGEA4 expression as indicated by Western blotting analysis. While p53 protein levels remained largely unchanged with both MAGEA4 knockdown and overexpression (Fig. 5A), c-Myc protein expression was significantly upregulated in MAGEA4-overexpressed cells and downregulated in MAGEA4-depleted cells (Fig. 5B). The RNA-seq data on transcriptomes of HGC-27 cells with MAGEA4 knockdown and control cells revealed 291 genes upregulated and 534 genes downregulated (Fig. 5C), however, no significant changes in c-Myc mRNA levels were observed (Fig. 5D), leading us to hypothesize that a post-transcriptional mechanism enhances c-Myc expression.\u003c/p\u003e\n\u003cp\u003ePrevious studies have reported that the RNA-binding protein STAU1 enhances the translation efficiency of c-Myc by binding to the 5\u0026apos; untranslated region (5\u0026apos;UTR) of its transcript(34, 35). Notably, our RNA-seq analysis identified STAU1 among the top 20 most significantly altered genes (Fig. 5E) with STAU1 protein levels decreasing significantly after MAGEA4 silencing and increasing after MAGEA4 overexpression (Fig. 5F). RT-qPCR analysis confirmed that STAU1 mRNA expression mirrored these changes while c-Myc mRNA levels remained stable (Fig. 5G).\u003c/p\u003e\n\u003cp\u003eTo determine whether MAGEA4 enhances c-Myc expression via STAU1, we performed STAU1 knockdown in both MAGEA4-overexpressed and control MKN-45 cells. Depletion of STAU1 eliminated the increase in c-Myc protein induced by MAGEA4 overexpression (Fig. 5H). In fact, c-Myc expression was also inhibited by STAU1 knockdown. These findings demonstrate that MAGEA4 upregulates c-Myc expression at the translational level via a STAU1-dependent pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAGEA4 promotes malignant phenotypes of GC cells by inducing CIN via the STAU1/c-Myc axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies reported c-Myc as driver of genomic instability (36) , and our results have established that MAGEA4 upregulates c-Myc expression at the translational level via STAU1. We further investigated the impact of the STAU1/c-Myc axis on CIN phenotypes and malignant behaviors in GC cells. Colony formation assays revealed that STAU1 knockdown not only reversed the enhanced proliferation induced by MAGEA4 overexpression in MKN-45 cells, but also suppressed the basal colony-forming ability in control cells (Fig. 6A). Similarly, in the Transwell migration and invasion assays, STAU1 depletion abolished the pro-migratory and pro-invasive effects induced by MAGEA4 overexpression, while impairing these capacities in wild-type MKN-45 cells (Fig. 6B and C). Thus, MAGEA4 promotes the malignant phenotypes of GC cells in a STAU1-dependent manner.\u003c/p\u003e\n\u003cp\u003eChromosome spread assays and DNA content analysis indicated that STAU1 knockdown markedly attenuated the increase in chromosomal numerical abnormalities resulting from MAGEA4 overexpression (Fig. 6D, F and H). Western blotting analysis and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eimmunofluorescence imaging showed that STAU1 silencing reduced \u0026gamma;H2AX expression in both MAGEA4-overexpressed and control cells (Fig. 5H and Fig. 6E, G). The frequencies of abnormal mitosis, multipolar mitosis, and micronuclei formation were similarly reduced (Fig. 6I-K). Representative images are provided in supplementary (fig. S2). These results indicated that MAGEA4 promotes the proliferation, metastasis and invasion of GC cells by inducing CIN through the STAU1/c-Myc signaling axis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, there has been growing interest in the role of MAGEA4 in promoting tumor progression. However, its specific mechanism in GC remains unclear. This study aimed to investigate the clinical significance, biological function, and molecular mechanism of MAGEA4 in the evolution of GC. Mechanistically, we found that MAGEA4 upregulates c-Myc expression at the translational level via STAU1, thereby inducing CIN and promoting GC development (Fig. 7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a cancer/testis antigen, MAGEA4 is highly expressed in tumor tissues such as esophageal squamous cell carcinoma, intrahepatic cholangiocarcinoma, and non-small cell lung cancer (NSCLC), where its elevated expression is associated with\u0026nbsp;adverse clinical outcomes\u0026nbsp;(37-39).\u0026nbsp;Evidence indicates that MAGEA4 can increase the DNA damage tolerance of cancer cells, thus supporting their survival (22).\u0026nbsp;The Cancer Genome Atlas (TCGA) database revealed that MAGEA4 is highly expressed in GC tissues but barely detectable in normal tissues, suggesting its potential as an ideal therapeutic target. To further explore its role in GC, we analyzed 40 pairs of matched clinical samples and observed significant upregulation of MAGEA4 in tumor tissues compared to adjacent normal tissues. Moreover, two tissue microarrays containing 195 GC samples and 71 normal gastric mucosa samples supported a strong correlation between high MAGEA4 expression and poor prognosis. Through gain‐\u0026nbsp;and loss‐of‐function experiments, we showed that MAGEA4 enhances the proliferation, migration, and invasion abilities of GC cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. These findings indicated that MAGEA4 plays a critical role in promoting GC progression.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To elucidate how MAGEA4 contributes to GC progression we performed RNA sequencing on transcriptomes of HGC-27 cells with MAGEA4 knockdown and control cells. Gene Ontology analysis revealed significant alterations in biological processes related to chromosome segregation, DNA replication, and\u0026nbsp;mitotic G2 DNA damage checkpoint signaling, all of which are closely associated with CIN. CIN is commonly observed in cancer patients and correlates with tumor aggressiveness and metastasis, serving as a key driver of tumor progression (7, 8, 40, 41). According to TCGA, GC is classified into four molecular subtypes: Epstein-Barr virus-positive (EBV+), microsatellite instability (MSI), genomically stable (GS), and chromosomal instability (CIN)(6). Among these, the CIN subtype is the most prevalent (42) and typically exhibits a higher mutation burden and more complex genomic features than the other subtypes (43, 44). We then conducted a series of experiments to examine whether MAGEA4 affects CIN in GC cells. The results showed that MAGEA4-overexpressed cells exhibited markedly increased CIN features, including \u0026gamma;H2AX foci accumulation, aneuploidy, abnormal mitosis, multipolar division, and micronucleus formation. Furthermore, in GC tissue sections, the frequency of these CIN-related markers was significantly higher in MAGEA4-high samples compared to MAGEA4-low samples. These results collectively support that MAGEA4 induces CIN in GC cells.\u003c/p\u003e\n\u003cp\u003eBuilding upon the established role of CIN in GC progression (14), our work focused on deciphering the molecular mechanism by which MAGEA4 induces this pathogenic state. The development of CIN involves a complex network of proteins, among which P53 and c-Myc are particularly prominent. Deficiencies in P53 function are well-documented to increase cellular susceptibility to chromosome misalignment and unequal segregation during mitosis (45, 46). Furthermore, the critical role of P53 in orchestrating DNA damage repair means its loss severely compromises this protective capacity, thereby amplifying CIN manifestations (47-49). In contrast, c-Myc drives CIN through two principal avenues: first, by accelerating cell proliferation via regulation of cell cycle-related genes, which increases the frequency of mitotic errors; and second, by impairing the normal function of the DNA repair system, thereby undermining genomic stability (31, 50-53). Our investigation into the expression of these key regulators in MAGEA4-modulated cell models revealed a distinct pattern: Western blotting showed that P53 protein levels remained largely unaltered in both MAGEA4 knockdown and overexpression models, whereas c-Myc protein expression exhibited a striking dependence on MAGEA4 status\u0026nbsp;despite\u0026nbsp;the absence of corresponding changes in c-Myc mRNA levels pointing toward post-transcriptional regulation. This hypothesis was subsequently validated through confirmation that MAGEA4 enhances c-Myc protein synthesis at the translational level via the RNA-binding protein STAU1.\u003c/p\u003e\n\u003cp\u003eTo establish the functional centrality of STAU1 within this pathway, we performed STAU1 knockdown experiments in both MAGEA4-overexpressed and control MKN-45 cell lines. The results showed that depleting STAU1 effectively reversed the CIN phenotype and other malignant characteristics induced by MAGEA4 overexpression. Notably, STAU1 knockdown in native MKN-45 cells was sufficient to significantly reduce baseline CIN and suppress the malignant phenotype. Together, these findings delineate a clear mechanistic pathway wherein MAGEA4 induces CIN via the STAU1/c-Myc signaling axis, thereby promoting the progression of GC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our findings establish MAGEA4 as a key driver of GC progression and a potential predictor of poor patient prognosis. By inducing chromosomal instability through the STAU1/c-Myc axis, MAGEA4 fuels tumor aggressiveness. Its highly restricted expression in tumor tissue positions MAGEA4 as a promising and specific therapeutic target. Future efforts should focus on elucidating the upstream mechanisms that govern MAGEA4\u0026apos;s control over STAU1.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eGC: gastric cancer\u003c/p\u003e\n\u003cp\u003eCIN: chromosomal instability\u003c/p\u003e\n\u003cp\u003eMAGEA4: Melanoma Antigen Gene A4\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments involving human participants in this study obtained written informed consent from all relevant individuals and were approved by the Clinical Research Ethics Committee of Anhui Medical University (Approval No.: PJ 2024-13-63). Experiments involving mice in this study were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (Approval No.: LLSC20200141).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll Authors agreed to the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data supporting the findings of this study are available within the article and its supplemental files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (82002545, 82573137, 81902451) and Shanghai Sailing Program (20YF1409200) and the Natural Science Research Project of Anhui Higher Education Institution (2025AHGXZK31473) and Clinical and Translational Research Project of Anhui Province (202427b10020121).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.D and ZW.L wrote the manuscript and participated in the entire experiment. CP.R analyzed and processed the data. CY.C and BB.Z collected the specimens. YS.J participated in data processing. CC.L and CY.F provided financial support and designed the experiment, and made substantial contributions to the writing of the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge Anhui Medical University for providing instrument support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLee K, Hwang H, Nam KT. Immune response and the tumor microenvironment: how they communicate to regulate gastric cancer. Gut and liver. 2014;8(2):131-9.\u003c/li\u003e\n\u003cli\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Proceedings of the National Academy of Sciences of the United States of America. 2003;100(17):9974-9.\u003c/li\u003e\n\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chromosomal instability, Gastric cancer, c-Myc, MAGEA4, STAU1","lastPublishedDoi":"10.21203/rs.3.rs-9348456/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9348456/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"This study aimed to investigate the clinical significance, biological function, and molecular mechanisms of MAGEA4 in the progression of gastric cancer (GC). Analysis of the TCGA database and 40 pairs of GC tissues and matched normal mucosa samples revealed that MAGEA4 expression was significantly upregulated in GC. Immunohistochemical analysis using two tissue microarrays containing 195 GC samples demonstrated that high MAGEA4 expression is significantly associated with advanced tumor stage, lymph node metastasis and unfavorable patient prognosis. Both in vitro and in vivo experiments confirmed that MAGEA4 significantly promoted the proliferation and metastasis of GC cells. Investigations of the underlying mechanisms revealed that MAGEA4 promotes GC progression by inducing chromosomal instability (CIN). This was evidenced by increased γH2AX (a sensitive DNA damage marker for double-strand breaks), abnormal chromosome numbers, abnormal mitosis, and other CIN features. At the molecular level, MAGEA4 enhances the translation efficiency of c-Myc through the RNA-binding protein STAU1, thereby inducing CIN. Knockdown of STAU1 effectively reversed the CIN phenotype and malignant phenotypes induced by MAGEA4 overexpression in GC cells. In summary, this research demonstrated the crucial role of MAGEA4 in promoting GC progression by inducing CIN via the STAU1/c-Myc axis. Consequently, MAGEA4 represents a highly promising novel therapeutic target for GC.","manuscriptTitle":"MAGEA4 promotes gastric cancer progression by inducing chromosomal instability through the STAU1/c-Myc axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 15:13:52","doi":"10.21203/rs.3.rs-9348456/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"29f89b9e-f959-40b4-a164-dafb63fcde2b","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T16:10:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 15:13:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9348456","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9348456","identity":"rs-9348456","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}