Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma

Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma Chong Chen, Hongyu Liu, Ailing Zhong, Zhenghao Lu, Jiaxin Li, and 31 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5298469/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Signet ring cell carcinoma (SRCC) is a lethal malignancy with unique histologic features, characterized with large vacuoles and compressed nuclei. Gastric SRCC is the most common SRCC, and its incidence is increasing recently. However, the driver genes of SRCC and the molecular mechanisms underlying its unusual histology remain unclear. Here, we developed a new type of gastric SRCC mouse models with gene-edited premalignant gastric organoids and validated ARID1A , one of the most frequently mutated genes in SRCC, as a bona fide tumor suppressor gene of gastric SRCC. Mechanistically, through CUT/Tag and ATAC-seq analyses, we found that Arid1a directly regulated the expressions of secretory factors Scin and on the other hand, Arid1a loss reprogrammed the genome binding of the SWI/SNF complexes and increased the expressions of mucin genes through the binding of Brd9 , a component of the noncanonical SWI/SNF complex. Inhibiting Brd9 reversed the pathology of Arid1a mutant SRCC. Thus, our studies revealed dual roles of ARID1A in restraining SRCC through both mucin production and secretion. These findings offer new insights into the susceptibilities of ARID1A deficient SRCC. Biological sciences/Cancer/Cancer models Biological sciences/Cancer/Gastrointestinal cancer Gastric signet ring cell carcinoma ARID1A mouse model pathology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Signet ring cell carcinoma (SRCC) is a histologically unique form of highly malignant adenocarcinoma, characterized by a large vacuole and the resulting cell periphery-localized nucleus, resembling a signet ring 1,2 , The most common SRCC tumors are found in gastric cancers and less frequently in colorectal cancers and others 3–5 . Gastric SRCC accounts for about 10% of total gastric cancers with increased frequencies in recent years and is associated with poor prognosis 6,7 . It is believed that this special histologic appearance is due to large amounts of mucins produced by SRCC cells and postulated their deficient ability to export them out of the cells 8,9 . However, the molecular mechanisms underlying these malignancy-associated abnormalities remain unclear. The recent progress in cancer genomics has revealed the genomic alterations in SRCC 10–12 . Besides TP53 , the most mutated gene in both SRCC and non-SRCC tumors, ARID1A and CDH1 are especially highly frequently mutated in SRCC. Other alterations, such as CLDN18-ARHGAP26/6 fusions, are also found in about 20% of SRCC patients 10,13,14 . CDH1 encodes the cell-cell adhesion molecular E-cadherin, and its heterozygous loss significantly promoted the formation of SRCC in mice treated with N-methyl-N-nitrosourea (MNU), while had less effect on tubular adenoma tumorigenesis 15 . Similarly, human gastric organoids with CDH1 knockout also displayed morphological changes similar to SRCC 16,17 . It has been suggested that downregulation of the Wnt signaling facilitated the SRCC histology driven by CDH1 mutations. CLDN18 is a tight junction gene and ARHGA26/6 are RHOA inhibitors and the CLDH18-ARHGAP26 fusion seemed to enhance the migration and chemoresistance of gastric cancer cells, which were consistent with the clinical features of SRCC patients 10 . The potential functions of other SRCC-associated genes, including ARID1A , are less understood. ARID1A , the core member of the SWI/SNF chromatin remodeling complex, is frequently mutated in various cancers, including ovarian clear cell carcinomas (OCC) (50%), uterine endometrioid carcinomas (29%), and gastric cancer (29%) 18,19 . Arid1a loss itself gave rise to invasive colon adenocarcinoma in mice, while, intriguingly, repressed the tumorigenesis in the small intestine of the Apc min mice 20 . And in liver cancer, ARID1A promoted cancer initiation but restrained the progression and metastasis at later stage 21 . ARID1A knockout could also transform human gastric organoids 22 . However, whether and how ARID1A deficiency plays functional roles in SRCC, especially related to its characteristic morphology need to be investigated. In this study, we developed a strategy to generate primary SRCC mouse models with genome-edited gastric organoids. We found that Arid1a loss promoted the formation of SRCC through both indirectly enhancing the expressions of mucin genes and directly impairing the secreting pathway. Results Arid1a deficient gastric organoids gave rise to SRCC We performed a genetic analysis of 171 samples of diffuse-type gastric carcinoma (DGC) and gastric signet ring cell carcinoma (SRCC) obtained from the cBioPortal database and found that the incorporation of shallow deletions escalates the frequency of ARID1A alterations to 39% (66/171). Notably, the analysis reveals that ARID1A variation frequently co-occur with TP53 (50%, 86/171) and PTEN (18%, 30/171) variations in these patients (Fig. 1 A and Supplementary Table 1). Consequently, we employed a gene-editing technique in organoids to establish an orthotopic SRCC model and investigate the carcinogenic role of Arid1a , in conjunction with Trp53 and Pten deletion, both in vitro and in vivo . Initially, we cultured gastric organoids from adult Trp53 −/− ; Cas9-EGFP mice. Subsequently, we delivered sgRNAs targeting Pten and Arid1a , fused with mCherry, into these organoids (Supplementary Table 2) 23,24 . The utilization of mCherry allowed for the facile identification of tumors originating from these organoids. Following this, we confirmed the mutations of Pten and Arid1a via a T7 endonuclease I mismatch detection assay (Supplementary Fig. 1A and Supplementary Table 2). The loss of ARID1A was confirmed in gastric organoids, as shown by Western blots (Fig. 1 B). Morphologically, TPA ( Trp53 −/− ; sg Pten ; sg Arid1a ) organoids exhibited greater hollow features, and the cavities of organoids were filled with mucin, which was identified via Alcian Blue PAS (AB-PAS) staining (Fig. 1 C). Furthermore, compared to those originating from TP ( Trp53 −/− ; sg Pten ) organoids, TPA organoids displayed a significantly increased size and proliferation capacity, accompanied by organoid morphological changes (Fig. 1 , D and E). In order to attain a more comprehensive understanding of the effects resulting from the absence of Arid1a in the early stages of tumorigenesis, we conducted RNA sequencing (RNA-seq) analysis to profile the transcriptomic differences of gastric organoids with or without Arid1a deficiency. As anticipated, Arid1a deficiency elicited extensive changes in gene expression (Fig. 1 F). Surprisingly, TPA organoids demonstrated a significantly greater number of upregulated genes in comparison to TP organoids (600 upregulated genes versus 335 downregulated genes, p 0.5, Fig. 1 G and Supplementary Table 3). Gene Ontology (GO) analysis indicated that the upregulated genes in the Arid1a -deficient group were significantly enriched in pathways relating to epithelial cell proliferation, extracellular structure organization, and the negative regulation of the Wnt signaling pathway, which may contribute to the characteristic pathological features of SRCC (Fig. 1 H). Moreover, we conducted subcutaneous xenograft assays to test the function of Arid1a in vivo . Arid1a loss dramatically accelerated tumor growth in the subcutaneous locus, whereas the control group demonstrated a meager growth advantage (Fig. 1 , I and J and Supplementary Fig. 1B). Pathological analysis revealed that the TPA tumors exhibited typical morphological features of SRCC, characterized by cytoplasmic filling with mucin and nuclei compression (Fig. 1 K). Furthermore, IHC staining confirmed the loss of ARID1A and overexpression of MUC1 in the TPA tumors (Fig. 1 K). In contrast, the TP group showed generalized hyperplasia (Fig. 1 K). A similar association between the loss of Arid1a and SRCC characteristics, along with increased organoid size, proliferation capacity, was observed in Arid1a -loss gastric organoids (Supplementary Fig. 1, C and E). Thus, our findings strongly suggest that the absence of Arid1a fosters the development of SRCC both in vitro and in vivo in mice. Orthotopic gastric SRCC with Arid1a loss displayed massive mucin production Subsequently, we induced the formation of primary and orthotopic SRCC by transplanting TPA gastric organoids into the submucosa of the recipient's stomach 25,26 . Recipient mice were monitored weekly and sacrificed once their body weights reduced to around 80% of the original level. Few of the mice with TP organoids developed tumors, and only one recipient mouse was shown to have colonized cells in the stomach after being sacrified. However, mice transplanted with TPA organoids developed gastric tumors. Notably, mice transplanted with TPA organoids much faster with shorter overall survival rates in comparison to the control recipient mice (TPA-1: median 67 days after transplantation; p = 1.0E-4 and TPA-2: median 142 days after transplantation; p = 4.4E-3, Fig. 2 A). Biopsy dissection confirmed that the TPA gastric organoids formed a single lesion in the stomach with specific mCherry expression, indicating their origin from the transplanted organoids (Fig. 2 B). Arid1a deficiency significantly enhanced tumor growth (Fig. 2 C), and the harvested gastric tumor cells from moribund mice presented a dramatic downregulation of ARID1A in the TPA group compared with the TP group (Fig. 2 D). Histologic analyses revealed that the lesions were mostly composed of tumor cells with prominent cytoplasmic vacuoles and a crescent-shaped nucleus eccentrically placed, suggesting that these tumors were typical SRCC (Fig. 2 E, Supplementary Fig. 2A). Meanwhile, we quantified the proportion of signet ring cells in each xenograft model and organoid culture and found that tumor tissues and primary SRCC tumor organoids demonstrated a higher proportion of signet ring cells (Supplementary Fig. 2B). To analyze which one or kinds of mucin genes work on it, we performed qPCR and observed a significant upregulation of mucin genes, including Muc1 and Muc20 (Fig. 2 F), and a consistent increase in MUC1 expression in Arid1a -deficient tumor cells. However, there was little difference in E-cadherin levels between TP and TPA tumor cells (Fig. 2 G). This observation contrasts with previous reports 27,28 and may indicate the involvement of a distinct mechanism. Furthermore, AB-PAS staining revealed mucin enrichment in the cytoplasm of most tumor cells (Fig. 2 H). Additionally, TPA tumors displayed a few metastatic competences at distal sites, while no detectable metastases were observed in TP tumors (Supplementary Fig. 2, C and D). Upon analysis of the transcriptome data of TP and TPA tumor organoids, distinct clusters were observed based on principal component analysis (Supplementary Fig. 2E). GSEA revealed that the upregulated and downregulated genes in TPA tumors, compared to the TP ones, were significantly positively and negatively enriched in SRCC and intestinal type gastric cancer (ITGC) patients, respectively (NES = 2.05, p = 0.00, and NES = -1.32, p = 0.04, respectively) (Fig. 2 I). Notably, TPA mice displayed comparable pathway enrichments to SRCC patients. We analyzed the molecular pathways significantly enriched in mouse TPA tumors and human SRCC, compared to mouse TP tumors and human ITGC, respectively. And the results showed that both collagen, metastasis, hypoxia, extracellular matrix associated pathways were upregulated, and DNA replication, ribosome, chromatid cohesion was downregulated in human SRCC and mice TPA (Supplementary Fig. 2, F and G). Moreover, a series of mucin genes were upregulated in SRCC patients relative to ITGC ones in multiple clinical cohorts and human cell lines, and this characteristic phenotype was also observed in our SRCC mice (Fig. 2 J and Supplementary Table 4). Collectively, these findings suggest that the orthotopic SRCC model accurately recapitulates the histological, pathological, and molecular features of the human SRCC. Multi-omics analyses revealed the molecular consequences of ARID1A loss in premalignant and malignant gastric epithelial cells Considering the role of ARID1A as a chromatin remodeler, we implemented Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) on TP and TPA gastric organoids to unravel epigenetic patterns and gain insight into the alterations occurring in Arid1a -deficient premalignant organoids. ATAC-seq analysis demonstrated that the absence of Arid1a caused widespread changes in the epigenome, resulting in significantly greater chromatin accessibility in TPA premalignant organoids relative to TP organoids (17474 peaks up versus 533 peaks down, p 0.5 and base-mean > 5, Fig. 3 A, Supplementary Fig. 3A and Supplementary Table 5), which was in line with transcriptome results. This result seemed paradoxical to the direct function of ARID1A on chromatin remodeling, which, together with similar previous reports on endometriosis and pancreatic ductal adenocarcinoma (PDAC) 29,30 , suggested potential complicate effect of ARID1A on chromosome remodeling through both direct and indirect regulations. And the highly accessible peaks in TPA organoids predominantly localized to promoter regions (53.14%), followed by distal intergenic regions (23.64%) and introns (12.45%), which differed from TP groups (62.29% distal intergenic, 24.95% intron, and 5.07% exon regions, Fig. 3 B, Supplementary Fig. 3B and Supplementary Table 5). Gene Ontology (GO) analyses suggested that TPA highly accessible genes were enriched in pathways relevant to extracellular matrix organization, cell-cell junction organization, regulation of epithelial mesenchymal transition, regulation of epithelial cell proliferation, actin filament organization, DNA repair and proteasomal protein catabolic process (Fig. 3 C). Moreover, motif analysis revealed that transcription factors such as Fra2 , Fosl2 , Junb , and Atf3 were highly enriched in TPA highly accessible sites, underscoring their potential role in SRCC development (Supplementary Fig. 3C). In terms of transcription level, TPA tumor organoids exhibited a much higher number of upregulated genes compared to the TP groups (779 downregulated genes versus 1545 upregulated genes, p 0.5, Fig. 3 D and Supplementary Table 3), which was consistent with the hyper-activation of genome accessibility and gene expression in Arid1a -loss organoids (Fig. 1 G and Fig. 3 A, Supplementary Table 3). GO analysis revealed that TPA downregulated genes were significantly enriched in protein acetylation, cellular protein localization, and the Wnt signaling pathway biological process (Supplementary Fig. 3D), while TPA upregulated genes were significantly enriched in cytokine production, extracellular matrix organization, ERK1 and ERK2 cascade, collagen fibril organization process, and mesenchyme development (Fig. 3 E). Through a comparative analysis of the transcriptome data from patients with SRCC and ITGC in the TCGA-STAD cohort, we identified a distinct set of genes that exhibit significantly higher expression levels in SRCC patients (Supplementary Table 1). This specific set of genes has been denoted as the SRCC signature. Most remarkably, we observed a significant upregulation of SRCC signature gene scores in TPA tumor organoids compared to TP ones based on RNA-seq data (Fig. 3 F). Notably, a cluster of mucin genes, including Muc1 , Muc5ac , and Muc20 , exhibited higher genome accessibility and expression levels in Arid1a deficient organoids compared to wildtype ones (Fig. 3 G). At the protein level, mass spectrometry-based (MS) proteomics analyses of SRCC ( Trp53 −/− ; sg Pten ; sg Arid1a ) and ITGC ( Trp53 −/− ; Myc ; sg Pten and sg Cdkn2b ), which from our reports previously 25 , tumor tissues revealed the downregulation of ARID1A and other subunit composition of BAF complex in Arid1a knockout tumors (Supplementary Fig. 3E). While a series of extracellular matrix-associated proteins, including MSLN, COL17A1, ITGA2/3/6, and ITGB6 were significantly upregulated in Arid1a loss SRCC tumors (Fig. 3 H and Supplementary Table 6). Multiple pathways related to cell adhesion and extracellular structure organization were significantly enriched in the SRCC tumors compared to the ITGC ones (Fig. 3 I). Consistent with transcriptome data, we also observed the SRCC signature gene scores elevated in Arid1a loss tumors at protein level (Fig. 3 J). Arid1a deficiency induces Scin downregulation disrupting vesicle secretion To investigate the molecular mechanisms by which Arid1a regulates the specific histology and pathology of SRCC, by analyzing the TCGA-STAD cohort data, we found that patients with ARID1A mutations exhibited considerably reduced expression levels of Scinderin ( SCIN ) 31 , which were pivotal components implicated in exocytosis (Fig. 4 A). Given that ARID1A acts as an epigenetic regulator critical for chromatin remodeling, we conducted a Cleavage Under Targets and Tagmentation (CUT&Tag)-based analysis of the direct targets regulated by ARID1A in TP gastric organoids. The results of CUT&Tag analysis revealed a predilection of ARID1A towards binding to the promoter (33.54%) and distal intergenic (30.53%) regions (Supplementary Fig. 4, A and B and Supplementary Table 7). By analyzing the RNAseq data, we found a significant negative enrichment of genes involved in the GO_ACTIN_FILAMENT_SEVERING pathway in TPA premalignant organoids relative to TP ones, this pathway holds significance for cellular secretion processes (Supplementary Fig. 4C). Further, by integrating the results of CUT&Tag and RNA-seq data, we identified Scin directly regulated by ARID1A (Fig. 4 , B and C). The downregulation of Scin expression levels in Arid1a -deficient tumor organoids was validated by RT-qPCR (Fig. 4 D). These findings demonstrated a regulatory relationship between ARID1A and the downstream targets SCIN . To elucidate the pivotal role of SCIN in the formation of signet ring cells, we utilized CRISPR/Cas9-mediated gene editing to introduce Scin mutation in TP tumor organoids and evaluated their impact on tumor cell morphology (Supplementary Fig. 4D). We noted a marked increase in the vacuolar phenotype and mucin overexpression in TP tumor organoids with Scin mutation (Fig. 4 E). In addition, the proportion of SRCC-like organoids displayed a significant rise in Scin mutant organoids compared to control groups (Fig. 4 F). In the subcutaneous transplantation model, Scin mutation recapitulated the phenotypic features associated with Arid1a loss, as confirmed by histological examination and MUC1 staining (Fig. 4 G). Further, we conducted transmission electron microscopy to examine secretory vesicles in TP and TP-sg Scin tumor cells. A greater number of secretory vesicles were observed in the Scin knockout group, consistent with the signet ring cell phenotype (Fig. 4 H). Moreover, Scin loss significantly increased the proportion of signet ring cells (Fig. 4 I) and accelerated tumor cell growth (Fig. 4 J). To further explore the mechanism of Scin in SRCC formation, we performed RNA-seq analyses to compare TP organoids with and without Scin . Firstly, the loss of Scin in TP tumor organoids significantly downregulated the GO_ACTIN_FILAMENT_SEVERING pathway (Supplementary Fig. 4E). This finding, consistent with the results comparing TPA and TP tumor organoids (Supplementary Fig. 4C), indicates that Scin loss leads to filament organization disorder and mediates exocytosis blockage. And the loss of Scin and Arid1a in TP tumor organoids led to a shared set of upregulated genes, which were significantly enriched in pathways related to mesenchymal cell differentiation, tissue remodeling, cell-substrate adhesion, integrin-mediated signaling, and glycoprotein biosynthesis. In contrast, downregulated genes were associated with protein transport, localization, and secretion (Supplementary Fig. 4, F and G). These transcriptomic findings provide new insights into the role of Scin loss in promoting SRCC tumorigenesis. Arid1a loss activated the Brd9 -containing non-classic BAF complex to enhance mucin production To unravel the underlying mechanisms driving tumorigenicity in SRCC associated with Arid1a mutations, we investigated the role of the mammalian SWI/SNF chromatin remodeling complexes. These complexes exist in three unique final-form assemblies: canonical BAF (cBAF), polybromo containing BAF (pBAF), and a recently characterized non-canonical complex (ncBAF) 32 . Previous studies have demonstrated that residual cBAF subcomplexes resulting from ARID1 scaffolding loss disrupt pBAF function but not ncBAF function in cBAF-perturbed cancers 33 . Based on this, we hypothesized that ncBAF complexes may maintain gene expression and function in ARID1A -loss states and promote the genesis of SRCC. Initially, analyzing the CUT&Tag data of BRD9, a core subunit of ncBAF complexes, which are not present in cBAF complexes, revealed a substantial alteration in BRD9 binding between TP and TPA gastric organoids. Specifically, TPA organoids displayed 1178 and 1467 peaks with significantly elevated and reduced binding levels compared to TP ones, respectively (Fig. 5 A and Supplementary Table 8). The binding was predominantly localized at distal intergenic regions (40.66% in TP and 40.42% in TPA, respectively) and promoter regions (10.95% in TP, 22.97% in TPA, respectively) (Fig. 5 B and Supplementary Table 8). Of particular note, the percentage of BRD9 binding in promoter regions was almost double in Arid1a loss organoids relative to wild type organoids (Fig. 5 B). Intensity curves demonstrated a stronger signal of BRD9 significantly increased binding regions in TPA gastric organoids compared to TP ones (Fig. 5 C). Consistently, these regions related genes that displayed elevated genome accessibility and expression levels in Arid1a loss organoids (Fig. 5 D and Supplementary Fig. 5A). Further functional analysis of the genes with significantly elevated BRD9 binding levels in Arid1a loss organoids revealed a marked enrichment of Gene Ontology terms related to locomotory behavior, stem cell proliferation, activation of MAPK activity and other related biological processes (Fig. 5 E). Interestingly, we observed enhanced BRD9 binding levels at promoter regions of mucin-related genes in TPA organoids compared to TP ones, as determined by examination of the BRD9 CUT&Tag data between Arid1a loss and wild-type gastric organoids (Fig. 5 F). To verify the role of BRD9 in the Arid1a -deficiency-induced elevation of mucinous production and cell proliferation, we are biologically and chemically depleted BRD9 using gRNA and dBRD9 chemical degradation strategies 33,34 . Brd9 knockout resulted in the downregulation of mucin gene expression in TPA tumor organoids (Supplementary Fig. 5, B and C). Through a side-by-side comparison of the response to dBRD9 treatment in TPA and TP tumor organoids in vitro , we found that TPA tumor organoids were more sensitive to dBRD9, while exhibiting reduced sensitivity to 5-Fluorouracil (5-Fu) treatment compared to TP organoids (Fig. 5 G and Supplementary Fig. 5D). And dBRD9 treatment led to significant regression in the histology and morphology of TPA tumor organoids, including reduced structural complexity, cribriform growth, and stratification (Fig. 5 H), but did not affect the TP tumor organoids (Supplementary Fig. 5E). The proportion of SRCC-like organoids in the dBRD9-treated groups was significantly decreased compared to that in control dimethyl sulfoxide (DMSO) groups, revealing a dose-dependent response (Fig. 5 I). And the gradual disappearance of vacuoles within signet ring cell-like organoids over time following treatment with dBRD9 (Supplementary Fig. 5G). Importantly, dBRD9 treatment led to the reversal of the aberrant transcriptional activation of SRCC signature genes in TPA tumor organoids (Fig. 5 J and Supplementary Table 1), including inhibition of mucin gene expression confirmed by RT-qPCR (Fig. 5 K), consistent with the Brd9 knockout results (Supplementary Fig. 5C), but had no influence of TP groups (Supplementary Fig. 5F). RNA-seq analysis revealed the key molecular differences between dBRD9-treated TP and TPA organoids. Specifically, genes such as Clip3 , Arhgef5 , and Exoc2 , which are involved in cytoskeletal organization, cell membrane localization, and exocytosis, were significantly up-regulated only in dBRD9-treated TPA tumor organoids. Conversely, genes like Lcf3f , Cryab , Abcc1 , which are involved in keratinization, stress response, and drug export from the cytoplasm, were significantly up-regulated in dBRD9-treated TP tumor organoids. GO enrichment analysis supports these findings, pathways related to actin filament organization and microtubule cytoskeleton organization were significantly enriched in dBRD9-treated TPA tumor organoids. In contrast, TP tumor organoids showed enrichment in various stress response pathways, including cellular response to glucose starvation, regulation of transcription from RNA polymerase II promoter in response to stress, and response to oxidative stress (Supplementary Fig. 5, H and I). These results indicate that dBRD9 primarily mediates cytoskeletal and microtubule remodeling in TPA tumor organoids to enhance cellular secretion functions, while in TP tumor organoids, it induces a stress response to the treatment. We further evaluated the effect of Arid1a loss on dBRD9 treatment in vivo (Fig. 5 L). TPA tumor organoids were subcutaneously transplanted into recipient mice, followed by daily treatment with either vehicle or dBRD9 (50 mg/kg). dBRD9 treatment significantly inhibited the growth of Arid1a -deficient tumors (Fig. 5 M). Histological analysis using H&E staining revealed that BRD9 inhibition reduced the proportion of signet ring cells compared to the vehicle group (Fig. 5 N). The data presented herein suggest that BRD9 inhibition represents a promising therapeutic strategy for improving the morphological and biological characteristics of SRCC. This finding is particularly significant given that current treatment guidelines for gastric adenocarcinoma lack effective strategies for the management of SRCC. Thus, targeting BRD9 may offer a novel approach to enhance therapeutic outcomes for patients suffering from this challenging subtype of gastric cancer. DISCUSSION Gastric SRCC is a subtype of gastric malignancies and its studies have been impeded by limited animal models. Here, we applied a recently developed strategy with genome-edited organoids to generate primary, orthotopic, and genetic drivers-defined SRCC models in mice. Similar strategies, called as organoid-initiated precision cancer models (OPCMs), have been applied for lung cancer, esophageal cancer, bladder cancer, and colon cancer 23,24,35,36 . Recently, we also developed OPCMs of gastric adenocarcinoma and found that different genetic alterations and microenvironments could significantly affect their progression and metastasis 25 . Compared to traditional genetically engineered mouse models (GEMMs), OPCMs of SRCC and other cancers are very convenient to investigate the functions of any new gene mutation. This advantage makes them of value to dissecting the functions of these thousands of human cancer-associated genes revealed through cancer genomics studies. With this strategy, we found that gastric organoids with Arid1a loss, together with Trp53 and Pten deficiencies, gave rise to classic SRCC both subcutaneously and orthotopically in stomach. These results would not only validate ARID1A , one of the most frequently mutated genes in SRCC, as a bona fide tumor suppressor of gastric SRCC in mice, but also provide a strategy to investigate the functions of other SRCC-associated genes. ARID1A is also frequently disrupted in various other types of human cancers 37–39 . However, its roles and underlying mechanisms in tumorigenesis and progression are complicated. For example, it has the opposite function in the initiation and progression of liver cancer through oxidative stress and chromatin accessibility, respectively 21 . And in liver cholangiocarcinoma, Arid1a loss cooperates with Kras mutation to promote tumorigenesis through repressing the TGF-β pathway 40 , while the YAP pathway is essential for Arid1a deficiency-induced pancreatic ductal adenocarcinoma 41 . Besides, in the urothelium, ARID1A loss caused a transcriptional-translational conflict resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression 42 . Because of the well-characterized roles of the ARID1A -containing SWI/SNF complex in chromosome remodeling and transcription regulation, it is expected that ARID1A loss would lead to a global reduction on chromatin accessibility 21,22,43 , however, accumulating evidence suggests that, at least in some tumors, ARID1A deficiency leads to increased chromatin accessibility, potentially due to the regulation of other chromatin remodeling factors by ARID1A 29,44,45 . Similarly, our studies have also shown that ARID1A knockout upregulates multiple gene expressions and increases chromatin accessibility in the promoter region in gastric cancer. Hence, in our study, we found that the expressions of secreting genes, Scin , directly enhanced and the expressions of mucin genes indirectly repressed in the Arid1a loss-induced SRCC. The mechanism is different from a previous study with ARID1A disruption in TP53 mutated human organoids, which didn’t display typical SRCC pathology 22 . These differences might be due to different genetic background. However, our data indicate that Arid1a loss could promote SRCC through both increasing mucin production and impaired secretion, which provide strong experimental evidence for the long-proposed hypothesis of SRCC formation. Given the prevalence and tumorigenic functions of ARID1A mutations, it has been attractive to explore potential treatment strategies for ARID1A altered tumors. It has been reported that ARID1A inactivated ovarian carcinoma has defects on the mevalonate pathway and thus, is sensitive to statin treatment by itself and together with immune checkpoint blockade 46 . Considering that ARID1A is a member of SWI/SNF complex which of subunits have interaction and mutations resulting in inflammation even tumors 32,47 . Through multi-omics analyses, it is found that ARID1A mutated gastric cancers might be responsive to bromodomain and NFKB inhibitions 48 . In SRCC, we find that Arid1a loss leads to Brd9 -mediated upregulation of mucin genes. Thus, we propose that targeting Brd9 or the non-classic BAF complex might be an effective strategy for SRCC, which is characterized by massive mucins. Our results are also consistent with the observations in several other types of cancers, such as sarcoma 33 , rhabdoid tumor 49 , and liver cancer 50 . It would be interesting to test potential inhibitors of non-classic BAF complex for patients with ARID1A mutations. However, we also notified that ARID1A might have complicated roles in both malignant cells and nonmalignant cells, such as its role in tumor-infiltrating T cells 51 . Thus, any treatment targeting ARID1A deficient tumors should be tested for its potential systematic effect. METHODS Mice . Trp53 −/− mice and Cas9-EGFP mice were purchased from Jackson Laboratories (Cat# 002101 and 026179, RRID: IMSR_JAX:026179, respectively). BALB/cA-nu mice were purchased from Beijing HuaFukang Biological Technology Co. Ltd (6-8-week-old, male). Mouse Organoid Culture . Mouse organoid cultures were established and maintained as described previously 25 . Specifically, normal mice stomach was mechanically minced into pieces of 5-mm 3 cubes and incubated in DPBS with 2.5 mM EDTA on ice. After incubation, the released gastric gland fractions were resuspended with Matrigel (Corning, Cat# 356237) and seeded in 48-wells plate. Tumor tissues were harvested and washed with ice-cold DPBS, and then minced into pieces. Incubating the minced tumor tissue in digestion medium containing 0.5 mg/ml collagenase IV (GIBCO, Cat# 17104-019) and 1.0 mg/ml collagenase I (GIBCO, Cat# 17100-017) in DMEM/F12 medium at 37°C until it dissociates into single cells or cell clusters. Tumor cells were collected by centrifugation and resuspended with Matrigel. Organoid culture medium was DMEM/F12 (GIBCO, Cat# C11330500BT) supplemented with 2 mM GlutaMAX (GIBCO, Cat# 35050-061), 1% Penicillin and Streptomycin (GIBCO, Cat# 15140-122), 1× B27 (GIBCO, Cat# A3582801), 1× N 2 (GIBCO, Cat# 17502048),10 mM Y-27632, 200ng/ml FGF10 (Peprotech, Cat# 100-26-1000), 500nM A-8301 (Peprotech, Cat# 9094360), 50ng/ml mouse recombinant EGF (Peprotech, Cat# AF-100-15-1000), 100 ng/ml mouse recombinant noggin (Peprotech, Cat# 120-10C-250), 10mM Nicotinamide (Sigma, Cat# N0636), 1 mM N-acetylcysteine (Sigma, Cat# A9165), 10% Wnt3a condition medium, 10% R-spondin1 condition medium). Organoid medium was refreshed every 2–3 days. Organoid genome editing . sgRNAs designed on the website of DNA2.0 Gene Design & Synthesis ( https://www.atum.bio/pipeline/dna ) were cloned into the lentiviral constructs U6-sgRNA-EFS-mCherry-P2A-puro. The sequences of gRNAs and primers targeting Pten , Arid1a , Scin and Brd9 are listed in Supplementary Table 2. Lentivirus packaging, infection and verification were done as previously reported 24 . Organoid Subcutaneous and Orthotopic Transplantation . Organoids were collected and aspirated into pre-chilled 29G insulin syringes. The organoid suspension (2 × 10 5 cells per mouse) was directly injected into 6 to 8-week-old male BALB/cA-nu mice subcutaneously or orthotopically. Refer to the previous article for detailed methods 25 . RNA-seq analysis . Transcriptome sequencing was conducted using the Illumina NovaSeq 6000 platform with 150 bp paired-end sequencing. After removing the adapters, ploy-N, and low-quality reads, clean data were aligned against the reference mouse genome (mm10) using STAR _2.6.0. Following this, raw counts were normalized and significance scores. as well as log2-fold change values, were calculated using the DESeq2 (v.1.22.2). The differentially expressed genes were used to perform Gene Ontology enrichment analyses with the R package clusterProfiler (v. 3.10.1) (RRID:SCR_016884). Furthermore, Gene Set Enrichment Analysis (GSEA) was employed to identify significantly enriched pathways. Preranked function was performed to clarify the overall transcriptome similarity between mouse models and tumor tissues of SRCC patients. Data visualization was conducted using the R packages ggpubr, ggplot2, pheatmap, and Vennerable. ATAC-seq analysis . The Illumina NovaSeq 6000 platform was utilized to sequence the library. To remove adapters from the 150 bp paired-end raw data, NGmerge was employed with the option "NGmerge -a -v -n 20". Bowtie2 software was used to construct an index for the mouse genome (mm10), followed by alignments using the option "--very-sensitive -X2000 -x mm10". Duplicates were removed using Picard's MarkDuplicates tool. Mitochondrial genome contamination was eliminated using SAMtools and awk commands, resulting in bam files. Normalized bw files were generated using deepTools with the option "bamCoverage -bs = 1 --normalizeUsing BPM" for subsequent visualization in the Integrated Genome Viewer (IGV). The HMMRATAC standard workflow was utilized for peak calling in ATAC-seq. The genomic distribution of accessibility sites was determined using ChIPseeker with the option "annotatePeak", and the TSS region "tssRegion" was set as (-3000, 3000). The "gappedPeak" files were transformed into GrangesList forms, and the peaks were converted into consensus counts. Data normalization and differential comparison were performed using DESeq2. Regions of differential accessibility were identified for downstream analyses using a p-value cutoff of 0.05 (Wald test). HOMER was used for motif analysis with the “findMotifsGenome.pl” function. CUT&Tag analysis . The Illumina NovaSeq 6000 platform was employed to perform genomic sequencing. Following quality control by the fastp standard workflow, the Bowtie2 software was utilized to construct an index for the mouse genome (mm10) and align paired-end clean data of 150 bp length using the options "--local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700". The SAMtools program was utilized to convert sam files to sorted bam files. Picard's MarkDuplicates tool was used to remove duplicates with the option "REMOVE_DUPLICATES = true". Normalized bw files were generated using deepTools with the option "bamCoverage -bs = 1 --normalizeUsing BPM" for further visualization in the Integrated Genome Viewer (IGV). Peak calling was performed using the MACS2 software with the option "macs2 callpeak --broad -q 1e-5 -f BAMPE -g mm --keep-dup all". ChIPseeker was employed to identify the genomic distribution of the peaks, with the TSS region "tssRegion" set as (-3000, 3000) for annotation. The "narrowPeak" files were transformed into GrangesList forms, and the peaks were converted into consensus counts. Data normalization and differential comparison were performed using DESeq2. Regions of differential modification were identified for downstream analyses using a p -value cutoff of 0.05 (Wald test). Statistical analysis . Organoid diameter and number assays, tumor measurements, RT-qPCR, morphological statistics, and in vitro treatment were analyzed for statistical significance using two-tailed unpaired parametric Student’s t -tests (Prism 9.0, GraphPad software). The data distribution was assumed to be normal, but this was not formally tested. Statistical test methods, sample sizes, and p values are indicated in the corresponding figure legends. For the in vitro treatment experiments, all samples were randomly assigned to vehicle or treatment groups. Tumor measurements were performed blindly. The measurements of organoid shapes were analyzed blindly. For other in vivo and in vitro experiments, the researchers were not blinded while performing the experiments. No data were excluded from the study. Statistical significance on RNA-seq, ATAC-seq and CUT&Tag data was determined using unpaired two-tailed t test, Wilcoxon signed-rank test, Wald test, hypergeometric distribution, or Likelihood-ratio test. Statistical significance was defined as p < 0.05. All the quantification and visualization of omics data analysis were performed on R v3.6. Study approval . All these mice were bred and kept in the SPF animal facility of Sichuan University, and no mouse was excluded in the experiments. All animal experiment protocols were approved by the Animal Care and Use Committee of Sichuan University. Declarations Author Contributions: Conceptualization, C.C.; Methodology: H.L., A.Z., Z.L., M.Z., J.L., Y.W., X.C., X.P., J.C., L.Z., L.G., S.H., X.W., Y.P., T.P., J.W., J.D., X.D., K.Y., K.L., X.C., X.C., Z.F., B.W., L.Z., W.Z. S.Y., Y.W., L.C., Z.X., C.Z., L.D.; Investigation: H.L., A.Z., Z.L.; Resources: L.G., T.P., K.Y., K.L., X.C., X.C., Z.F., L.Z., W.Z., S.Y., Y.W., L.C., Z.X., C.Z., Y.L., F.N.; Writing-Original Draft: H.L., A.Z., Z.L., J.H., Y.L., C.C.; Writing-Review & Editing: H.L., A.Z., Z.L., J.C., J.H., Y.L., C.C.; Funding Acquisition: B.W., Y.L., F.N., Y.L., C.C.; Supervision: F.N., J.H., Y.L., C.C. Competing Interest Statement: The authors disclose no conflicts. Data availability . The RNA-seq, ATAC-seq, and CUT&Tag data in this study are deposited in NCBI GEO: GSE232292. The secure token: mnunqagozjsjzur. Supplemental information More detailed information relevant to this study can be found in the supplementary tables, methods, and figures. Acknowledgments We thank Dr. Yuquan Wei for generous support. We thank all the CC-LY lab members and Fangfang Wang (Institute of hematology, West China Hospital) for discussion and technical support. We thank the Core Facilities of West China Hospital and the Chengdu OrganoidMed Medical Laboratory for technical support. Funding We thank the members of the CC-LY laboratory for their technical support and suggestions. This work was supported by the National Natural Science Foundation of China (2017YFA0505601 and 82130007, Y. Liu.; 82102779, J.C.; T2221004, C.C. and S.Y.), the Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJC2023010004), the 1.3.5. Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC21003, Y. Lu; ZYYC20004, C.C.; ZYGD22012, Y. Liu; ZYJC21009, Y. 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A. et al. Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. Cancer Cell 40 , 768-786 e767, doi:10.1016/j.ccell.2022.06.001 (2022). Additional Declarations There is NO Competing Interest. Supplementary Files 1020SupplementaryFile.docx Table1.xlsx Supplementary Table 1. The alteration frequencies of cancer associated genes, the co-occurrence analysis of ARID1A , TP53 and PTEN in diffuse-type gastric carcinoma and gastric signet ring cell carcinoma samples from the cBioPortal dataset, as well as the SRCC signature gene list, related to Figure 1, Figure 3, Figure 5. Table2.xlsx Supplementary Table 2. The sequences of sgRNAs, PCR and RT-qPCR primer, related to Figure 2, Figure 4, Figure 5, Supplementary Figure 1, Supplementary Figure 4 and Supplementary Figure 5. Table3.xlsx Supplementary Table 3. Differential expression genes in premalignant, tumor organoids with sgScr or sg Arid1a , and tumor organoids treated with DMSO or dBRD9, related to Figure 1-5 and Supplementary Figure 1-5. Table4.xlsx Supplementary Table 4. The expression levels of mucin genes in ITGC and SRCC patients from the TCGA-STAD cohort, the Korea University cohort (GSE26899) and the CCLE database, related to Figure 2 and Supplementary Figure 2. Table5.xlsx Supplementary Table 5. Differential genome accessible regions between TPA and TP gastric organoids, related to Figure 3, Figure 5 and Supplementary Figure 3. Table6.xlsx Supplementary Table 6. The protein mass spectrometry analysis results of SRCC and ITGC gastric tumor tissues, related to Figure 3. Table7.xlsx Supplementary Table 7. ARID1A binding regions in TP gastric organoids, related to Figure 4 and Supplementary Figure 4. Table8.xlsx Supplementary Table 8. Differential BRD9 binding regions between TPA and TP gastric organoids, related to Figure 5. 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China","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Yang","suffix":""},{"id":376324714,"identity":"d46f6225-4afe-4729-848e-bce6e41dcbd2","order_by":20,"name":"Kai Liu","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery and Laboratory of Gastric Cancer, State Key Laboratory of Biotherapy","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Liu","suffix":""},{"id":376324715,"identity":"6c111285-3c7c-4972-9c0a-fdb5c4058eb1","order_by":21,"name":"Xin-Zu Chen","email":"","orcid":"https://orcid.org/0000-0002-7619-6244","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xin-Zu","middleName":"","lastName":"Chen","suffix":""},{"id":376324716,"identity":"76e03e73-d944-46b9-a570-dfafeb04fe89","order_by":22,"name":"Xiaolong Chen","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery and Laboratory of Gastric Cancer, State Key Laboratory of Biotherapy","correspondingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Chen","suffix":""},{"id":376324717,"identity":"6de4354c-c164-443e-acdd-d138897c740a","order_by":23,"name":"Zhe Feng","email":"","orcid":"","institution":"Department of Gastroenterology, West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Feng","suffix":""},{"id":376324718,"identity":"2fd73db2-cad1-453b-9cc0-add3f4d47411","order_by":24,"name":"Baohong Wu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Baohong","middleName":"","lastName":"Wu","suffix":""},{"id":376324719,"identity":"3a1fcbb9-423b-49d9-869e-354cfb368ef1","order_by":25,"name":"Linyong Zhao","email":"","orcid":"","institution":"Collaborative Innovation Center of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China.","correspondingAuthor":false,"prefix":"","firstName":"Linyong","middleName":"","lastName":"Zhao","suffix":""},{"id":376324720,"identity":"0481f540-0f1b-4053-878a-f25285432b4b","order_by":26,"name":"Weihan Zhang","email":"","orcid":"","institution":"West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Weihan","middleName":"","lastName":"Zhang","suffix":""},{"id":376324721,"identity":"6c454cf4-fc0f-40b7-8761-68caeeb2ae0b","order_by":27,"name":"Shengyong Yang","email":"","orcid":"https://orcid.org/0000-0001-5147-3746","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Shengyong","middleName":"","lastName":"Yang","suffix":""},{"id":376324722,"identity":"a440e17a-878b-4a8b-b09b-7898b17fe2bf","order_by":28,"name":"Yuan Wang","email":"","orcid":"https://orcid.org/0000-0002-6324-6134","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Wang","suffix":""},{"id":376324723,"identity":"fbb43ac7-81de-454d-a4a1-962be38d0f36","order_by":29,"name":"Lu Chen","email":"","orcid":"https://orcid.org/0000-0002-1083-9729","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Chen","suffix":""},{"id":376324724,"identity":"66d1c827-90f2-4069-a484-dde7a8329b35","order_by":30,"name":"Chengjian Zhao","email":"","orcid":"","institution":"sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Chengjian","middleName":"","lastName":"Zhao","suffix":""},{"id":376324725,"identity":"a4006787-cebc-4254-9556-fc10a74c3f52","order_by":31,"name":"Zhihong Xue","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Xue","suffix":""},{"id":376324726,"identity":"df09e0ad-cc2a-4bbb-9b01-b0ba089ea4c9","order_by":32,"name":"Lunzhi Dai","email":"","orcid":"https://orcid.org/0000-0002-3003-8910","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Lunzhi","middleName":"","lastName":"Dai","suffix":""},{"id":376324727,"identity":"d061262e-1578-4a9b-9655-99ccd7c150fe","order_by":33,"name":"Feifei Na","email":"","orcid":"","institution":"Lab of Aging Research and Cancer Drug Targets, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Na","suffix":""},{"id":376324728,"identity":"a7109f1e-f3cc-4370-8db0-2168112201f1","order_by":34,"name":"Jiankun Hu","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery, West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jiankun","middleName":"","lastName":"Hu","suffix":""},{"id":376324729,"identity":"1bd4cd0e-3a13-479a-bf23-55ffc7549f51","order_by":35,"name":"Yu Liu","email":"","orcid":"https://orcid.org/0000-0002-1193-7830","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-10-20 13:00:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5298469/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5298469/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68787661,"identity":"9f444b2a-61b1-4e9c-8316-3a032e4fae6d","added_by":"auto","created_at":"2024-11-12 04:31:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1707394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eArid1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency elicits the manifestation of SRCC features both \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA) The OncoPrint (top) showing the variation frequencies (including mutations: truncating, in frame, missense and splice site mutation; copy number variations: shallow deletion and deep deletion) of \u003cem\u003eARID1A\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e in diffuse-type gastric carcinoma and gastric signet ring cell carcinoma samples (n = 171). The co-occurrence analysis (bottom) of \u003cem\u003eARID1A\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e variations in diffuse-type gastric carcinoma and gastric SRCC samples (n = 171). Data were analyzed from cBioPortal public datasets. The significance levels of co-occurrence were calculated by the statistical method Mutual Exclusivity Modules, provided by the cBioPortal.\u003c/p\u003e\n\u003cp\u003eB) Western blotting analysis of ARID1A protein levels in TP and TPA gastric organoids. 1 and 2 represented two different sgRNAs.\u003c/p\u003e\n\u003cp\u003eC) Representative bright-field (top), H\u0026amp;E (middle) and AB-PAS staining (bottom) in TP (\u003cem\u003eTrp53\u003c/em\u003e-/-; sgPt\u003cem\u003een\u003c/em\u003e), TPA (\u003cem\u003eTrp53\u003c/em\u003e-/-; sg\u003cem\u003ePten\u003c/em\u003e; sg\u003cem\u003eArid1a\u003c/em\u003e) mouse gastric organoids. Scale bars, 100μm and 20μm. 1 and 2 represented two different sgRNAs.\u003c/p\u003e\n\u003cp\u003eD) The diameters of TP and TPA gastric organoids. Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-value was calculated by unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003eE) The number of TP and TPA gastric organoids (n = 5). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-value was calculated by unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003eF) The dot plot showing principal component analysis result of RNA-seq data from TP and TPA gastric organoids.\u003c/p\u003e\n\u003cp\u003eG) \u0026nbsp;The volcano map showing differential expression genes between TP and TPA gastric organoids (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 and absolute log\u003csub\u003e2\u003c/sub\u003efold-change \u0026gt; 0.5).\u003c/p\u003e\n\u003cp\u003eH) Gene ontology enrichment results of the TPA up-regulated genes compared with TP gastric organoids (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 and log\u003csub\u003e2\u003c/sub\u003efold-change \u0026gt; 0.5).\u003c/p\u003e\n\u003cp\u003eI) The bright field images of subcutaneously transplanted TP and TPA tumor (n = 3). Scale bar, 500μm.\u003c/p\u003e\n\u003cp\u003eJ) The growth curves of subcutaneously transplanted TP and TPA tumor (n = 3). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et-\u003c/em\u003etest.\u003c/p\u003e\n\u003cp\u003eK) Representative H\u0026amp;E (left), ARID1A (middle) and MUC1 (right) staining in the TP (up) and TPA (bottom) subcutaneous tumors. Scale bar, 20μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/36da91350b3319d63bd1f1d0.png"},{"id":68787659,"identity":"8b275a2e-baa3-4ef2-ae8c-335d66153897","added_by":"auto","created_at":"2024-11-12 04:31:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1597268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eArid1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency markedly promotes the development of gastric SRCC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Survival curve of orthotopically transplanted TP (n = 9), TPA-1 (n = 6) and TPA-2 (n = 6) mice. 1 and 2 represented two different sgRNAs.\u003c/p\u003e\n\u003cp\u003eB) Representative bright-field (top) and red fluorescence (bottom) images of the orthotopically transplanted TP (left), TPA-1 (middle) and TPA-2 (right) tumors. Scale bar, 2mm.\u003c/p\u003e\n\u003cp\u003eC) The stomach weight of orthotopically transplanted TP (n = 6), TPA-1(n = 6) and TPA-2 (n = 4) mice. Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et-\u003c/em\u003etest.\u003c/p\u003e\n\u003cp\u003eD) Western blotting analysis of ARID1A protein levels in TP and TPA tumor organoids.\u003c/p\u003e\n\u003cp\u003eE) Representative H\u0026amp;E staining in the orthotopically transplanted TP and TPA tumors. Scale bar, 20μm.\u003c/p\u003e\n\u003cp\u003eF) Relative expression levels of \u003cem\u003eMuc1\u003c/em\u003e, \u003cem\u003eMuc20\u003c/em\u003e in TP and TPA-1 tumor organoids, measured by RT-qPCR (n = 3). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003eG) Western blotting showing the protein levels of MUC1 and E-Cadherin in TP and TPA tumor organoids.\u003c/p\u003e\n\u003cp\u003eH) Representative AB-PAS staining in the orthotopically transplanted TP and TPA tumors. Scale bar, 20μm.\u003c/p\u003e\n\u003cp\u003eI) GSEA showing the TPA up-regulated (top), and down-regulated (bottom) gene sets compared with TP tumor organoids, significantly enriched in SRCC patients (n = 12) and ITGC patients (n = 74), respectively.\u003c/p\u003e\n\u003cp\u003eJ) The heatmap showing the expression levels of mucin genes between SRCC and ITGC patients in the TCGA-STAD cohort, the Korea university cohort (GSE26899), the CCLE database, TP and TPA tumor organoids.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/57836de1f10fb80bb7878592.png"},{"id":68788470,"identity":"82983ee6-3481-41a5-8b6b-e920782eadff","added_by":"auto","created_at":"2024-11-12 04:47:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":526585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMulti-omics analyses elucidated the molecular characters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArid1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deficiency in both premalignant and malignant gastric epithelial cells, impacting the epigenome, transcriptome, and proteome levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) The volcano plot showing the significant accessible peaks between TP and TPA gastric organoids (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05, absolute log\u003csub\u003e2\u003c/sub\u003efold-change \u0026gt; 0.5 and base-mean \u0026gt; 5).\u003c/p\u003e\n\u003cp\u003eB) The pie charts showing the distribution of TPA significantly highly closed (top) and opened (bottom) peaks in annotated regions of the genome compared with TP gastric organoids.\u003c/p\u003e\n\u003cp\u003eC) Gene ontology enrichment results of genes with significantly high genome accessibility levels in TP and TPA gastric organoids.\u003c/p\u003e\n\u003cp\u003eD) The volcano plot showing differential expression genes between TP and TPA tumor organoids (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 and absolute log\u003csub\u003e2\u003c/sub\u003efold-change \u0026gt; 0.5).\u003c/p\u003e\n\u003cp\u003eE) Gene ontology enrichment results of genes with significantly high expression levels in TPA tumor organoids compared with TP ones.\u003c/p\u003e\n\u003cp\u003eF) The box plot showing the SRCC signature score levels between TP and TPA tumor organoids in RNA-seq data. Likelihood-ratio test was performed to determine the significant level.\u003c/p\u003e\n\u003cp\u003eG) Integrative Genomics Viewer showing the genome accessibility and expression levels of mucin related gene locus between TP and TPA organoids.\u003c/p\u003e\n\u003cp\u003eH) The heatmap showing genes with up-regulated protein levels in SRCC tumors compared with ITGC ones, detected by MS proteomics. Crucial proteins are labeled on the right.\u003c/p\u003e\n\u003cp\u003eI) Gene ontology enrichments of the upregulated genes in SRCC tumors compared with ITGC tumors, measured by MS proteomics assay.\u003c/p\u003e\n\u003cp\u003eJ) The box plot showing the SRCC signature score levels between ITGC and SRCC tumors in MS proteomics data. Likelihood-ratio test was performed to determine the significant level.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/f25b2a10096da20361e9a7ac.png"},{"id":68788269,"identity":"d4709835-b679-418f-9219-5724dafa5ae9","added_by":"auto","created_at":"2024-11-12 04:39:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1641790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eArid1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e suppresses SRCC by regulating the expression of secretory genes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eScin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) \u0026nbsp;Normalized expression levels of \u003cem\u003eSCIN \u003c/em\u003e\u0026nbsp;in \u003cem\u003eARID1A\u003c/em\u003e wild-type (n = 285) and mutant (n = 90) GC patients in the TCGA-STAD cohort. \u003cem\u003eP\u003c/em\u003e-values were determined by the Wald test.\u003c/p\u003e\n\u003cp\u003eB) The Venn diagram showing the overlap of ARID1A binding genes and down-regulated genes in TPA gastric organoids compared with TP ones. \u003cem\u003eP\u003c/em\u003e values was determined by hypergeometric distribution.\u003c/p\u003e\n\u003cp\u003eC) Integrative Genomics Viewer showing the ARID1A binding levels in TP gastric organoids on \u003cem\u003eScin \u003c/em\u003elocus.\u003c/p\u003e\n\u003cp\u003eD) The relative mRNA levels of \u003cem\u003eScin\u003c/em\u003e in TP (n = 2) or TPA (n = 3) tumor organoids, measured by RT-qPCR. Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et\u003c/em\u003e-test with Welch’s correction.\u003c/p\u003e\n\u003cp\u003eE) Representative bright-field (top), H\u0026amp;E (middle) and AB-PAS staining (bottom) in TP tumor-sgScr, TP tumor-sg\u003cem\u003eScin\u003c/em\u003e-1, TP tumor-sg\u003cem\u003eScin\u003c/em\u003e-2 organoids. Scale bar, 20μm. 1 and 2 represented two different sgRNAs.\u003c/p\u003e\n\u003cp\u003eF) The percentage of SRCC-like organoids in TP tumor-sgScr, TP tumor-sg\u003cem\u003eScin\u003c/em\u003e-1, TP tumor-sg\u003cem\u003eScin\u003c/em\u003e-2 (n = 5). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003eG) Representative bright-field (top), H\u0026amp;E (middle) and MUC1 (bottom) staining of the subcutaneous TP tumor-sgScr, TP tumor-sg\u003cem\u003eScin\u003c/em\u003e tissues. Scale bars, 1mm (top), 20μm (middle) and 20μm (bottom).\u003c/p\u003e\n\u003cp\u003eH) The transmission electron microscopy images of TP tumor tissues with sgScr or sg\u003cem\u003eScin\u003c/em\u003e. Scale bar, 1mm.\u003c/p\u003e\n\u003cp\u003eI) The percentage of signet ring cells in subcutaneous TP tumor tissues with sgScr, sg\u003cem\u003eScin\u003c/em\u003e-1, or sg\u003cem\u003eScin\u003c/em\u003e-2 (n = 3). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003eJ) The growth curves of the subcutaneous TP tumors with sgScr, sg\u003cem\u003eScin\u003c/em\u003e-1, or sg\u003cem\u003eScin\u003c/em\u003e-2 (n = 3). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-values were calculated by unpaired \u003cem\u003et-\u003c/em\u003etest.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/5a6bf90621f4eba446f17938.png"},{"id":68788270,"identity":"8f480828-2f14-40ae-a777-0e52d82841bd","added_by":"auto","created_at":"2024-11-12 04:39:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1010729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe loss of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArid1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e led to the activation of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBrd9\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-containing non-classic BAF complex, increasing mucin production.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Tornado plots showing the differential binding levels of BRD9 in TP and TPA gastric organoids (\u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 and absolute log\u003csub\u003e2\u003c/sub\u003efold change \u0026gt; 0.5).\u003c/p\u003e\n\u003cp\u003eB) The pie charts showing the distribution of significantly decreased (top) and increased (bottom) binding peaks of BRD9 in annotated regions of the genome in TPA gastric organoids compared to TP ones.\u003c/p\u003e\n\u003cp\u003eC) Intensity curves showing the average signal levels of BRD9 significantly increased binding regions between TP and TPA gastric organoids.\u003c/p\u003e\n\u003cp\u003eD) The box plot showing the genome accessibility levels of genes with significantly increased BRD9 binding levels in TPA organoids compared to TP ones.\u003c/p\u003e\n\u003cp\u003eE) The bar plot showing Gene ontology enrichment results of genes with significantly increased BRD9 binding levels in TPA organoids compared to TP ones.\u003c/p\u003e\n\u003cp\u003eF) Integrative Genomics Viewer showing the BRD9 binding levels in TP and TPA gastric organoids on mucin related gene locus.\u003c/p\u003e\n\u003cp\u003eG) Dose-response curves of TP and TPA tumor organoids treated with dBRD9.\u003c/p\u003e\n\u003cp\u003eH) Representative H\u0026amp;E images of TP and TPA tumor organoids treated with DMSO or 10μM dBRD9. Scale bar, 20μm.\u003c/p\u003e\n\u003cp\u003eI) The percentage of SRCC-like organoids in TPA tumor organoids treated with DMSO, 1μM, 10μM, and 100μM dBRD9 (n = 3).\u003c/p\u003e\n\u003cp\u003eJ) The box plots showing the SRCC signature gene scores in DMSO and dBRD9 treated TPA tumor organoids. Likelihood-ratio test was performed to determine the significant level.\u003c/p\u003e\n\u003cp\u003eK) The relative mRNA levels of \u003cem\u003eMuc1\u003c/em\u003e, \u003cem\u003eMuc5ac\u003c/em\u003e, and \u003cem\u003eMuc20\u003c/em\u003e in TPA tumor organoids treated with DMSO or 10μM dBRD9, measured by RT-qPCR (n = 3). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-value was calculated by unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e\n\u003cp\u003e(L) Schematic diagram of the strategy for dBRD9 treatment in TPA tumor-bearing mice.\u003c/p\u003e\n\u003cp\u003e(M) The curves showing the volume of TPA subcutaneous tumor treated with vehicle or dBRD9 (n = 4). Data presented as the means ± the SEM, \u003cem\u003ep\u003c/em\u003e-value was calculated by two-tailed Mann Whitney test.\u003c/p\u003e\n\u003cp\u003e(N) Representative H\u0026amp;E image of mouse TPA tumors treated with dBRD9 and Vehicle. Scale bar, 20μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/13df66cf2e11b1e8edd61873.png"},{"id":72045945,"identity":"b8774c86-b971-4f07-bb6e-0bcbb7d57588","added_by":"auto","created_at":"2024-12-21 03:58:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8576138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/9ace0e82-d339-4f86-b7b8-8d071c0462ae.pdf"},{"id":68787672,"identity":"7ed8c70c-be46-4505-96fe-db82ab63da3e","added_by":"auto","created_at":"2024-11-12 04:31:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3933995,"visible":true,"origin":"","legend":"","description":"","filename":"1020SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/4e4ce3b63ff4f67251b957dc.docx"},{"id":68787664,"identity":"cbeb9215-bb57-4c23-a8ff-a26fbedbed97","added_by":"auto","created_at":"2024-11-12 04:31:58","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":68256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe alteration frequencies of cancer associated genes, the co-occurrence analysis of \u003cem\u003eARID1A\u003c/em\u003e, \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003ePTEN\u003c/em\u003e in diffuse-type gastric carcinoma and gastric signet ring cell carcinoma samples from the cBioPortal dataset, as well as the SRCC signature gene list, related to Figure 1, Figure 3, Figure 5.\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/7880f648a24e406d8f18f797.xlsx"},{"id":68787660,"identity":"754ef33c-eaf0-4935-a294-1dddc2a21a4f","added_by":"auto","created_at":"2024-11-12 04:31:57","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences of sgRNAs, PCR and RT-qPCR primer, related to Figure 2, Figure 4, Figure 5, Supplementary Figure 1, Supplementary Figure 4 and Supplementary Figure 5.\u003c/p\u003e","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/a807efde8b3f3e6ef26b292e.xlsx"},{"id":68787663,"identity":"97d93bb5-3fab-4fca-9ffb-9f23d5d8b3ca","added_by":"auto","created_at":"2024-11-12 04:31:58","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":955962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential expression genes in premalignant, tumor organoids with sgScr or sg\u003cem\u003eArid1a\u003c/em\u003e, and tumor organoids treated with DMSO or dBRD9, related to Figure 1-5 and Supplementary Figure 1-5.\u003c/p\u003e","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/53a074f00a07e0b20daf2f4e.xlsx"},{"id":68787667,"identity":"cb7bdc9c-d881-4e27-ac84-b1598afb789b","added_by":"auto","created_at":"2024-11-12 04:31:58","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":40824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of mucin genes in ITGC and SRCC patients from the TCGA-STAD cohort, the Korea University cohort (GSE26899) and the CCLE database, related to Figure 2 and Supplementary Figure 2.\u003c/p\u003e","description":"","filename":"Table4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/7c092500d4f9eb50e3e54200.xlsx"},{"id":68787673,"identity":"d82fbb0c-92fb-48a0-80ab-9d9a2a5382f4","added_by":"auto","created_at":"2024-11-12 04:31:58","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4117690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential genome accessible regions between TPA and TP gastric organoids, related to Figure 3, Figure 5 and Supplementary Figure 3.\u003c/p\u003e","description":"","filename":"Table5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/9ca3b525861c0e6c78401b01.xlsx"},{"id":68788471,"identity":"8185f1a9-7a23-4e12-9700-6b5b9d7cafd4","added_by":"auto","created_at":"2024-11-12 04:47:58","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":583030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein mass spectrometry analysis results of SRCC and ITGC gastric tumor tissues, related to Figure 3.\u003c/p\u003e","description":"","filename":"Table6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/4cfb21e61c3bec15609364e2.xlsx"},{"id":68788272,"identity":"cf6b2c2b-2ce4-48f3-8f6a-2812b277221c","added_by":"auto","created_at":"2024-11-12 04:39:58","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":3914026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 7.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eARID1A binding regions in TP gastric organoids, related to Figure 4 and Supplementary Figure 4.\u003c/p\u003e","description":"","filename":"Table7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/9a667f672cb73ff358642f5b.xlsx"},{"id":68787670,"identity":"12283387-564b-478d-82d3-ebc9d11017ed","added_by":"auto","created_at":"2024-11-12 04:31:58","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":670177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 8.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential BRD9 binding regions between TPA and TP gastric organoids, related to Figure 5.\u003c/p\u003e","description":"","filename":"Table8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5298469/v1/53cdb1da5d9ac04ba3dcb30b.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSignet ring cell carcinoma (SRCC) is a histologically unique form of highly malignant adenocarcinoma, characterized by a large vacuole and the resulting cell periphery-localized nucleus, resembling a signet ring\u003csup\u003e1,2\u003c/sup\u003e, The most common SRCC tumors are found in gastric cancers and less frequently in colorectal cancers and others\u003csup\u003e3\u0026ndash;5\u003c/sup\u003e. Gastric SRCC accounts for about 10% of total gastric cancers with increased frequencies in recent years and is associated with poor prognosis\u003csup\u003e6,7\u003c/sup\u003e. It is believed that this special histologic appearance is due to large amounts of mucins produced by SRCC cells and postulated their deficient ability to export them out of the cells\u003csup\u003e8,9\u003c/sup\u003e. However, the molecular mechanisms underlying these malignancy-associated abnormalities remain unclear.\u003c/p\u003e \u003cp\u003eThe recent progress in cancer genomics has revealed the genomic alterations in SRCC\u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. Besides \u003cem\u003eTP53\u003c/em\u003e, the most mutated gene in both SRCC and non-SRCC tumors, \u003cem\u003eARID1A\u003c/em\u003e and \u003cem\u003eCDH1\u003c/em\u003e are especially highly frequently mutated in SRCC. Other alterations, such as CLDN18-ARHGAP26/6 fusions, are also found in about 20% of SRCC patients\u003csup\u003e10,13,14\u003c/sup\u003e. \u003cem\u003eCDH1\u003c/em\u003e encodes the cell-cell adhesion molecular E-cadherin, and its heterozygous loss significantly promoted the formation of SRCC in mice treated with N-methyl-N-nitrosourea (MNU), while had less effect on tubular adenoma tumorigenesis\u003csup\u003e15\u003c/sup\u003e. Similarly, human gastric organoids with \u003cem\u003eCDH1\u003c/em\u003e knockout also displayed morphological changes similar to SRCC\u003csup\u003e16,17\u003c/sup\u003e. It has been suggested that downregulation of the Wnt signaling facilitated the SRCC histology driven by \u003cem\u003eCDH1\u003c/em\u003e mutations. \u003cem\u003eCLDN18\u003c/em\u003e is a tight junction gene and \u003cem\u003eARHGA26/6\u003c/em\u003e are RHOA inhibitors and the CLDH18-ARHGAP26 fusion seemed to enhance the migration and chemoresistance of gastric cancer cells, which were consistent with the clinical features of SRCC patients\u003csup\u003e10\u003c/sup\u003e. The potential functions of other SRCC-associated genes, including \u003cem\u003eARID1A\u003c/em\u003e, are less understood.\u003c/p\u003e \u003cp\u003e \u003cem\u003eARID1A\u003c/em\u003e, the core member of the SWI/SNF chromatin remodeling complex, is frequently mutated in various cancers, including ovarian clear cell carcinomas (OCC) (50%), uterine endometrioid carcinomas (29%), and gastric cancer (29%)\u003csup\u003e18,19\u003c/sup\u003e. \u003cem\u003eArid1a\u003c/em\u003e loss itself gave rise to invasive colon adenocarcinoma in mice, while, intriguingly, repressed the tumorigenesis in the small intestine of the \u003cem\u003eApc\u003c/em\u003e\u003csup\u003emin\u003c/sup\u003e mice\u003csup\u003e20\u003c/sup\u003e. And in liver cancer, \u003cem\u003eARID1A\u003c/em\u003e promoted cancer initiation but restrained the progression and metastasis at later stage\u003csup\u003e21\u003c/sup\u003e. \u003cem\u003eARID1A\u003c/em\u003e knockout could also transform human gastric organoids\u003csup\u003e22\u003c/sup\u003e. However, whether and how \u003cem\u003eARID1A\u003c/em\u003e deficiency plays functional roles in SRCC, especially related to its characteristic morphology need to be investigated.\u003c/p\u003e \u003cp\u003eIn this study, we developed a strategy to generate primary SRCC mouse models with genome-edited gastric organoids. We found that \u003cem\u003eArid1a\u003c/em\u003e loss promoted the formation of SRCC through both indirectly enhancing the expressions of mucin genes and directly impairing the secreting pathway.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eArid1a\u003c/b\u003e \u003cb\u003edeficient gastric organoids gave rise to SRCC\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe performed a genetic analysis of 171 samples of diffuse-type gastric carcinoma (DGC) and gastric signet ring cell carcinoma (SRCC) obtained from the cBioPortal database and found that the incorporation of shallow deletions escalates the frequency of \u003cem\u003eARID1A\u003c/em\u003e alterations to 39% (66/171). Notably, the analysis reveals that \u003cem\u003eARID1A\u003c/em\u003e variation frequently co-occur with \u003cem\u003eTP53\u003c/em\u003e (50%, 86/171) and \u003cem\u003ePTEN\u003c/em\u003e (18%, 30/171) variations in these patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Table\u0026nbsp;1). Consequently, we employed a gene-editing technique in organoids to establish an orthotopic SRCC model and investigate the carcinogenic role of \u003cem\u003eArid1a\u003c/em\u003e, in conjunction with \u003cem\u003eTrp53\u003c/em\u003e and \u003cem\u003ePten\u003c/em\u003e deletion, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInitially, we cultured gastric organoids from adult \u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e; Cas9-EGFP mice. Subsequently, we delivered sgRNAs targeting \u003cem\u003ePten\u003c/em\u003e and \u003cem\u003eArid1a\u003c/em\u003e, fused with mCherry, into these organoids (Supplementary Table\u0026nbsp;2) \u003csup\u003e23,24\u003c/sup\u003e. The utilization of mCherry allowed for the facile identification of tumors originating from these organoids. Following this, we confirmed the mutations of \u003cem\u003ePten\u003c/em\u003e and \u003cem\u003eArid1a\u003c/em\u003e via a T7 endonuclease I mismatch detection assay (Supplementary Fig.\u0026nbsp;1A and Supplementary Table\u0026nbsp;2). The loss of ARID1A was confirmed in gastric organoids, as shown by Western blots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Morphologically, TPA (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; sg\u003cem\u003ePten\u003c/em\u003e; sg\u003cem\u003eArid1a\u003c/em\u003e) organoids exhibited greater hollow features, and the cavities of organoids were filled with mucin, which was identified via Alcian Blue PAS (AB-PAS) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, compared to those originating from TP (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; sg\u003cem\u003ePten\u003c/em\u003e) organoids, TPA organoids displayed a significantly increased size and proliferation capacity, accompanied by organoid morphological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, D and E).\u003c/p\u003e \u003cp\u003eIn order to attain a more comprehensive understanding of the effects resulting from the absence of \u003cem\u003eArid1a\u003c/em\u003e in the early stages of tumorigenesis, we conducted RNA sequencing (RNA-seq) analysis to profile the transcriptomic differences of gastric organoids with or without \u003cem\u003eArid1a\u003c/em\u003e deficiency. As anticipated, \u003cem\u003eArid1a\u003c/em\u003e deficiency elicited extensive changes in gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Surprisingly, TPA organoids demonstrated a significantly greater number of upregulated genes in comparison to TP organoids (600 upregulated genes versus 335 downregulated genes, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and absolute log\u003csub\u003e2\u003c/sub\u003e-fold change\u0026thinsp;\u0026gt;\u0026thinsp;0.5, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and Supplementary Table\u0026nbsp;3). Gene Ontology (GO) analysis indicated that the upregulated genes in the \u003cem\u003eArid1a\u003c/em\u003e-deficient group were significantly enriched in pathways relating to epithelial cell proliferation, extracellular structure organization, and the negative regulation of the Wnt signaling pathway, which may contribute to the characteristic pathological features of SRCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eMoreover, we conducted subcutaneous xenograft assays to test the function of \u003cem\u003eArid1a in vivo\u003c/em\u003e. \u003cem\u003eArid1a\u003c/em\u003e loss dramatically accelerated tumor growth in the subcutaneous locus, whereas the control group demonstrated a meager growth advantage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, I and J and Supplementary Fig.\u0026nbsp;1B). Pathological analysis revealed that the TPA tumors exhibited typical morphological features of SRCC, characterized by cytoplasmic filling with mucin and nuclei compression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Furthermore, IHC staining confirmed the loss of ARID1A and overexpression of MUC1 in the TPA tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). In contrast, the TP group showed generalized hyperplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). A similar association between the loss of \u003cem\u003eArid1a\u003c/em\u003e and SRCC characteristics, along with increased organoid size, proliferation capacity, was observed in \u003cem\u003eArid1a\u003c/em\u003e-loss gastric organoids (Supplementary Fig.\u0026nbsp;1, C and E). Thus, our findings strongly suggest that the absence of \u003cem\u003eArid1a\u003c/em\u003e fosters the development of SRCC both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e in mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOrthotopic gastric SRCC with\u003c/b\u003e \u003cb\u003eArid1a\u003c/b\u003e \u003cb\u003eloss displayed massive mucin production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSubsequently, we induced the formation of primary and orthotopic SRCC by transplanting TPA gastric organoids into the submucosa of the recipient's stomach \u003csup\u003e25,26\u003c/sup\u003e. Recipient mice were monitored weekly and sacrificed once their body weights reduced to around 80% of the original level. Few of the mice with TP organoids developed tumors, and only one recipient mouse was shown to have colonized cells in the stomach after being sacrified. However, mice transplanted with TPA organoids developed gastric tumors. Notably, mice transplanted with TPA organoids much faster with shorter overall survival rates in comparison to the control recipient mice (TPA-1: median 67 days after transplantation; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.0E-4 and TPA-2: median 142 days after transplantation; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.4E-3, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Biopsy dissection confirmed that the TPA gastric organoids formed a single lesion in the stomach with specific mCherry expression, indicating their origin from the transplanted organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). \u003cem\u003eArid1a\u003c/em\u003e deficiency significantly enhanced tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the harvested gastric tumor cells from moribund mice presented a dramatic downregulation of ARID1A in the TPA group compared with the TP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Histologic analyses revealed that the lesions were mostly composed of tumor cells with prominent cytoplasmic vacuoles and a crescent-shaped nucleus eccentrically placed, suggesting that these tumors were typical SRCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Supplementary Fig.\u0026nbsp;2A). Meanwhile, we quantified the proportion of signet ring cells in each xenograft model and organoid culture and found that tumor tissues and primary SRCC tumor organoids demonstrated a higher proportion of signet ring cells (Supplementary Fig.\u0026nbsp;2B). To analyze which one or kinds of mucin genes work on it, we performed qPCR and observed a significant upregulation of mucin genes, including \u003cem\u003eMuc1\u003c/em\u003e and \u003cem\u003eMuc20\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), and a consistent increase in MUC1 expression in \u003cem\u003eArid1a\u003c/em\u003e-deficient tumor cells. However, there was little difference in E-cadherin levels between TP and TPA tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). This observation contrasts with previous reports \u003csup\u003e27,28\u003c/sup\u003e and may indicate the involvement of a distinct mechanism. Furthermore, AB-PAS staining revealed mucin enrichment in the cytoplasm of most tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Additionally, TPA tumors displayed a few metastatic competences at distal sites, while no detectable metastases were observed in TP tumors (Supplementary Fig.\u0026nbsp;2, C and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon analysis of the transcriptome data of TP and TPA tumor organoids, distinct clusters were observed based on principal component analysis (Supplementary Fig.\u0026nbsp;2E). GSEA revealed that the upregulated and downregulated genes in TPA tumors, compared to the TP ones, were significantly positively and negatively enriched in SRCC and intestinal type gastric cancer (ITGC) patients, respectively (NES\u0026thinsp;=\u0026thinsp;2.05, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00, and NES = -1.32, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Notably, TPA mice displayed comparable pathway enrichments to SRCC patients. We analyzed the molecular pathways significantly enriched in mouse TPA tumors and human SRCC, compared to mouse TP tumors and human ITGC, respectively. And the results showed that both collagen, metastasis, hypoxia, extracellular matrix associated pathways were upregulated, and DNA replication, ribosome, chromatid cohesion was downregulated in human SRCC and mice TPA (Supplementary Fig.\u0026nbsp;2, F and G). Moreover, a series of mucin genes were upregulated in SRCC patients relative to ITGC ones in multiple clinical cohorts and human cell lines, and this characteristic phenotype was also observed in our SRCC mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and Supplementary Table\u0026nbsp;4). Collectively, these findings suggest that the orthotopic SRCC model accurately recapitulates the histological, pathological, and molecular features of the human SRCC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMulti-omics analyses revealed the molecular consequences of\u003c/b\u003e \u003cb\u003eARID1A\u003c/b\u003e \u003cb\u003eloss in premalignant and malignant gastric epithelial cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eConsidering the role of \u003cem\u003eARID1A\u003c/em\u003e as a chromatin remodeler, we implemented Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) on TP and TPA gastric organoids to unravel epigenetic patterns and gain insight into the alterations occurring in \u003cem\u003eArid1a\u003c/em\u003e-deficient premalignant organoids. ATAC-seq analysis demonstrated that the absence of \u003cem\u003eArid1a\u003c/em\u003e caused widespread changes in the epigenome, resulting in significantly greater chromatin accessibility in TPA premalignant organoids relative to TP organoids (17474 peaks up versus 533 peaks down, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, absolute log\u003csub\u003e2\u003c/sub\u003e-fold change\u0026thinsp;\u0026gt;\u0026thinsp;0.5 and base-mean\u0026thinsp;\u0026gt;\u0026thinsp;5, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;3A and Supplementary Table\u0026nbsp;5), which was in line with transcriptome results. This result seemed paradoxical to the direct function of \u003cem\u003eARID1A\u003c/em\u003e on chromatin remodeling, which, together with similar previous reports on endometriosis and pancreatic ductal adenocarcinoma (PDAC)\u003csup\u003e29,30\u003c/sup\u003e, suggested potential complicate effect of \u003cem\u003eARID1A\u003c/em\u003e on chromosome remodeling through both direct and indirect regulations. And the highly accessible peaks in TPA organoids predominantly localized to promoter regions (53.14%), followed by distal intergenic regions (23.64%) and introns (12.45%), which differed from TP groups (62.29% distal intergenic, 24.95% intron, and 5.07% exon regions, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;3B and Supplementary Table\u0026nbsp;5). Gene Ontology (GO) analyses suggested that TPA highly accessible genes were enriched in pathways relevant to extracellular matrix organization, cell-cell junction organization, regulation of epithelial mesenchymal transition, regulation of epithelial cell proliferation, actin filament organization, DNA repair and proteasomal protein catabolic process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, motif analysis revealed that transcription factors such as \u003cem\u003eFra2\u003c/em\u003e, \u003cem\u003eFosl2\u003c/em\u003e, \u003cem\u003eJunb\u003c/em\u003e, and \u003cem\u003eAtf3\u003c/em\u003e were highly enriched in TPA highly accessible sites, underscoring their potential role in SRCC development (Supplementary Fig.\u0026nbsp;3C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn terms of transcription level, TPA tumor organoids exhibited a much higher number of upregulated genes compared to the TP groups (779 downregulated genes versus 1545 upregulated genes, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and absolute log\u003csub\u003e2\u003c/sub\u003e-fold change\u0026thinsp;\u0026gt;\u0026thinsp;0.5, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Supplementary Table\u0026nbsp;3), which was consistent with the hyper-activation of genome accessibility and gene expression in \u003cem\u003eArid1a\u003c/em\u003e-loss organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplementary Table\u0026nbsp;3). GO analysis revealed that TPA downregulated genes were significantly enriched in protein acetylation, cellular protein localization, and the Wnt signaling pathway biological process (Supplementary Fig.\u0026nbsp;3D), while TPA upregulated genes were significantly enriched in cytokine production, extracellular matrix organization, ERK1 and ERK2 cascade, collagen fibril organization process, and mesenchyme development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Through a comparative analysis of the transcriptome data from patients with SRCC and ITGC in the TCGA-STAD cohort, we identified a distinct set of genes that exhibit significantly higher expression levels in SRCC patients (Supplementary Table\u0026nbsp;1). This specific set of genes has been denoted as the SRCC signature. Most remarkably, we observed a significant upregulation of SRCC signature gene scores in TPA tumor organoids compared to TP ones based on RNA-seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Notably, a cluster of mucin genes, including \u003cem\u003eMuc1\u003c/em\u003e, \u003cem\u003eMuc5ac\u003c/em\u003e, and \u003cem\u003eMuc20\u003c/em\u003e, exhibited higher genome accessibility and expression levels in \u003cem\u003eArid1a\u003c/em\u003e deficient organoids compared to wildtype ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eAt the protein level, mass spectrometry-based (MS) proteomics analyses of SRCC (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e; sg\u003cem\u003ePten\u003c/em\u003e; sg\u003cem\u003eArid1a\u003c/em\u003e) and ITGC (\u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e; \u003cem\u003eMyc\u003c/em\u003e; sg\u003cem\u003ePten\u003c/em\u003e and sg\u003cem\u003eCdkn2b\u003c/em\u003e), which from our reports previously\u003csup\u003e25\u003c/sup\u003e, tumor tissues revealed the downregulation of ARID1A and other subunit composition of BAF complex in \u003cem\u003eArid1a\u003c/em\u003e knockout tumors (Supplementary Fig.\u0026nbsp;3E). While a series of extracellular matrix-associated proteins, including MSLN, COL17A1, ITGA2/3/6, and ITGB6 were significantly upregulated in \u003cem\u003eArid1a\u003c/em\u003e loss SRCC tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and Supplementary Table\u0026nbsp;6). Multiple pathways related to cell adhesion and extracellular structure organization were significantly enriched in the SRCC tumors compared to the ITGC ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Consistent with transcriptome data, we also observed the SRCC signature gene scores elevated in \u003cem\u003eArid1a\u003c/em\u003e loss tumors at protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003cb\u003eArid1a\u003c/b\u003e \u003cb\u003edeficiency induces\u003c/b\u003e \u003cb\u003eScin\u003c/b\u003e \u003cb\u003edownregulation disrupting vesicle secretion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the molecular mechanisms by which \u003cem\u003eArid1a\u003c/em\u003e regulates the specific histology and pathology of SRCC, by analyzing the TCGA-STAD cohort data, we found that patients with \u003cem\u003eARID1A\u003c/em\u003e mutations exhibited considerably reduced expression levels of Scinderin (\u003cem\u003eSCIN\u003c/em\u003e)\u003csup\u003e31\u003c/sup\u003e, which were pivotal components implicated in exocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Given that \u003cem\u003eARID1A\u003c/em\u003e acts as an epigenetic regulator critical for chromatin remodeling, we conducted a Cleavage Under Targets and Tagmentation (CUT\u0026amp;Tag)-based analysis of the direct targets regulated by ARID1A in TP gastric organoids. The results of CUT\u0026amp;Tag analysis revealed a predilection of ARID1A towards binding to the promoter (33.54%) and distal intergenic (30.53%) regions (Supplementary Fig.\u0026nbsp;4, A and B and Supplementary Table\u0026nbsp;7). By analyzing the RNAseq data, we found a significant negative enrichment of genes involved in the GO_ACTIN_FILAMENT_SEVERING pathway in TPA premalignant organoids relative to TP ones, this pathway holds significance for cellular secretion processes (Supplementary Fig.\u0026nbsp;4C). Further, by integrating the results of CUT\u0026amp;Tag and RNA-seq data, we identified \u003cem\u003eScin\u003c/em\u003e directly regulated by ARID1A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B and C). The downregulation of \u003cem\u003eScin\u003c/em\u003e expression levels in \u003cem\u003eArid1a\u003c/em\u003e-deficient tumor organoids was validated by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings demonstrated a regulatory relationship between \u003cem\u003eARID1A\u003c/em\u003e and the downstream targets \u003cem\u003eSCIN\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the pivotal role of \u003cem\u003eSCIN\u003c/em\u003e in the formation of signet ring cells, we utilized CRISPR/Cas9-mediated gene editing to introduce \u003cem\u003eScin\u003c/em\u003e mutation in TP tumor organoids and evaluated their impact on tumor cell morphology (Supplementary Fig.\u0026nbsp;4D). We noted a marked increase in the vacuolar phenotype and mucin overexpression in TP tumor organoids with \u003cem\u003eScin\u003c/em\u003e mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In addition, the proportion of SRCC-like organoids displayed a significant rise in \u003cem\u003eScin\u003c/em\u003e mutant organoids compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In the subcutaneous transplantation model, \u003cem\u003eScin\u003c/em\u003e mutation recapitulated the phenotypic features associated with \u003cem\u003eArid1a\u003c/em\u003e loss, as confirmed by histological examination and MUC1 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Further, we conducted transmission electron microscopy to examine secretory vesicles in TP and TP-sg\u003cem\u003eScin\u003c/em\u003e tumor cells. A greater number of secretory vesicles were observed in the \u003cem\u003eScin\u003c/em\u003e knockout group, consistent with the signet ring cell phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Moreover, \u003cem\u003eScin\u003c/em\u003e loss significantly increased the proportion of signet ring cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI) and accelerated tumor cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). To further explore the mechanism of \u003cem\u003eScin\u003c/em\u003e in SRCC formation, we performed RNA-seq analyses to compare TP organoids with and without \u003cem\u003eScin\u003c/em\u003e. Firstly, the loss of \u003cem\u003eScin\u003c/em\u003e in TP tumor organoids significantly downregulated the GO_ACTIN_FILAMENT_SEVERING pathway (Supplementary Fig.\u0026nbsp;4E). This finding, consistent with the results comparing TPA and TP tumor organoids (Supplementary Fig.\u0026nbsp;4C), indicates that \u003cem\u003eScin\u003c/em\u003e loss leads to filament organization disorder and mediates exocytosis blockage. And the loss of \u003cem\u003eScin\u003c/em\u003e and \u003cem\u003eArid1a\u003c/em\u003e in TP tumor organoids led to a shared set of upregulated genes, which were significantly enriched in pathways related to mesenchymal cell differentiation, tissue remodeling, cell-substrate adhesion, integrin-mediated signaling, and glycoprotein biosynthesis. In contrast, downregulated genes were associated with protein transport, localization, and secretion (Supplementary Fig.\u0026nbsp;4, F and G). These transcriptomic findings provide new insights into the role of \u003cem\u003eScin\u003c/em\u003e loss in promoting SRCC tumorigenesis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eArid1a\u003c/b\u003e \u003cb\u003eloss activated the\u003c/b\u003e \u003cb\u003eBrd9\u003c/b\u003e\u003cb\u003e-containing non-classic BAF complex to enhance mucin production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo unravel the underlying mechanisms driving tumorigenicity in SRCC associated with \u003cem\u003eArid1a\u003c/em\u003e mutations, we investigated the role of the mammalian SWI/SNF chromatin remodeling complexes. These complexes exist in three unique final-form assemblies: canonical BAF (cBAF), polybromo containing BAF (pBAF), and a recently characterized non-canonical complex (ncBAF)\u003csup\u003e32\u003c/sup\u003e. Previous studies have demonstrated that residual cBAF subcomplexes resulting from ARID1 scaffolding loss disrupt pBAF function but not ncBAF function in cBAF-perturbed cancers\u003csup\u003e33\u003c/sup\u003e. Based on this, we hypothesized that ncBAF complexes may maintain gene expression and function in \u003cem\u003eARID1A\u003c/em\u003e-loss states and promote the genesis of SRCC.\u003c/p\u003e \u003cp\u003eInitially, analyzing the CUT\u0026amp;Tag data of BRD9, a core subunit of ncBAF complexes, which are not present in cBAF complexes, revealed a substantial alteration in BRD9 binding between TP and TPA gastric organoids. Specifically, TPA organoids displayed 1178 and 1467 peaks with significantly elevated and reduced binding levels compared to TP ones, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Table\u0026nbsp;8). The binding was predominantly localized at distal intergenic regions (40.66% in TP and 40.42% in TPA, respectively) and promoter regions (10.95% in TP, 22.97% in TPA, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Supplementary Table\u0026nbsp;8). Of particular note, the percentage of BRD9 binding in promoter regions was almost double in \u003cem\u003eArid1a\u003c/em\u003e loss organoids relative to wild type organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Intensity curves demonstrated a stronger signal of BRD9 significantly increased binding regions in TPA gastric organoids compared to TP ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Consistently, these regions related genes that displayed elevated genome accessibility and expression levels in \u003cem\u003eArid1a\u003c/em\u003e loss organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;5A). Further functional analysis of the genes with significantly elevated BRD9 binding levels in \u003cem\u003eArid1a\u003c/em\u003e loss organoids revealed a marked enrichment of Gene Ontology terms related to locomotory behavior, stem cell proliferation, activation of MAPK activity and other related biological processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Interestingly, we observed enhanced BRD9 binding levels at promoter regions of mucin-related genes in TPA organoids compared to TP ones, as determined by examination of the BRD9 CUT\u0026amp;Tag data between \u003cem\u003eArid1a\u003c/em\u003e loss and wild-type gastric organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify the role of BRD9 in the \u003cem\u003eArid1a\u003c/em\u003e-deficiency-induced elevation of mucinous production and cell proliferation, we are biologically and chemically depleted BRD9 using gRNA and dBRD9 chemical degradation strategies\u003csup\u003e33,34\u003c/sup\u003e. \u003cem\u003eBrd9\u003c/em\u003e knockout resulted in the downregulation of mucin gene expression in TPA tumor organoids (Supplementary Fig.\u0026nbsp;5, B and C). Through a side-by-side comparison of the response to dBRD9 treatment in TPA and TP tumor organoids \u003cem\u003ein vitro\u003c/em\u003e, we found that TPA tumor organoids were more sensitive to dBRD9, while exhibiting reduced sensitivity to 5-Fluorouracil (5-Fu) treatment compared to TP organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and Supplementary Fig.\u0026nbsp;5D). And dBRD9 treatment led to significant regression in the histology and morphology of TPA tumor organoids, including reduced structural complexity, cribriform growth, and stratification (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), but did not affect the TP tumor organoids (Supplementary Fig.\u0026nbsp;5E). The proportion of SRCC-like organoids in the dBRD9-treated groups was significantly decreased compared to that in control dimethyl sulfoxide (DMSO) groups, revealing a dose-dependent response (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). And the gradual disappearance of vacuoles within signet ring cell-like organoids over time following treatment with dBRD9 (Supplementary Fig.\u0026nbsp;5G). Importantly, dBRD9 treatment led to the reversal of the aberrant transcriptional activation of SRCC signature genes in TPA tumor organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and Supplementary Table\u0026nbsp;1), including inhibition of mucin gene expression confirmed by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK), consistent with the \u003cem\u003eBrd9\u003c/em\u003e knockout results (Supplementary Fig.\u0026nbsp;5C), but had no influence of TP groups (Supplementary Fig.\u0026nbsp;5F). RNA-seq analysis revealed the key molecular differences between dBRD9-treated TP and TPA organoids. Specifically, genes such as \u003cem\u003eClip3\u003c/em\u003e, \u003cem\u003eArhgef5\u003c/em\u003e, and \u003cem\u003eExoc2\u003c/em\u003e, which are involved in cytoskeletal organization, cell membrane localization, and exocytosis, were significantly up-regulated only in dBRD9-treated TPA tumor organoids. Conversely, genes like \u003cem\u003eLcf3f\u003c/em\u003e, \u003cem\u003eCryab\u003c/em\u003e, \u003cem\u003eAbcc1\u003c/em\u003e, which are involved in keratinization, stress response, and drug export from the cytoplasm, were significantly up-regulated in dBRD9-treated TP tumor organoids. GO enrichment analysis supports these findings, pathways related to actin filament organization and microtubule cytoskeleton organization were significantly enriched in dBRD9-treated TPA tumor organoids. In contrast, TP tumor organoids showed enrichment in various stress response pathways, including cellular response to glucose starvation, regulation of transcription from RNA polymerase II promoter in response to stress, and response to oxidative stress (Supplementary Fig.\u0026nbsp;5, H and I). These results indicate that dBRD9 primarily mediates cytoskeletal and microtubule remodeling in TPA tumor organoids to enhance cellular secretion functions, while in TP tumor organoids, it induces a stress response to the treatment. We further evaluated the effect of Arid1a loss on dBRD9 treatment \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). TPA tumor organoids were subcutaneously transplanted into recipient mice, followed by daily treatment with either vehicle or dBRD9 (50 mg/kg). dBRD9 treatment significantly inhibited the growth of \u003cem\u003eArid1a\u003c/em\u003e-deficient tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Histological analysis using H\u0026amp;E staining revealed that BRD9 inhibition reduced the proportion of signet ring cells compared to the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). The data presented herein suggest that BRD9 inhibition represents a promising therapeutic strategy for improving the morphological and biological characteristics of SRCC. This finding is particularly significant given that current treatment guidelines for gastric adenocarcinoma lack effective strategies for the management of SRCC. Thus, targeting BRD9 may offer a novel approach to enhance therapeutic outcomes for patients suffering from this challenging subtype of gastric cancer.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eGastric SRCC is a subtype of gastric malignancies and its studies have been impeded by limited animal models. Here, we applied a recently developed strategy with genome-edited organoids to generate primary, orthotopic, and genetic drivers-defined SRCC models in mice. Similar strategies, called as organoid-initiated precision cancer models (OPCMs), have been applied for lung cancer, esophageal cancer, bladder cancer, and colon cancer\u003csup\u003e23,24,35,36\u003c/sup\u003e. Recently, we also developed OPCMs of gastric adenocarcinoma and found that different genetic alterations and microenvironments could significantly affect their progression and metastasis\u003csup\u003e25\u003c/sup\u003e. Compared to traditional genetically engineered mouse models (GEMMs), OPCMs of SRCC and other cancers are very convenient to investigate the functions of any new gene mutation. This advantage makes them of value to dissecting the functions of these thousands of human cancer-associated genes revealed through cancer genomics studies. With this strategy, we found that gastric organoids with \u003cem\u003eArid1a\u003c/em\u003e loss, together with \u003cem\u003eTrp53\u003c/em\u003e and \u003cem\u003ePten\u003c/em\u003e deficiencies, gave rise to classic SRCC both subcutaneously and orthotopically in stomach. These results would not only validate \u003cem\u003eARID1A\u003c/em\u003e, one of the most frequently mutated genes in SRCC, as a \u003cem\u003ebona fide\u003c/em\u003e tumor suppressor of gastric SRCC in mice, but also provide a strategy to investigate the functions of other SRCC-associated genes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eARID1A\u003c/em\u003e is also frequently disrupted in various other types of human cancers\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. However, its roles and underlying mechanisms in tumorigenesis and progression are complicated. For example, it has the opposite function in the initiation and progression of liver cancer through oxidative stress and chromatin accessibility, respectively\u003csup\u003e21\u003c/sup\u003e. And in liver cholangiocarcinoma, \u003cem\u003eArid1a\u003c/em\u003e loss cooperates with \u003cem\u003eKras\u003c/em\u003e mutation to promote tumorigenesis through repressing the TGF-β pathway\u003csup\u003e40\u003c/sup\u003e, while the YAP pathway is essential for \u003cem\u003eArid1a\u003c/em\u003e deficiency-induced pancreatic ductal adenocarcinoma\u003csup\u003e41\u003c/sup\u003e. Besides, in the urothelium, \u003cem\u003eARID1A\u003c/em\u003e loss caused a transcriptional-translational conflict resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBecause of the well-characterized roles of the \u003cem\u003eARID1A\u003c/em\u003e-containing SWI/SNF complex in chromosome remodeling and transcription regulation, it is expected that \u003cem\u003eARID1A\u003c/em\u003e loss would lead to a global reduction on chromatin accessibility\u003csup\u003e21,22,43\u003c/sup\u003e, however, accumulating evidence suggests that, at least in some tumors, \u003cem\u003eARID1A\u003c/em\u003e deficiency leads to increased chromatin accessibility, potentially due to the regulation of other chromatin remodeling factors by \u003cem\u003eARID1A\u003c/em\u003e\u003csup\u003e29,44,45\u003c/sup\u003e. Similarly, our studies have also shown that \u003cem\u003eARID1A\u003c/em\u003e knockout upregulates multiple gene expressions and increases chromatin accessibility in the promoter region in gastric cancer. Hence, in our study, we found that the expressions of secreting genes, \u003cem\u003eScin\u003c/em\u003e, directly enhanced and the expressions of mucin genes indirectly repressed in the \u003cem\u003eArid1a\u003c/em\u003e loss-induced SRCC. The mechanism is different from a previous study with \u003cem\u003eARID1A\u003c/em\u003e disruption in \u003cem\u003eTP53\u003c/em\u003e mutated human organoids, which didn\u0026rsquo;t display typical SRCC pathology\u003csup\u003e22\u003c/sup\u003e. These differences might be due to different genetic background. However, our data indicate that \u003cem\u003eArid1a\u003c/em\u003e loss could promote SRCC through both increasing mucin production and impaired secretion, which provide strong experimental evidence for the long-proposed hypothesis of SRCC formation.\u003c/p\u003e \u003cp\u003eGiven the prevalence and tumorigenic functions of \u003cem\u003eARID1A\u003c/em\u003e mutations, it has been attractive to explore potential treatment strategies for \u003cem\u003eARID1A\u003c/em\u003e altered tumors. It has been reported that \u003cem\u003eARID1A\u003c/em\u003e inactivated ovarian carcinoma has defects on the mevalonate pathway and thus, is sensitive to statin treatment by itself and together with immune checkpoint blockade\u003csup\u003e46\u003c/sup\u003e. Considering that \u003cem\u003eARID1A\u003c/em\u003e is a member of SWI/SNF complex which of subunits have interaction and mutations resulting in inflammation even tumors\u003csup\u003e32,47\u003c/sup\u003e. Through multi-omics analyses, it is found that \u003cem\u003eARID1A\u003c/em\u003e mutated gastric cancers might be responsive to bromodomain and NFKB inhibitions\u003csup\u003e48\u003c/sup\u003e. In SRCC, we find that \u003cem\u003eArid1a\u003c/em\u003e loss leads to \u003cem\u003eBrd9\u003c/em\u003e-mediated upregulation of mucin genes. Thus, we propose that targeting \u003cem\u003eBrd9\u003c/em\u003e or the non-classic BAF complex might be an effective strategy for SRCC, which is characterized by massive mucins. Our results are also consistent with the observations in several other types of cancers, such as sarcoma\u003csup\u003e33\u003c/sup\u003e, rhabdoid tumor\u003csup\u003e49\u003c/sup\u003e, and liver cancer\u003csup\u003e50\u003c/sup\u003e. It would be interesting to test potential inhibitors of non-classic BAF complex for patients with \u003cem\u003eARID1A\u003c/em\u003e mutations. However, we also notified that \u003cem\u003eARID1A\u003c/em\u003e might have complicated roles in both malignant cells and nonmalignant cells, such as its role in tumor-infiltrating T cells\u003csup\u003e51\u003c/sup\u003e. Thus, any treatment targeting \u003cem\u003eARID1A\u003c/em\u003e deficient tumors should be tested for its potential systematic effect.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e. \u003cem\u003eTrp53\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and Cas9-EGFP mice were purchased from Jackson Laboratories (Cat# 002101 and 026179, RRID: IMSR_JAX:026179, respectively). BALB/cA-nu mice were purchased from Beijing HuaFukang Biological Technology Co. Ltd (6-8-week-old, male).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse Organoid Culture\u003c/strong\u003e. Mouse organoid cultures were established and maintained as described previously\u003csup\u003e25\u003c/sup\u003e. Specifically, normal mice stomach was mechanically minced into pieces of 5-mm\u003csup\u003e3\u003c/sup\u003e cubes and incubated in DPBS with 2.5 mM EDTA on ice. After incubation, the released gastric gland fractions were resuspended with Matrigel (Corning, Cat# 356237) and seeded in 48-wells plate. Tumor tissues were harvested and washed with ice-cold DPBS, and then minced into pieces. Incubating the minced tumor tissue in digestion medium containing 0.5 mg/ml collagenase IV (GIBCO, Cat# 17104-019) and 1.0 mg/ml collagenase I (GIBCO, Cat# 17100-017) in DMEM/F12 medium at 37\u0026deg;C until it dissociates into single cells or cell clusters. Tumor cells were collected by centrifugation and resuspended with Matrigel. Organoid culture medium was DMEM/F12 (GIBCO, Cat# C11330500BT) supplemented with 2 mM GlutaMAX (GIBCO, Cat# 35050-061), 1% Penicillin and Streptomycin (GIBCO, Cat# 15140-122), 1\u0026times; B27 (GIBCO, Cat# A3582801), 1\u0026times; N\u003csub\u003e2\u003c/sub\u003e (GIBCO, Cat# 17502048),10 mM Y-27632, 200ng/ml FGF10 (Peprotech, Cat# 100-26-1000), 500nM A-8301 (Peprotech, Cat# 9094360), 50ng/ml mouse recombinant EGF (Peprotech, Cat# AF-100-15-1000), 100 ng/ml mouse recombinant noggin (Peprotech, Cat# 120-10C-250), 10mM Nicotinamide (Sigma, Cat# N0636), 1 mM N-acetylcysteine (Sigma, Cat# A9165), 10% Wnt3a condition medium, 10% R-spondin1 condition medium). Organoid medium was refreshed every 2\u0026ndash;3 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrganoid genome editing\u003c/strong\u003e. sgRNAs designed on the website of DNA2.0 Gene Design \u0026amp; Synthesis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.atum.bio/pipeline/dna\u003c/span\u003e\u003c/span\u003e) were cloned into the lentiviral constructs U6-sgRNA-EFS-mCherry-P2A-puro. The sequences of gRNAs and primers targeting \u003cem\u003ePten\u003c/em\u003e, \u003cem\u003eArid1a\u003c/em\u003e, \u003cem\u003eScin\u003c/em\u003e and \u003cem\u003eBrd9\u003c/em\u003e are listed in Supplementary Table 2. Lentivirus packaging, infection and verification were done as previously reported\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrganoid Subcutaneous and Orthotopic Transplantation\u003c/strong\u003e. Organoids were collected and aspirated into pre-chilled 29G insulin syringes. The organoid suspension (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per mouse) was directly injected into 6 to 8-week-old male BALB/cA-nu mice subcutaneously or orthotopically. Refer to the previous article for detailed methods\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq analysis\u003c/strong\u003e. Transcriptome sequencing was conducted using the Illumina NovaSeq 6000 platform with 150 bp paired-end sequencing. After removing the adapters, ploy-N, and low-quality reads, clean data were aligned against the reference mouse genome (mm10) using STAR _2.6.0. Following this, raw counts were normalized and significance scores. as well as log2-fold change values, were calculated using the DESeq2 (v.1.22.2). The differentially expressed genes were used to perform Gene Ontology enrichment analyses with the R package clusterProfiler (v. 3.10.1) (RRID:SCR_016884). Furthermore, Gene Set Enrichment Analysis (GSEA) was employed to identify significantly enriched pathways. Preranked function was performed to clarify the overall transcriptome similarity between mouse models and tumor tissues of SRCC patients. Data visualization was conducted using the R packages ggpubr, ggplot2, pheatmap, and Vennerable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATAC-seq analysis\u003c/strong\u003e. The Illumina NovaSeq 6000 platform was utilized to sequence the library. To remove adapters from the 150 bp paired-end raw data, NGmerge was employed with the option \u0026quot;NGmerge -a -v -n 20\u0026quot;. Bowtie2 software was used to construct an index for the mouse genome (mm10), followed by alignments using the option \u0026quot;--very-sensitive -X2000 -x mm10\u0026quot;. Duplicates were removed using Picard\u0026apos;s MarkDuplicates tool. Mitochondrial genome contamination was eliminated using SAMtools and awk commands, resulting in bam files. Normalized bw files were generated using deepTools with the option \u0026quot;bamCoverage -bs\u0026thinsp;=\u0026thinsp;1 --normalizeUsing BPM\u0026quot; for subsequent visualization in the Integrated Genome Viewer (IGV). The HMMRATAC standard workflow was utilized for peak calling in ATAC-seq.\u0026nbsp;The genomic distribution of accessibility sites was determined using ChIPseeker with the option \u0026quot;annotatePeak\u0026quot;, and the TSS region \u0026quot;tssRegion\u0026quot; was set as (-3000, 3000). The \u0026quot;gappedPeak\u0026quot; files were transformed into GrangesList forms, and the peaks were converted into consensus counts. Data normalization and differential comparison were performed using DESeq2. Regions of differential accessibility were identified for downstream analyses using a p-value cutoff of 0.05 (Wald test). HOMER was used for motif analysis with the \u0026ldquo;findMotifsGenome.pl\u0026rdquo; function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCUT\u0026amp;Tag analysis\u003c/strong\u003e. The Illumina NovaSeq 6000 platform was employed to perform genomic sequencing. Following quality control by the fastp standard workflow, the Bowtie2 software was utilized to construct an index for the mouse genome (mm10) and align paired-end clean data of 150 bp length using the options \u0026quot;--local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700\u0026quot;. The SAMtools program was utilized to convert sam files to sorted bam files. Picard\u0026apos;s MarkDuplicates tool was used to remove duplicates with the option \u0026quot;REMOVE_DUPLICATES\u0026thinsp;=\u0026thinsp;true\u0026quot;. Normalized bw files were generated using deepTools with the option \u0026quot;bamCoverage -bs\u0026thinsp;=\u0026thinsp;1 --normalizeUsing BPM\u0026quot; for further visualization in the Integrated Genome Viewer (IGV). Peak calling was performed using the MACS2 software with the option \u0026quot;macs2 callpeak --broad -q 1e-5 -f BAMPE -g mm --keep-dup all\u0026quot;. ChIPseeker was employed to identify the genomic distribution of the peaks, with the TSS region \u0026quot;tssRegion\u0026quot; set as (-3000, 3000) for annotation. The \u0026quot;narrowPeak\u0026quot; files were transformed into GrangesList forms, and the peaks were converted into consensus counts. Data normalization and differential comparison were performed using DESeq2. Regions of differential modification were identified for downstream analyses using a \u003cem\u003ep\u003c/em\u003e-value cutoff of 0.05 (Wald test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e. Organoid diameter and number assays, tumor measurements, RT-qPCR, morphological statistics, and \u003cem\u003ein vitro\u003c/em\u003e treatment were analyzed for statistical significance using two-tailed unpaired parametric Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests (Prism 9.0, GraphPad software). The data distribution was assumed to be normal, but this was not formally tested. Statistical test methods, sample sizes, and \u003cem\u003ep\u003c/em\u003e values are indicated in the corresponding figure legends. For the \u003cem\u003ein vitro\u003c/em\u003e treatment experiments, all samples were randomly assigned to vehicle or treatment groups. Tumor measurements were performed blindly. The measurements of organoid shapes were analyzed blindly. For other \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments, the researchers were not blinded while performing the experiments. No data were excluded from the study. Statistical significance on RNA-seq, ATAC-seq and CUT\u0026amp;Tag data was determined using unpaired two-tailed t test, Wilcoxon signed-rank test, Wald test, hypergeometric distribution, or Likelihood-ratio test. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All the quantification and visualization of omics data analysis were performed on R v3.6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy approval\u003c/strong\u003e. All these mice were bred and kept in the SPF animal facility of Sichuan University, and no mouse was excluded in the experiments. All animal experiment protocols were approved by the Animal Care and Use Committee of Sichuan University.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, C.C.; Methodology: H.L., A.Z., Z.L., M.Z., J.L., Y.W., X.C., X.P., J.C., L.Z.,\u0026nbsp;L.G., S.H., X.W., Y.P., T.P., J.W., J.D., X.D., K.Y., K.L., X.C., X.C., Z.F.,\u0026nbsp;B.W., L.Z., W.Z. S.Y., Y.W., L.C., Z.X., C.Z., L.D.; Investigation: H.L., A.Z., Z.L.; Resources: L.G., T.P., K.Y., K.L., X.C., X.C., Z.F.,\u0026nbsp;L.Z., W.Z., S.Y., Y.W., L.C., Z.X., C.Z., Y.L., F.N.; Writing-Original Draft: H.L., A.Z., Z.L., J.H., Y.L., C.C.; Writing-Review \u0026amp; Editing: H.L., A.Z., Z.L., J.C., J.H., Y.L., C.C.; Funding Acquisition: B.W., Y.L., F.N.,\u0026nbsp;Y.L., C.C.; Supervision: F.N.,\u0026nbsp;J.H., Y.L., C.C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement:\u0026nbsp;\u003c/strong\u003eThe authors disclose no conflicts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe RNA-seq, ATAC-seq, and CUT\u0026amp;Tag data in this study are deposited in NCBI GEO: GSE232292. The secure token: mnunqagozjsjzur.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMore detailed information relevant to this study can be found in the supplementary tables, methods, and figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Yuquan Wei for generous support. We thank all the CC-LY lab members\u0026nbsp;and\u0026nbsp;Fangfang Wang (Institute of hematology, West China Hospital) \u0026nbsp;for discussion and technical support. We thank the Core Facilities of West China Hospital and the Chengdu OrganoidMed Medical Laboratory for technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the members of the CC-LY laboratory for their technical support and suggestions. This work was supported by the National Natural Science Foundation of China (2017YFA0505601 and 82130007, Y. Liu.; 82102779, J.C.; T2221004, C.C. and S.Y.), the Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJC2023010004), the 1.3.5. Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC21003, Y. Lu; ZYYC20004, C.C.; ZYGD22012, Y. Liu; ZYJC21009, Y. Liu), the Sichuan Science and Technology Program (2020YFQ0059, C.C.; 2022YFS0205, F.N.), the Post-Doctor Research Project, West China Hospital, Sichuan University (2023HXBH019), National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University (Z2024JC001, C.C.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNagtegaal, I. 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A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence. \u003cem\u003eCancer Cell\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 768-786 e767, doi:10.1016/j.ccell.2022.06.001 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gastric signet ring cell carcinoma, ARID1A, mouse model, pathology","lastPublishedDoi":"10.21203/rs.3.rs-5298469/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5298469/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSignet ring cell carcinoma (SRCC) is a lethal malignancy with unique histologic features, characterized with large vacuoles and compressed nuclei. Gastric SRCC is the most common SRCC, and its incidence is increasing recently. However, the driver genes of SRCC and the molecular mechanisms underlying its unusual histology remain unclear. Here, we developed a new type of gastric SRCC mouse models with gene-edited premalignant gastric organoids and validated \u003cem\u003eARID1A\u003c/em\u003e, one of the most frequently mutated genes in SRCC, as a \u003cem\u003ebona fide\u003c/em\u003e tumor suppressor gene of gastric SRCC. Mechanistically, through CUT/Tag and ATAC-seq analyses, we found that \u003cem\u003eArid1a\u003c/em\u003e directly regulated the expressions of secretory factors \u003cem\u003eScin\u003c/em\u003e and on the other hand, \u003cem\u003eArid1a\u003c/em\u003e loss reprogrammed the genome binding of the SWI/SNF complexes and increased the expressions of mucin genes through the binding of \u003cem\u003eBrd9\u003c/em\u003e, a component of the noncanonical SWI/SNF complex. Inhibiting \u003cem\u003eBrd9\u003c/em\u003e reversed the pathology of \u003cem\u003eArid1a\u003c/em\u003e mutant SRCC. Thus, our studies revealed dual roles of \u003cem\u003eARID1A\u003c/em\u003e in restraining SRCC through both mucin production and secretion. These findings offer new insights into the susceptibilities of \u003cem\u003eARID1A\u003c/em\u003e deficient SRCC.\u003c/p\u003e","manuscriptTitle":"Dual roles of ARID1A in both mucin production and secretion suggest susceptibilities of gastric signet ring cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-12 04:31:53","doi":"10.21203/rs.3.rs-5298469/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c09b9609-4822-44dd-8482-a2d7952c3a13","owner":[],"postedDate":"November 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":40058371,"name":"Biological sciences/Cancer/Cancer models"},{"id":40058372,"name":"Biological sciences/Cancer/Gastrointestinal cancer"}],"tags":[],"updatedAt":"2026-04-15T14:20:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-12 04:31:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5298469","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5298469","identity":"rs-5298469","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}