Abstract Gastric cancer remains among the most common and lethal malignancies worldwide, and survival rate for stage IV patients is still significantly low, underscoring the need for novel preventive and therapeutic strategies. Recent studies using patient-derived organoids indicate that, in many gastric cancers, Wnt signaling is activated through an exogenous, ligand-dependent mechanism despite the absence of APC or CTNNB1 mutations. Here, we investigated how ligand-dependent Wnt signaling contributes to gastric cancer development and metastasis. We generated genetically engineered mouse models (KTP mice) carrying Kras G12D, Tgfbr2 -/-, Trp53 R270H mutations in the gastric epithelial cells, as well as with the same mutations plus Wnt1 expression (WKTP mice). Whereas KTP mice developed gastric metaplasia, WKTP mice developed dysplastic tumors, suggesting that ligand-dependent Wnt signaling promotes primary tumorigenesis. In metastasis analysis, organoids derived from WKTP mouse tumors formed liver metastases after splenic transplantation, while KTP organoids did not. Notably, genetic disruption of Apc was insufficient to confer metastatic capacity on KTP cells, suggesting that Wnt signaling activation in stromal cells is critical for metastasis. Mechanistically, tumor-derived Wnt ligands cooperated with TGF-beta induce Has2 expression in stromal fibroblasts (CAFs), resulting in hyaluronan accumulation within the liver metastatic niche. Importantly, enforced hyaluronidase expression in cancer cells markedly suppressed liver metastasis. These results indicate a pivotal role of ligand-dependent Wnt signaling in the CAFs in gastric cancer metastasis through Has2-mediated hyaluronan deposition. Therefore, targeting Wnt signaling/Has2-hyaluronan axis may be a potential therapeutic strategy against metastatic gastric cancer. Citation Format: Hiroko Oshima, Yuichiro Furutani, Noriyuki Inaki, Nick Barker, Masanobu Oshima. Ligand-dependent Wnt signaling drives metastatic niche formation in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB304.

The majority of gastric cancer cells proliferate in a Wnt ligand-dependent manner. To investigate this, we generated mice harboring Kras, Tgfbr2, and Trp53 (KTP) as well as with the same mutations plus Wnt1 expression (WKTP) in gastric mucosa. While KTP mice develop gastric metaplasia, WKTP mice develop dysplastic tumors, highlighting the role of ligand-dependent Wnt signaling in primary tumorigenesis. Organoids derived from WKTP mice form liver metastases following splenic transplantation, whereas KTP organoids do not. Notably, Apc disruption fails to induce metastasis of KTP cells, suggesting that stromal Wnt signaling promotes metastasis. Mechanistically, tumor-derived Wnt ligands cooperate with TGFβ signaling to induce Has2 expression in cancer-associated fibroblasts (CAFs), leading to hyaluronan accumulation in the metastatic microenvironment. Strikingly, hyaluronidase expression in WKTP cells significantly suppresses liver metastasis. Here we show the critical role of ligand-dependent Wnt signaling and Has2-mediated hyaluronan deposition in metastasis, offering potential therapeutic strategy against gastric cancer metastasis.

Abstract Missense-type p53 mutations have shown to acquire novel oncogenic roles through a gain-of-function mechanism. However, there is intratumor heterogeneity in stabilization of mutant p53 protein, and it has not been well understood about the interaction between p53-stabilized and -destabilized cells in the same tumors. We established mouse intestinal tumor–derived organoids carrying ApcΔ716, KrasG12D, and Tgfbr2−/− mutations with Trp53R270H or Trp53Null mutation (AKTPR270H and AKTPNull, respectively). Using these organoids, we found that the activation level of Wnt/β-catenin signaling is significantly higher in AKTPR270H cells compared with AKTPNull cells. Notably, Wnt activation in the AKTPNull cells was significantly increased when co-cultured with AKTPR270H cells. Expression analysis revealed that COX-2 is significantly upregulated in AKTPR270H but not in AKTPNull cells, suggesting that mutant p53 induces the COX-2/prostaglandin E2 (PGE2) pathway. Importantly, Wnt activation in co-cultured AKTPNull cells with AKTPR270H was significantly suppressed when treated with the inhibitor of COX-2 or PGE2 receptors EP2/EP4. Furthermore, stimulation with PGE2 increased Wnt signaling activity in AKTPNull cells. These results indicate that the COX-2/PGE2 pathway is activated in the p53-stabilized cells in the missense-type p53-mutant cancer, and secreted PGE2 may transactivate Wnt/β-catenin signaling in neighboring p53-destabilized tumor cells in the intratumor microenvironment. Therefore, targeting stabilized mutant p53 or the COX-2/PGE2 pathway may suppress Wnt/β-catenin signaling of both mutant p53–stabilized and –destabilized cells; thus, this can be a possible preventive or therapeutic strategy. Significance: There is intratumor heterogeneity in the stabilization of missense mutant p53, and it has been thought that only cells with nuclear accumulation of mutant p53 have oncogenic function. However, using mouse intestinal tumor–derived organoids, we show that mutant p53–stabilized cells transactivate Wnt/β-catenin signaling in neighboring p53-destabilized cells through activating the COX-2/PGE2 pathway. These results suggest that both p53-stabilized cells and p53-destabilized cells contribute to malignant progression through interaction within the intratumor microenvironment.

IPA results for AKTP R270 vs AKTP Null cells

Promotion of Wnt/β-catenin signaling in missense-type p53-mutant colon tumors. A, Luciferase activities of NF-κΒ reporter assay are shown as a bar graph (mean ± SD). B, Luciferase activities of Wnt/β-catenin reporter assay (TOPFlash) are shown as a bar graph (mean ± SD). Three independent AKTPNull and AKTPR270H organoid lines for each were used in the reporter assays (A and B). Statistical analysis was performed using the one-way ANOVA test (A and B). C, Luciferase activities of Wnt/β-catenin reporter assay for control-transfected and Trp53 siRNA–transfected AKTPR270H organoid cells are shown as a bar graph (mean ± SD). The immunoblotting result for p53 is shown. GAPDH was used as the internal control. Each dot in A–C graphs indicates an independent experiment. P values are indicated. D, Representative confocal microscopy images of fluorescence immunocytochemistry for β-catenin (green) and E-cadherin (red) in AKTPNull and AKTPR270H organoids. Arrowheads indicate cells with β-catenin stabilization. Bars, 25 μm. E, Representative photographs of fluorescence IHC for β-catenin (green) and E-cadherin (red) of AKTPNull and AKTPR270H liver metastatic tumors. Insets show enlarged images. Bars, 100 μm. Note that β-catenin accumulation was predominantly found in AKTPR270H tumors. F, Immunoblotting results for p53 in colorectal cancer cell lines. β-Actin was used as the internal control. G, Luciferase activities of Wnt/β-catenin reporter assay (TOPFlash) for colorectal cancer cell lines are shown as a bar graph (mean ± SD). Each dot indicates an independent experiment. The P value is provided. H, Representative photographs of immunostaining for p53 (left) and β-catenin (right) of p53-positive and β-catenin nuclear-accumulated (top) and p53-negative and β-catenin not nuclear–accumulated (bottom) human colorectal cancer. Insets show enlarged images of the boxed area. Arrowheads indicate p53 stabilization (left) and β-catenin nuclear accumulation (right). Bars, 200 μm. I, Ratio of β-catenin nuclear accumulation in p53-positive (+) and p53-negative (−) colorectal cancer scored using the IHC results in H. The P value is indicated.

Heterogeneity of p53 and β-catenin stabilization in tumor cells. A, Representative photographs of IHC for p53 of human colon cancer (left) and AKTPR270H liver metastatic tumor (right). Insets show enlarged images. Arrowheads indicate p53-negative (destabilized) tumor cells. Bars, 100 μm. B, Ratio of p53-positive (+) cells in primary and metastatic colorectal cancer (CRC; squares) and AKTPR270H liver metastatic tumors of two independent lines (circles). Each dot indicates the results of the independent microscopic fields. C, Representative photographs of fluorescence IHC for p53 (green, left), β-catenin (red, center), and merged images (right) of AKTPR270H liver metastasis (top) and human colon cancer (bottom) Insets show enlarged images. Arrowheads and arrows indicate double-positive and single-positive cells, respectively. Asterisks indicate double-negative cells. Bars, 100 μm. D, Relative proportions (%) of p53-positive, β-catenin–positive, and double-negative cells in the liver metastasis of AKTPR270H cells (left), AKTPNull cells (right), and human colorectal cancer (center) are shown as bar graphs. Three biologically independent data for each are shown.

Activation of Wnt/β-catenin signaling in p53-negative cells through the COX-2/PGE2 pathway. A, Schematic drawing of Wnt/β-catenin reporter assays (TOPFlash) of co-cultured AKTPNull-bottom cells with AKTPR270H-top cells or AKTPNull-top cells. B, Luciferase activities of Wnt reporter assays (TOPFlash) of AKTPNull-bottom cells co-cultured with AKTPNull-top cells or AKTPR270H-top cells are shown as a bar graph (mean ± SD). Each dot represents an independent experiment. The P value is provided. C, RNA-seq data for Ptgs2 in three independent AKTFPR270H/+ and AKTFPR270H/LOH organoid lines. fragments per kilobase of exon per million reads mapped, the mean fold change, and P value are provided. D, Immunoblotting results for p53 and COX-2 in three independent AKTPNull and AKTPR270H lines. GAPDH was used as the internal control. E, Representative fluorescence immunocytochemistry images for p53 (green) and COX-2 (red) of AKTPNull (left) and AKTPR270H (right) organoids. F, Representative image of fluorescence IHC for p53 (green) and COX-2 (red) of AKTPR270H liver metastatic tumor. Insets in E and F show enlarged images. Bars in E and F, 100 μm. G, Representative images of fluorescence IHC for COX-2 (red) and F4/80 (macrophages, green) or αSMA (myofibroblasts, green). Merged images are shown (right). Bars, 50 μm. H, Luciferase activities of Wnt reporter assays (TOPFlash) of AKTPNull-bottom cells in co-cultured AKTPNull-top or AKTPR270H-top cells or control AKTPNull-bottom monoculture cells in the presence or absence of COX-2 inhibitor, celecoxib, are shown as a bar graph (mean ± SD). Each dot represents an independent experiment. The P value is indicated. ns, not significant. I and J, Luciferase activities of Wnt/β-catenin reporter assays in AKTPNull cells treated with PGE2 (I) or AKTPNull cells co-cultured with AKTPR270H treated with EP2/EP4 inhibitors (J) are shown as bar graphs (mean ± SD). Data from two independent AKTPNull lines (#1 and #2) are shown. Each dot indicates an independent experiment. P values are provided.

Circulating tumor cell (CTC) clusters are often detected in blood samples of patients with high-grade tumor and are associated with tumor metastasis and poor prognosis. However, the underlying mechanisms by which cancer cell clusters are released from primary tumors beyond blood vessel barriers remain unclear. In this study, a three-dimensional (3D) in vitro culture system was developed to visualize tumor intravasation by positioning tumor organoids with distinct genetic backgrounds to surround microvessels. We visualized tumor intravasation in a cluster unit, including collective migration toward microvessels, vessel co-option, and the release of CTC clusters-an invasion mechanism not previously reported. Furthermore, elevated levels of transforming growth factor β (TGF-β) and activin expression in endothelial cells within the coculture microenvironment were pivotal for facilitating tumor cell intravasation, which was associated with endothelial-to-mesenchymal transition (EndoMT) in microvessels. Our 3D in vitro system can be used to develop therapeutic strategies for tumor metastasis by targeting the release of CTC clusters.