Objective  To investigate the mechanism by which Helicobacter pylori cooperates with Yes-associated protein (YAP) in regulating autophagy and gastric cancer stemness, and elucidate its impact on the metastatic and invasive potential of gastric cancer.

Methods H. pylori and the autophagy inhibitor chloroquine (CQ), either alone or in combination, were co-cultured with HGC-27 gastric cancer cells transfected with a YAP-overexpressing plasmid (H2) or an empty vector (HN2). Wound-healing and Transwell assays were performed to evaluate changes in cell migration and invasion. All in vitro experiments were conducted with three technical replicates per group and independently repeated for three times. Quantification was performed using ImageJ, and differences were analyzed by one-way ANOVA or three-way ANOVA. For in vivo assays, liver metastasis models were established by tail vein injection of H2 or HN2 cells into female SPF-grade BALB/c-nu mice. After 8 weeks, livers samples were collected for hematoxylin and eosin (H&E) staining to assess histopathological changes. Immunohistochemistry was used to detect the expression of EpCAM/CD326, Claudin 18, LC3BⅡ, and P62, while quantitative real-time PCR was conducted to measure the mRNA transcription levels of gastric cancer stem cell markers CD133, Lgr5, and CD44

Results H. pylori infection markedly improved the migratory and invasive abilities of gastric cancer cells. Wound-healing assays showed that the migration rate of the HN2+H. pylori group increased by 26.88% compared with the control, while Transwell assays revealed a 34.91% increase in migrated cells. YAP overexpression further amplified these pro-metastatic effects, with the H2+H. pylori group exhibiting a 48.59% higher migration rate and 85.00% more invasive cells compared with the HN2+H. pylori group. Treatment with the autophagy inhibitor CQ significantly reversed these effects, reducing migration to 32.39 ± 1.19% and invasion by 24.68% in the H2+H. pylori+CQ group, confirming a central role of autophagy. In vivo, immunohistochemistry demonstrated positive expression of EpCAM/CD326 and Claudin 18 in liver tissues of all groups except controls, indicating successful establishment of liver metastasis models. H. pylori infection significantly increased metastatic potential, as evidenced by CD326 and Claudin 18 expression levels of 38.39% and 19.73% in the HN2+H. pylori group, respectively. YAP overexpression further enhanced this effect, with EpCAM/CD326 and Claudin 18 expression rising to 60.85% and 66.80% in the H2+H. pylori group. CQ intervention partially reversed these changes, lowering expression to 44.17% and 16.66%, respectively. Histopathological analysis revealed that H. pylori infection aggravated liver injury, including sinusoidal dilatation, inflammatory infiltration, and disorganized cell arrangement, while YAP overexpression combined with infection caused more severe damage, such as pronounced congestion, necrosis, and degeneration. CQ treatment alleviated these pathological alterations.Immunohistochemistry showed that H. pylori infection and YAP overexpression synergistically promoted autophagy, indicated by increased LC3BⅡ and decreased P62 expression, whereas CQ effectively blocked autophagic flux. Moreover, H. pylori and YAP cooperatively upregulated the mRNA levels of gastric cancer stem cell markers CD133, Lgr5, and CD44, which were partially reversed by CQ. Three-way ANOVA (2×2×2 factorial design) revealed significant main effects of YAP, H. pylori, and CQ across all 10 parameters. Significant interactions were observed between YAP and H. pylori (migration, invasion, stemness markers, EpCAM/CD326, Claudin 18, LC3BⅡ, and P62), between H. pylori and CQ (invasion and stemness markers), and among all three factors (migration, invasion, EpCAM/CD326, Claudin 18, and P62).

Conclusion H. pylori synergizes with YAP to regulate autophagy and improve gastric cancer stemness, ultimately promoting the metastasis and invasion of gastric cancer.