Peritoneal dissemination is a major cause of mortality in gastric cancer (GC), yet its molecular underpinnings remain incompletely defined. By integrating single-cell transcriptomic profiling of primary tumors and peritoneal lesions with functional and mechanistic studies, we identified a hypoxia-sensitive GC cell population that emerges during peritoneal metastasis and exhibits heightened autophagic activity and metastatic potential. Hypoxic stress robustly induced the deubiquitinase USP33 in GC cells, and high USP33 expression correlated with adverse clinical outcome. Gain- and loss-of-function assays demonstrated that USP33 enhances autophagy and promotes proliferation, invasion, and survival of GC cells. Proteomic and biochemical analyses revealed that USP33 directly interacts with the autophagy regulator ATG101 and stabilizes it by removing K48-linked polyubiquitin chains at lysine residues 106 and 191, thereby sustaining autophagic flux and facilitating peritoneal colonization. Upstream, hypoxia activated EPAS1/HIF2A, which bound to and transcriptionally upregulated USP33, establishing a hypoxia-responsive EPAS1-USP33-ATG101 axis. Disruption of this axis, either by USP33 silencing or pharmacological inhibition of EPAS1, suppressed autophagy-dependent peritoneal metastasis and prolonged survival in vivo. Collectively, our findings define a mechanistic link between hypoxia, oncogenic autophagy, and peritoneal dissemination in GC, and highlight the EPAS1-USP33-ATG101 axis as a promising therapeutic target in advanced disease.
