HIF1α-dependent GGT5 expression promotes gastric cancer progression under hypoxia

The hypoxic tumor microenvironment fosters rapid growth and metastasis of carcinomas. Glutathione (GSH) plays a pivotal role in tumor metabolism. Understanding the mechanisms underlying GSH metabolism, including the role of hypoxia-inducible factor 1-alpha (HIF1α) in gastric cancer (GC) under hypoxic conditions, is crucial. Key hypoxia-associated genes involved in GSH metabolism in GC were identified using bioinformatic analyses and validated in clinical specimens. Quantitative PCR (qPCR) and western blotting were used to assess GGT5 expression and its regulation by HIF1α. Chromatin immunoprecipitation (ChIP)-qPCR and dual-luciferase reporter assays were performed to identify HIF1α binding sites within the GGT5 promoter. In vitro and in vivo assays, including CCK-8, flow cytometry, transwell migration, and tumor xenograft models, were used to evaluate cell viability, apoptosis, and migration under hypoxic conditions. Cellular experiments were performed under a combined hypoxia-mimetic condition consisting of 1% O₂ and 200 µM CoCl₂. The DTNB method was used to measure the intracellular GSH content and GSH/GSSG ratio. GGT5 was identified as a key hypoxia-associated gene, with high expression associated with advanced T stage, older age, and shorter survival in unadjusted analyses. GGT5 expression was significantly higher under the combined hypoxia-mimetic condition than that of normoxic conditions, and knockdown of HIF1α reduced GGT5 levels in GC cells under the combined hypoxia-mimetic condition. ChIP-qPCR and dual-luciferase assays revealed that HIF1α directly binds to a specific site (− 903 to − 896 bp) of the GGT5 promoter. Silencing GGT5 under hypoxic conditions suppressed GC cell proliferation and migration, increased apoptosis, and reduced both intracellular GSH levels and the GSH/GSSG ratio. GGT5 knockdown diminished the tumorigenic capacity of xenograft models in vivo. Our findings support direct transcriptional regulation of GGT5 by HIF1α under the combined hypoxia-mimetic condition. Loss-of-function experiments suggest that GGT5 may contribute to malignant phenotypes and altered glutathione status in GC cells. However, these findings do not establish the specificity of the observed effects to oxygen deprivation or a causal GSH-dependent mechanism.