The MDM2‐IL‐33 Axis Promotes Gastric Tumorigenesis via K63 Ubiquitination‐Dependent ERK/JNK Activation

This study uncovers a novel post-translational regulatory mechanism through which MDM2 stabilizes IL-33 via K63-linked ubiquitination, thus facilitating the progression of gastric cancer. Using MKN45 and AGS gastric cancer cells, we conducted MDM2 knockdown followed by qPCR and Western blot to evaluate IL-33 regulation. Co-IP and cycloheximide chase assays verified MDM2-IL-33 binding and stabilization. Ubiquitination assays using WT and mutant (K63R/K48-only) ubiquitin identified MDM2-dependent K63-linked polyubiquitination of IL-33. A subcutaneous xenograft tumor model in nude mice was established to validate the oncogenic role of the MDM2-IL-33 axis in vivo. Functional assays (CCK-8, colony formation, Transwell, and wound healing) revealed MDM2's requirement for IL-33-mediated oncogenic effects. Western blots showed MDM2-IL-33 axis modulates apoptosis (Bcl-2/Bax/Caspase-3) and ERK signaling (p-ERK/p-JNK). We have demonstrated that knocked down MDM2 significantly decreases IL-33 protein levels without influencing its mRNA expression, suggesting that the regulation occurred at the post-translational stage. Co-immunoprecipitation assays have confirmed the physical interaction between MDM2 and IL-33 in gastric cancer cell lines, namely MKN45 and AGS. Meanwhile, ubiquitination assays have revealed that MDM2 specifically mediated the K63-linked polyubiquitination of IL-33, thereby enhancing its stability. Functionally, overexpressing IL-33 promoted malignant phenotypes in gastric cancer cells, such as increased cell viability, enhanced clonogenic growth, and augmented invasion and migration capabilities. Intriguingly, depleting MDM2 effectively abolished these promoting effects. Mechanistically, the MDM2-IL-33 axis exerted its influence by suppressing apoptosis through modulating the expression of Bcl-2/Bax/Caspase-3, and by activating the oncogenic ERK/JNK signaling pathway. Crucially, MDM2 overexpression promoted tumor growth, which was effectively reversed by transfection with si-MDM2. These findings firmly established MDM2 as a crucial regulator of IL-33 stability and highlighted the significant therapeutic potential of targeting the MDM2-IL-33 interaction to impede gastric cancer progression.