Therapeutic targeting of the USP2-E2F4 axis inhibits autophagic machinery essential for zinc homeostasis in cancer progression

Macroautophagy (autophagy) is a conserved cellular process linked to tumorigenesis and cancer aggressiveness, though the mechanisms regulating the expression of autophagic genes in cancer remain unclear. In this study, we identified E2F4 (E2F transcription factor 4) as a novel transcriptional activator of cytoprotective autophagy, crucial for maintaining zinc homeostasis in cancer cells. Both gain- and loss-of-function experiments revealed that E2F4 promotes autophagy in a cell cycle-dependent manner, facilitating the degradation of metallothionein (MT) proteins, enhancing Zn²⁺ distribution within autophagosomes, reducing labile intracellular zinc, and promoting the growth, invasion, and metastasis of gastric cancer cells. Mechanistically, E2F4 directly activated the transcription of key autophagy-related genes, ATG2A (autophagy related 2A) and ULK2 (unc-51 like autophagy activating kinase 2), leading to the autophagic degradation of MT1E, MT1M, and MT1X. Additionally, USP2 (ubiquitin-specific peptidase 2) stabilized E2F4 protein levels through a physical interaction and deubiquitination, thereby enhancing its transactivation activity in cancer cells. Rescue experiments demonstrated that USP2 exerts oncogenic effects by facilitating E2F4-driven autophagy and zinc homeostasis. The autophagy inhibitor emetine blocked the interaction between USP2 and E2F4, increased labile intracellular zinc, and suppressed tumorigenesis and cancer aggressiveness. In clinical gastric cancer samples, both USP2 and E2F4 were upregulated and associated with poor patient prognosis. These findings suggest that targeting the USP2-E2F4 axis may offer a therapeutic strategy to inhibit autophagy and disrupt zinc homeostasis,Milciclib critical factors in cancer progression.