TRIM21 promotes colorectal cancer malignancy by coupling USP4/TGF-β signaling to ferroptosis-related homeostasis.

Colorectal cancer is a major cause of cancer-related mortality, and its clinical management is still limited by recurrence, metastasis, and therapy resistance. Although ferroptosis is increasingly recognized as a therapeutically relevant vulnerability in colorectal cancer, the upstream regulators that link malignant signaling to ferroptosis-related homeostasis remain poorly defined. TRIM21 is a multifunctional E3 ubiquitin ligase with emerging roles in cancer biology, but its involvement in USP4/TGF-β-associated regulation and ferroptosis-related phenotypes in colorectal cancer is not well understood. Public transcriptomic datasets were analyzed to evaluate the clinical significance of TRIM21 expression across different clinicopathological categories. Gain- and loss-of-function approaches were applied in HCT116 cells to evaluate proliferation, migration, and invasion using CCK-8, wound-healing, and Transwell assays. We examined the relationship between TRIM21 and USP4 by co-immunoprecipitation and a rescue design involving TRIM21 silencing with USP4 re-expression. Ferroptosis-associated markers (SLC7A11 and GPX4) were evaluated using immunoblotting, and ferroptosis-related biochemical indices (Fe(2+), SOD activity, and MDA content) were quantified. Ferrostatin-1 was used to pharmacologically investigate ferroptosis under TRIM21/USP4 perturbations. TRIM21 was clinically associated with advanced clinicopathological features and was elevated in colorectal cancer cell models. Functional studies revealed that TRIM21 promotes proliferative and invasive/migratory phenotypes, accompanied by coordinated changes in USP4 and TGFB1 transcript levels. USP4 re-expression in TRIM21-silenced cells partially restored malignant traits and reshaped ferroptosis-associated molecular and biochemical readouts. Pharmacological inhibition of ferroptosis modulated TRIM21/USP4-linked phenotypes and corresponding ferroptosis-related indices. These findings identify TRIM21 as a clinically relevant regulator that links USP4/TGF-β-associated signaling to ferroptosis-related homeostasis, thereby promoting malignant behaviors in colorectal cancer and providing a targetable vulnerability for therapeutic development.