ISGylation-mediated stabilization of CYP4Z1 fuels breast cancer initiation and progression

Tumor-initiating cells (TICs) are central to cancer progression and metastasis; however, they remain challenging to target therapeutically. Here, using a breast-specific CYP4Z1-transgenic MMTV-PyMT mouse model, we show that CYP4Z1 notably accelerates tumor initiation, enlarges tumor size, promotes metastasis, and expands TIC populations. Multi-omics analyses reveal that CYP4Z1 + epithelial cells exhibit enhanced stemness and reduced differentiation compared with CYP4Z1 − counterparts. Notably, ISG15-mediated ISGylation of CYP4Z1 at lysine residues K259, K279, and K502 stabilizes the protein, augments its enzymatic function, promotes endoplasmic reticulum localization, and prevents ubiquitination, thereby amplifying TIC-like properties. This modification further upregulates triglyceride synthesis and promotes lipid droplet formation. Finally, we develop BM-51, a specific CYP4Z1 inhibitor that attenuates TIC-like traits and enhances chemotherapy efficacy. Our findings highlight the therapeutic potential of targeting CYP4Z1 and its ISGylation to disrupt TIC-driven breast cancer progression.