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. The ISGylation of CYP4Z1 is controlled by enzymes associated with ISGylation, including E1 enzyme UBE1L, E2 enzyme UBCH8, E3 enzyme Herc5, and the de-ISGylation enzyme USP18. The ISGylation of CYP4Z1 enhances the tumorigenic properties of CYP4Z1 in two key ways. First, the ISGylation of CYP4Z1 inhibits its ubiquitination and degradation, resulting in elevated CYP4Z1 protein levels. Second, the ISGylation of CYP4Z1 facilitates the translocation of CYP4Z1 to the endoplasmic reticulum (ER), which promotes downstream triglyceride synthesis and lipid droplet formation. These processes contribute to tumor initiation, metastasis, and chemoresistance. The inhibitor BM-51, designed explicitly for CYP4Z1, can counteract the tumor-promoting effects of CYP4Z1 by suppressing its enzymatic activity. TAG, triglyceride; UB, ubiquitin.