ZFPL1 Promotes Colorectal Cancer Progression by Stabilizing ASS1 to Drive the Urea Cycle and M2 Macrophage‐Mediated Metastatic Colonization

Abstract Colorectal cancer (CRC) progression is driven by diverse molecular mechanisms, underscoring the urgent need for novel therapeutic strategies, especially for liver metastases. Through an integrated analysis of multiple single‐cell RNA sequencing databases, zinc finger protein‐like 1 (ZFPL1) is identified as a gene specifically enriched in malignant cells from both primary and metastatic CRC. Multi‐omics investigations demonstrate that high ZFPL1 expression correlates with aggressive clinicopathological features and poor survival. Functionally, ZFPL1 promotes tumor proliferation, invasion, and migration both in vivo and in vitro. Mechanistically, ZFPL1 directly binds argininosuccinate synthase 1 (ASS1), shielding its K57 residue from tripartite motif containing 33‐mediated ubiquitination to prevent proteasomal degradation. This stabilization activates urea cycle metabolism, driving CRC progression. Crucially, ZFPL1 deficiency remodels the tumor microenvironment by reducing immunosuppressive populations‐M2 macrophages, and promoting pro‐inflammatory M1 polarization. Virtual screening identifies Salvianolic acid B (Sal B) as a ZFPL1 inhibitor, which disrupts ZFPL1‐ASS1 binding, triggering ASS1 ubiquitination and degradation. In vivo, Sal B synergized with anti‐PD‐1 therapy, significantly reducing tumor burden versus monotherapy. These findings establish ZFPL1 as a key regulator of CRC progression through ASS1‐dependent urea cycle activation and immunomodulation, nominating the ZFPL1‐ASS1 axis as a therapeutic target, with Sal B demonstrating combinatorial potential with immunotherapy.