Targeting CAD with a tumor microenvironment-responsive nano-heterojunction for synergistic induction of cuproptosis and inhibition of colorectal cancer progression.

Carbamoyl-phosphate synthetase II/Aspartate transcarbamylase/Dihydroorotase (CAD) is a multifunctional enzyme with demonstrated oncogenic potential across various cancers, emerging as a promising therapeutic target. However, its functional role and therapeutic relevance in colorectal cancer (CRC) remain unclear. Here, we demonstrate for the first time that elevated CAD expression promotes CRC malignancy by enhancing proliferation, motility, and apoptosis resistance. Clinically, high CAD expression correlates with poor survival in CRC patients. Leveraging these findings, we developed a tumor microenvironment (TME)-responsive nickel‑copper nano‑heterojunction (NCSH@siCAD&AVT‑18A) that selectively releases hydrogen sulfide and copper ions in the acidic, reducing TME. This platform induces intracellular acidification and redox stress, leading to synergistic tumor suppression through a trimodal strategy that integrates CAD gene silencing, cuproptosis induction, and immune modulation. In vivo studies confirmed potent antitumor efficacy across subcutaneous, orthotopic, and metastatic CRC models, without systemic toxicity. Our work identifies CAD as a key prognostic marker and therapeutic target in CRC, and presents a novel combinatory regimen that integrates gene silencing with metal ion-mediated therapy, offering a mechanistically innovative and clinically viable strategy for CRC treatment with superior efficacy and minimal toxicity.