CAD manipulates tumor intrinsic DHO/UBE4B/NF-κB pathway and fuels macrophage cross-talk, promoting hepatocellular carcinoma metastasis

Background and Aims: Portal vein tumor thrombosis (PVTT), an indicator of clinical metastasis, significantly shortens hepatocellular carcinoma (HCC) patients’ lifespan, and no effective treatment has been established. We aimed to illustrate mechanisms underlying PVTT formation and tumor metastasis, and identified potential targets for clinical intervention. Approach and Results: Multi-omics data of 159 HCC patients (including 37 cases with PVTT) was analyzed to identify contributors to PVTT formation and tumor metastasis. In vitro and in vivo experiments were performed to confirm the critical role of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) in HCC metastasis. Metabolomics and transcriptomics techniques, single-cell RNA sequencing, combined with experimental verification were complemented to illustrate mechanisms underlying CAD induced pro-metastatic efficacy. Analysis of proteogenomic data of HCC cohort identified CAD as the key contributor to PVTT formation and tumor metastasis in HCC. Further experiments confirmed that high CAD expression could significantly promote HCC metastasis, and vice versa. Mechanistically, CAD manipulated de novo pyrimidine anabolism, leading to dihydroorotic acid (DHO) accumulation which directly bound to ubiquitination factor E4B (UBE4B). UBE4B subsequently regulated JAK1 ubiquitination and activated the NF-κB pathway to promote epithelial-mesenchymal transition (EMT) of HCC cells. Additionally, CAD generated an immunosuppressive milieu conducive to HCC metastasis by recruiting and reprogramming macrophages into a “pro-tumor” phenotype. Consequently, the metastatic capability of HCC was remarkably enhanced. Conclusion: Therapy targeting CAD may offer a promising approach to curb HCC metastasis by reducing tumor cells’ metastatic potential and also shifting the tumor microenvironment towards a less pro-metastatic state.