ASS1 inhibits liver cancer by promoting CAD ubiquitination and reversing the urea cycle and pyrimidine synthesis imbalance

Background: The urea cycle and pyrimidine synthesis occur mainly in the liver and undergo opposite changes during hepatocarcinogenesis. Argininosuccinate synthase 1 (ASS1) and carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) are key enzymes in the urea cycle and pyrimidine synthesis, respectively, and compete for the common substrate, aspartate. Moreover, ASS1 is lowly expressed in certain cancers, while CAD is highly expressed. However, the role of ASS1 and CAD in liver cancer still remains unclear. Methods: ASS1 and CAD expression in liver cancer were detected by tissue microarrays. Overexpression of ASS1 and CAD was achieved via lentivirus methods. All in vitro experiments were conducted in cells. The interactions of ASS1 and CAD were detected by co-immunoprecipitation (Co-IP) and GST-pull down. The in vivo study was conducted in a BALB/c nude mouse model. Intracellular metabolites were detected by LC–MS/MS. Results: ASS1 was lowly expressed in liver cancer, while CAD was highly expressed. In patients with recurrent liver cancer, ASS1 and CAD were significantly negatively correlated. Moreover, liver cancer patients with low ASS1 expression and high CAD expression had a poor prognosis. ASS1 and CAD interacted directly and promoted CAD ubiquitination through STUB1. In addition, Overexpression of CAD attenuated the tumor-suppressive effect of ASS1 in liver cancer cells. Pyrimidine supplementation enhanced the growth of liver cancer cells with ASS1 overexpression. Conclusions: ASS1 deficiency causes an imbalance in the urea cycle and pyrimidine synthesis in liver cancer. ASS1 directly controls the ubiquitination of CAD via STUB1, rather than just competing with aspartate, thereby suppressing liver cancer. Thus, ASS1 has potential as a druggable target in liver cancer.