ASS1 downregulation enhances pyrimidine synthesis that drives cancer cell proliferation.
Major finding: ASS1 downregulation enhances pyrimidine synthesis that drives cancer cell proliferation.
Mechanism: Decreased ASS1 promotes pyrimidine synthesis via CAD by increasing cytosolic aspartate availability.
Impact: Inhibition of thymidylate synthase or CAD reduces ASS1-deficient cell proliferation.
Somatic loss of argininosuccinate synthase 1 (ASS1), which encodes a urea cycle enzyme required for the production of arginine from aspartate and citrulline, is correlated with poor prognosis in patients with cancer. Aspartate is a substrate for the synthesis of arginine via ASS1 as well as pyrimidine nucleotides via carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), suggesting that reduced ASS1 activity may promote proliferation by increasing aspartate levels for pyrimidine synthesis. Consistent with this idea, Rabinovich, Adler, and colleagues found that fibroblasts derived from patients with citrullinemia type I, a urea cycle disorder caused by ASS1 deficiency, exhibited enhanced pyrimidine synthesis and proliferation compared with cells isolated from patients with citrullinemia type II, which is caused by inactivation of the mitochondrial aspartate transporter citrin (encoded by SLC25A13). Furthermore, ASS1 deficiency in cancer cell lines was associated with upregulation of CAD, increased pyrimidine synthesis and proliferation rate, and more rapid tumor growth in vivo. Overexpression of ASS1 in ASS1-deficient cancer cells was sufficient to induce diversion of aspartate away from pyrimidine synthesis and to decrease proliferation, which was restored by supplementation with nucleic acids. Similarly, reduction in cytosolic aspartate availability in ASS1-deficient cancer cells via knockdown of citrin resulted in decreased pyrimidine levels and diminished proliferation. Indeed, analysis of The Cancer Genome Atlas database showed that tumors with decreased ASS1 and elevated SLC25A13 levels were associated with worse prognosis. ASS1 deficiency in cancer cells also resulted in increased ribosomal protein S6 kinase 1–mediated phosphorylation of CAD downstream of mTOR activation, which was reversed by citrin knockdown. In line with this finding, decreasing pyrimidine synthesis using thymidylate synthase inhibitors or by CAD inhibition via treatment with the mTOR inhibitor rapamycin reduced ASS1-deficient cell proliferation. These results indicate that ASS1 downregulation drives cancer cell proliferation via modulation of aspartate availability for CAD and suggest potential therapeutic strategies for ASS1-deficient tumors.