Significant Down-Regulation of Urea Cycle Generates Clinically Relevant Proteomic Signature in Hepatocellular Carcinoma Patients with Macrovascular Invasion.

Vascular invasion is considered as the critical risk factor of hepatocellular carcinoma (HCC). To reveal the molecular mechanisms underlying macrovascular invasion (MaVI) in HCC, we performed an iTRAQ based proteomic study to identify notably dysregulated proteins from eight HCC patients with differential vascular invasion and further confirmed them in the other 53 HCC patients. Forty-seven proteins were found significantly down-regulated in HCC with MaVI. More importantly, 30 of them were not changed in HCC without MaVI. Gene ontology analysis of these 47 proteins shows the top three enriched biological processes are urea cycle, gluconeogenesis, and arginine biosynthetic process. We validated nine remarkably dysregulated candidates in HCC patients with MaVI by Western blot including eight down-regulated proteins (CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6, and CES1) and one up-regulated protein (CKAP4). Furthermore, dysregulation of CPS1, ASL, and ARG1, key enzymes involved in urea cycle, together with Annexin A6 and CES1, major proteins in regulating cholesterol homeostasis and fatty acid ester metabolism, was verified using immunohistochemical staining. The significant down-regulation of urea cycle generates clinically relevant proteomic signature in HCC patients with macrovascular invasion, which may provide possible insights into the molecular mechanisms of metastasis and new therapeutic targets of HCC.