Defective urea cycle promotes oncogenesis of hepatocellular carcinoma

PROJECT SUMMARY In mammals, majority of nitrogen waste is produced by gut microbiome in the form of ammonia. Ammonia is a neurotoxin and is cleared by two major pathways: 1) conversion via the urea cycle enzymes (UCEs) into nontoxic urea for excretion; and 2) assimilation into glutamate (Glu) by glutamate dehydrogenase (GDH) then

into glutamine (Gln) by glutamate ammonium ligase (GLUL), also termed glutamine synthetase (GS). In recent years, UCEs and Glu/Gln synthesis have been found to be involved in tumor growth/development in various tissues. Partially elevated UCE activities are implicated in tumor promotion via mechanisms such as enhanced

pyrimidine and polyamine production. Likewise, Glu/Gln biosynthesis is generally thought to be tumor- promoting in various cancers. Curiously, while the liver is the main organ that handles ammonia waste, the roles of the two ammonia handling pathways in liver cancer remain systematically unexplored. In

hepatocellular carcinoma (HCC), the major histological subtype of liver cancer, UCE expression is often down- regulated and correlates with worse prognosis, and GS expression is frequently elevated yet correlates with better outcomes. Using mouse models of HCC driven by oncogenic -catenin, we recently reported that -

catenin led to decreased expression of UCEs and increased expression of GS. Increased ammonia levels were observed in the plasma and tumor interstitial fluids, which were exacerbated by the ablation of hepatic GS. These data indicate that oncogenic -catenin leads to the disruption of nitrogen homeostasis, and that

defective ammonia-clearance promotes HCC growth. The main goals of this application are to study the roles of the UCEs in HCC, and to test the hypothesis that lowering ammonia burden can be a therapeutic strategy for HCC treatment/prevention. We propose three Specific Aims. Aim1: Test the hypothesis that suppression of

UCE expression contributes to the growth of HCC driven by -catenin. This will be done by 1) overexpressing or knocking down UCEs in various HCC mouse models; 2) studying the mechanism how down-regulation of UCEs may promote HCC. Aim 2: Study how oncogenic -catenin regulates UCE transcription. We will test how

-catenin interacts with other transcription factors such as C/EBP and HNF4a, and test how they interact with the UCE promoter/enhancer regions to regulate expression. Aim 3: testing that lowering ammonia burden can be a therapeutic/preventive strategy for HCC. This will be done by feeding mice with low protein diet and test

the effect on growth signaling and HCC progression in vivo. Successful accomplishment of the project will determine the role of UCEs in HCC, identify hyperammonemia as a risk factor, and help establish dietary intervention as a strategy for treating/preventing HCC.