PROJECT SUMMARY Glutamine (Gln) is a multifaceted amino acid that serves as important carbon and nitrogen sources. In a situation where circulatory supply of Gln is limited, such as in poorly vascularized tumors, cell autonomous Gln synthesis is turned on to assimilate the inorganic ammonia into Gln to support the nitrogen anabolic processes. In mammals, Gln synthesis is catalyzed by glutamine synthetase (GS) that condenses ammonium with glutamate to produce Gln. GS has been shown to enhance cell survival/growth/repairs in various cancers by promoting de novo glutamine synthesis and subsequent nitrogen anabolism. In the liver, where blood supply of Gln is abundant, GS-mediated Gln synthesis serves as a mechanism for ammonia detoxification next to the urea cycle. Interestingly, in hepatocellular carcinoma (HCC), the major histological subtype of liver malignancy, GS expression is frequently elevated whereas urea cycle enzymes (UCEs) down-regulated. Both elevated GS and decreased UCEs correlate well with -catenin activation, a prevalent oncogenic driving event in HCC. While this seems to be consistent with the common thought that GS promotes tumor growth, surprisingly, using the Glulflox/flox mouse, we recently reported that genetic ablation of hepatic GS accelerated tumor growth in several HCC models that involve -catenin6. Echoing the scenario in clinic, oncogenic -catenin suppressed the expression of the UCEs while induced the expression of GS. It was then speculated that the suppression of UCEs led to defective ammonia clearance, and GS was upregulated to help alleviate the hyperammonemia situation. GS ablation exacerbated the ammonia burden and facilitated the production of Glu-derived non- essential amino acids (NEAAs) that subsequently stimulated mTORC1. These findings prompt us to form the hypothesis that GS-mediated ammonia clearance functions to suppress tumor growth in -catenin-driven HCC. We propose two Specific Aims: 1) Study the theory that defective ammonia clearance promotes HCC. We will first determine whether GS suppresses HCC growth in a cell-intrinsic fashion, study how hyperammonemia promotes HCC, and take unbiased approach to uncover other mechanisms that may be responsible for enhanced HCC growth resulting from hyperammonemia. 2) Study how -catenin regulates GS expression in the liver. GS transcription is activated by Wnt/-catenin during liver zonal development and upon oncogenic transformation of hepatocytes. However, the underlying mechanism remains elusive. Our preliminary data suggest that GS expression is regulated by a liver-specific cis-acting distal enhancer. We will identify and characterize the liver-specific distal enhancer and study the underlying mechanism for GS expression regulated by the novel transcription factors that are associated with the enhancer. Accomplishing these Aims will help establish a novel theory that GS-mediated ammonia clearance is a tumor-suppressing mechanism in HCC, and will uncover ...