L-Asparaginase and Inhibitors of Glutamine Synthetase DiscloseGlutamine Addiction of Beta-Catenin-Mutated Human HepatocellularCarcinoma Cells

Abstract: Selected oncogenic mutations support unregulated growth enhancing glutamine availability but increasing the dependence of tumor cells on the amino acid. Data from literature indicate that a subset of HepatoCellular Carcinomas (HCC) is characterized by mutations of beta-catenin and overexpression of Glutamine Synthetase (GS). To assess if this phenotype may constitute an example of glutamine addiction, we treated four human HCC lines with the enzyme LAsparaginase (ASNase), a glutaminolytic drug. ASNase had a significant antiproliferative effect only in the beta-catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and alpha-ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2alpha phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the beta-catenin wild type cell line Huh-7. When used with ASNase, the GS inhibitor methionine sulfoximine (MSO) emptied cell glutamine pool, rresting proliferation in ASNase-insensitive Huh-7 cells and activating caspase-3 and apoptosis in HepG2 cells. Compared with Huh-7 cells, HepG2 cells accumulated much higher levels of glutamine and MSO, due to the higher expression and activity of SNAT2, a concentrative transporter for neutral amino acids, but were much more sensitive to glutamine withdrawal from the medium. In the presence of ASNase, MSO caused a paradoxical maintenance of rapamycin-sensitive mTOR activity in both HepG2 and Huh-7 cells. Beta-catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another beta-catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, beta-catenin mutated HCC cells are more sensitive to glutamine depletion and accumulate higher levels of GS inhibitors. These results indicate that glutamine deprivation may constitute a targeted therapy for beta-catenin-mutated HCC cells addicted to the amino acid