L-Asparaginase and inhibitors of glutamine synthetase disclose glutamine addiction of β-catenin-mutated human hepatocellular carcinoma 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 β-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 L-Asparaginase (ASNase), a glutaminolytic drug. ASNase had a significant antiproliferative effect only in the β-catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and α-ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2α phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the β-catenin wild type cell line Huh-7. When used with ASNase, the GS inhibitor methionine sulfoximine (MSO) emptied cell glutamine pool, arresting 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. β-catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another β-catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, β-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 β-catenin-mutated HCC cells addicted to the amino acid.