title: 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.
authors.affiliation: Unit of General and Clinical Pathology, Department of Experimental Medicine, Università degli Studi di Parma, Via Volturno 39, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Tardito
authors.forename: M
authors.initials: M
authors.lastname: Chiu
authors.forename: J
authors.initials: J
authors.lastname: Uggeri
authors.forename: A
authors.initials: A
authors.lastname: Zerbini
authors.forename: F
authors.initials: F
authors.lastname: Da Ros
authors.forename: V
authors.initials: V
authors.lastname: Dall'Asta
authors.forename: G
authors.initials: G
authors.lastname: Missale
authors.forename: O
authors.initials: O
authors.lastname: Bussolati
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001215:Asparaginase / Q000378:metabolism / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020717:Eukaryotic Initiation Factor-2B / Q000378:metabolism; D020868:Gene Silencing; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D058990:Molecular Targeted Therapy; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D020717:Eukaryotic Initiation Factor-2B; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D051176:beta Catenin; D005973:Glutamine; D001215:Asparaginase; D005974:Glutamate-Ammonia Ligase