Current Cancer Drug Targets, 2011, 11, 000-000 1 1568-0096/11 $58.00+.00 © 2011 Bentham Science Publishers L-Asparaginase and Inhibitors of Glutamine Synthetase Disclose Glutamine Addiction of -Catenin-Mutated Human Hepatocellular Carcinoma Cells S. Tardito 1 , M. Chiu 1,2 , J. Uggeri 3 , A. Zerbini 4 , F. Da Ros 1 , V. Dall’Asta 1 , G. Missale 4 and O. Bussolati* ,1 1 Unit of General and Clinical Pathology and 3 Unit of Histology, Department of Experimental Medicine, Università degli Studi di Parma, Via Volturno 39, Italy 2 Doctorate School in Molecular Medicine, University of Milan, Italy 4 Laboratory of Viral Immunopathology, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci 14, 43125 Parma, Italy 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 -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 -catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and -ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2 phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the -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. -catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another -catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, -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 -catenin-mutated HCC cells addicted to the amino acid. Keywords: L-Asparaginase, Cancer Metabolism, -Catenin, Glutamine, Hepatocellular Carcinoma. INTRODUCTION Hepatocellular carcinoma (HCC) is the major primary liver cancer and accounts for more than half a million deaths each year [1]. One of the reasons for this heavy death toll consists in the high level of chemoresistance presented by most HCC. Hence, novel therapeutic approaches are urgently needed. A common alteration observed in HCC is the activation of the Wnt--catenin pathway [2]. This pathway controls the intracellular levels of the multifunctional protein -catenin, which is both a cadherin-associated cytoskeletal component and a transcription factor. In the absence of Wnt signalling, the protein is rapidly phosphorylated by the “-catenin degradation machinery”, consisting of Apc, GSK3, Axin1 and 2, ubiquitinylated and then degraded by the proteasomes [3]. Wnt activation promotes the inactivation of GSK3 and, hence, a marked increase of the shelf life of -catenin. The free protein accumulates in the cytoplasm to migrate into the *Address correspondence to this author at the Unit of General and Clinical Pathology, Department of Experimental Medicine, Via Volturno 39 - 43100 Parma, Italy; Tel: +39 0521 033783; Fax: +39 0521 033742; E-mail: ovidio.bussolati@unipr.it nucleus where it complexes with members of the T-cell factor/ lymphocyte enhancer-binding factor (Tcf/LEF) family and promotes the transcription of several genes [4]. Several components of the -catenin pathway have been found mutated in HCC, the most frequently observed of which are CTNNB1 (for -catenin) and AXIN1 [5-7]. However, it has been reported that up to 60% of HCC have evident nuclear positivity for -catenin and cytoplasmic accumulation of the protein, the hallmark of Wnt pathway activation, although many of these tumors do not present mutations of either CTNNB1 or AXIN1 [8]. These observations suggest that also upstream components of Wnt cascade, such as the Frizzled-7 receptor, may be activated in HCC [9]. The gene GLUL codes for the enzyme Glutamine Synthetase (GS), which catalyzes glutamine (Gln) synthesis from glutamate and NH 4 + , and is among the best characterized targets of Wnt--catenin in HCC cells [10, 11]. Gln, the prevalent amino acid in human plasma, is used in several metabolic pathways, such as the biosynthesis of purine nucleotides. Moreover, Gln is an important organic osmolyte [12] and recent results suggest that it constitutes a major metabolic fuel for some types of tumor cells [13]. Therefore, it is not surprising that Gln depletion represents a