Deletion of Amino Acid Transporter ASCT2 (SLC1A5) Reveals an Essential Role for Transporters SNAT1 (SLC38A1) and SNAT2 (SLC38A2) to Sustain Glutaminolysis in Cancer Cells

Abstract

Many cancer cells depend on glutamine as they use the glutaminolysis pathway to generate building blocks and energy for anabolic purposes. As a result, glutamine transporters are essential for cancer growth and are potential targets for cancer chemotherapy with ASCT2 (SLC1A5) being investigated most intensively. Here we show that HeLa epithelial cervical cancer cells and 143B osteosarcoma cells express a set of glutamine transporters including SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT4 (SLC38A4), LAT1 (SLC7A5), and ASCT2 (SLC1A5). Net glutamine uptake did not depend on ASCT2 but required expression of SNAT1 and SNAT2. Deletion of ASCT2 did not reduce cell growth but caused an amino acid starvation response and up-regulation of SNAT1 to replace ASCT2 functionally. Silencing of GCN2 in the ASCT2(؊/؊) background reduced cell growth, showing that a combined targeted approach would inhibit growth of glutamine-dependent cancer cells.Differentiated, non-dividing cells generate energy by metabolizing nutrients via the TCA 2 cycle and respiratory chain, resulting in the production of carbon dioxide and water (1). Synthesis of macromolecules is limited to essential turnover of proteins and polynucleotides. Rapidly dividing cells, by contrast, synthesize significant amounts of protein, DNA, RNA, and membrane lipids de novo. Synthesis of these compounds withdraws large amounts of metabolites from metabolic pathways. Cyclic pathways, such as the TCA cycle, are very sensitive to withdrawal of metabolites because depletion of TCA cycle intermediates will render the cycle non-functional and compromise the capacity to generate energy (2). Linear pathways, by contrast, increase the flow-through to adapt to high metabolic demand. Converting the TCA cycle into a linear pathway is of considerable advantage to rapidly dividing cells, such as cancer cells, activated lymphocytes, and stem cells. This linearized version of the TCA cycle has been termed glutaminolysis, allowing generation of energy and metabolic building blocks (2, 3). Glutamine, the starting substrate of this pathway, is imported by the cell and deaminated to glutamate. Glutamate is converted into 2-oxoglutarate by glutamate dehydrogenase or by a transaminase reaction. After going through several steps of the TCA cycle, malate or oxaloacetate is produced. Oxaloacetate can be converted into aspartate, which is a major precursor for nucleotide biosynthesis. Both oxaloacetate and malate can also be converted into pyruvate, which can be used to generate alanine, lactate, or acetyl-CoA. As a result, many cancer cells depend on glutamine unless they express glutamine synthetase (4). It is worth noting that glutaminolysis through the conversion of 2-oxoglutarate to oxaloacetate produces two NADH, one FADH, and one GTP and therefore provides the cell with significant energy. Activation of the glutaminolysis pathway in cancer cells is accompanied by a reduced entry of pyruvate into the TCA cycle (5).The adaptation of metabolism to cellular growth is associated with transcription fa...

Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2

“…To address this question a HeLa SNX27 KO cell line (18) were examined. We initially examined any changes of ASCT2 along with SNAT1 (SLC38A1) and SNAT2 (SLC38A2), the two other amino acid transporters implicated in cellular glutamine uptake (19), at the transcriptional level. Quantitative real time PCR analysis by TaqMAN assay demonstrated that the KO of SNX27 did not affect the gene expressions levels of these transporters ( Fig.2A).…”

Identification of novel inhibitors of the amino acid transporter B0AT1 (SLC6A19), a potential target to induce protein restriction and to treat type 2 diabetes

“…To this end, glutamine uptake was measured in the presence and absence of Na + , and compared to the same transport activities in the presence of inhibitor. In Figure 5B the inhibition of net Na + -dependent glutamine transport is shown, which is largely mediated by ASCT2 (Broer et al, 2016). Leucine transport in MCF-7 cells, by contrast, is largely mediated by system L (molecular isoforms LAT1 or LAT2), which can be inhibited by the amino acid analogue 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH, Figure 5C) and α-methyl-tyrosine.…”

The potential of inhibiting glutamine uptake as a therapeutic target for multiple myeloma

“…Unfortunately, both compounds are less than optimal. Although employed to suppress ASCT2 activity, benzylserine has been recently found ineffective in inhibiting the transporter in Xenopus oocytes [9], while it directly inhibits LAT1 in the same model [10]. Conversely, in the same model, GPNA exhibited a marked inhibitory effect on ASCT2, but the inhibitor also suppressed the activity of several SNAT transporters [9].…”

“…Although employed to suppress ASCT2 activity, benzylserine has been recently found ineffective in inhibiting the transporter in Xenopus oocytes [9], while it directly inhibits LAT1 in the same model [10]. Conversely, in the same model, GPNA exhibited a marked inhibitory effect on ASCT2, but the inhibitor also suppressed the activity of several SNAT transporters [9]. In spite of lack of a complete structural characterization of the carrier, other ASCT2 inhibitors have been proposed and used in experimental therapy [11], but their specificity awaits confirmation.…”

“…Until this objective is achieved, the aim to employ this transporter as a specific therapeutic target appears hardy feasible. Although the role of ASCT2 in Gln accumulation has been recently discussed [9], its specific inhibition remains a valuable objective, also considering the relationships between its activity and mTORC1 activation. However, the role of the various transporters involved in Gln transport in MM cells, in particular SNAT1, still awaits a complete characterization, especially upon Gln shortage, when this transporter may be upregulated, or under the hypoxic conditions encountered in BM in vivo.…”