Aliases: AAAT, ATBO
Gene name: Solute carrier family 1 member 5 (SLC1A5)
ASCT2 is a ubiquitously expressed, broad-specificity, sodium-dependent neutral amino acid exchanger. Along with LAT1, a sodium-independent amino acid antiporter, ASCT2 is thought to be involved in the “harmonization” of extracellular and intracellular amino acid pools. ASCT2 displays functional asymmetry, i.e. it transports some substrates only inwardly while others bidirectionally. Cysteine, initially believed to be a substrate, later turned out to be a non-transported modulator. The prime function of ASCT2 is the cellular import of the conditionally essential amino acid glutamine. In a ternary antiport mechanism, extracellular glutamine accompanied by Na+ is exchanged with another neutral amino acid from the intracellular pool. Glutamine is a versatile metabolite that can be utilized both as an energy source and a biosynthetic building block, and rapidly proliferating cells often vastly overconsume this amino acid. Physiological processes like T-cell activation, as well as the growth of many cancer types heavily depend on ASCT2-mediated glutamine uptake. ASCT2 and LAT1 are coordinately overexpressed in multiple forms of cancer, and at least in some of them a functional cooperation between the two is required to maintain mTORC1 signaling. Inhibition of ASCT2 has effectively attenuated tumor growth in preclinical models, and the modulation of ASCT2 is regarded as a promising anticancer strategy. Although selective and potent inhibitors of ASCT2 have been identified and are being sought after, no currently approved drug is known to interfere with ASCT2 function, and the FDA or EMA guidelines contain no recommendations on testing interactions of NCEs with ASCT2.
Under physiological conditions, ASCT2 is distributed ubiquitously in the body, with protein expression detected in the lung, skeletal muscle, large intestine, kidney, testis, T cells, brain, and adipose tissue . In polarized epithelia such as intestinal or renal proximal tubule epithelial cells, ASCT2 is localized to the apical membrane domain . Aberrant overexpression of ASCT2, often in parallel with upregulation of LAT1, was documented in a wide variety of malignant tumors (see Clinical significance). Very recently, a shorter transcript variant of ASCT2 has been shown to localize to the mitochondria in cancer cells .
Function, physiology, and clinically significant polymorphisms
Both mouse and human ASCT2 were cloned and characterized in 1996 [4, 5]. Amino acid identity of human ASCT2 with the rat and mouse orthologs is 79 and 82%, respectively. The human SCL1A5 gene is located at 19q13.3 and contains 8 exons. The longest of three alternative transcripts encodes a 541-amino acid polypeptide with 8 transmembrane helices. Beside this extensively investigated major isoform, a shorter transcript variant lacking exon 1, encoding a 339-amino acid protein with a mitochondrial targeting signal, has recently been shown to be expressed in multiple cancer types under the control of HIF-2α . As comparatively little is known about the mitochondrial variant, the rest of this text refers to the plasma membrane isoform of ASCT2.
Cryo-EM imaging has confirmed that monomers of ASCT2, each organized into a fixed (scaffold) and an elevator (transport) domain, assemble into homotrimers in the membrane . N-glycosylation of ASCT2 is critical for trafficking to the membrane but not for intrinsic transport function .
Functional studies conducted with the purified rat protein have elucidated a peculiar three-substrate antiport mechanism, whereby the exchange of an extracellular neutral amino acid with an intracellular one is accompanied by the concomitant uptake of a Na+ ion with a 1:1:1 stoichiometry . ASCT2 exhibits both functional and kinetic asymmetry . Functional asymmetry refers to its ability to transport glutamine, serine, asparagine, and threonine in both directions, but alanine, valine, and methionine in the inward direction only. Kinetic asymmetry denotes a differential affinity to its substrates on the extracellular (Km in the micromolar range) versus the intracellular (Km in the millimolar range) side, which is in good agreement with the physiological concentrations of each substrate. Contrary to initial assumptions, cysteine is a modulator and not a substrate of ASCT2. Extracellular cysteine is not only a potent competitive inhibitor of glutamine uptake, but at higher concentrations it triggers unidirectional glutamine efflux without itself being inwardly transported. This modulatory function of cysteine, exerted through a putative allosteric site, is speculated to endow ASCT2 with a redox sensing capacity .
The major function of ASCT2 is the import of extracellular glutamine by exchange with another neutral amino acid such as serine, asparagine, or threonine from the intracellular pool. Glutamine is a conditionally essential amino acid: albeit cells can synthesize it de novo, few cell types are self-sufficient on glutamine and most tissues, especially those under increased metabolic demand, depend on external supply. Once in the cell, glutamine can be utilized in a variety of ways : it can enter the Krebs cycle to produce energy, or converted via reductive carboxylation into citrate to support lipid synthesis; its carbon chain and nitrogens can be incorporated into nucleobases or other amino acids; and it contributes in multiple ways to the biosynthesis of glutathione, a redox buffer that protects cells against oxidative stress. On the organismal level, glutamine also acts as a nitrogen carrier that shuttles ammonium from peripheral tissues, especially skeletal muscle, to the kidney. In accordance with such a multiplicity of roles, glutamine is the most abundant amino acid in human plasma with a steady-state concentration of 0.5-0.8 mM.
Since glutamine uptake is too important to be trusted on a single protein, this task is shared among multiple classes of broad-specificity amino acid transporters, and the likely reason why Slc1a5-null mice display no obvious phenotype  is compensation by functionally overlapping glutamine uptake systems, primarily the SLC38 family of sodium-coupled neutral amino acid transporters (SNATs). Thus, ASCT2 is probably dispensable in most mammalian tissues under healthy conditions. The few physiological processes in which ASCT2-mediated transport has been obligatorily implicated include T-cell activation  and the glutamine-glutamate cycle in the brain  and the placenta . Still in the brain, ASCT1 and 2 were shown to transport D-serine, a co-agonist of NMDA-type glutamate receptors .
Independently from its transporter function, ASCT2 is a receptor for retroviruses in nonhuman mammals, and mediates syncytium formation induced by fusogenic retroviral envelope proteins . In humans, ASCT2 has retained this function as a receptor of syncytin-1, an endogenized retroviral envelope protein that promotes placental syncytiotrophoblast formation  and osteoclast fusion .
Over 4000 SNPs of SLC1A5 are reported in the NCBI dbSNP database; however, apart from two variants suggested to associate with cancer [20, 21] and two other variants positively linked to human longevity , the significance of most SLC1A5 SNPs is unclear.
ASCT2 has been most extensively studied for its role in cancer. Metabolic reprogramming of cancer often involves increased utilization of glutamine both as an energy source and a biosynthetic building block, which is also referred to as “glutamine addiction”. Overexpression of ASCT2, usually in parallel with LAT1, is driven by oncogenes like MYC  and was observed in cancers of the prostate, lung, breast, kidney, the gastrointestinal tract and liver, the female reproductive tract, and the nervous system . ASCT2 was also shown to be critical for leukemia formation in a mouse model, and was suggested as a potential therapeutic target .
According to a model originally proposed by Fuchs and Bode  and experimentally confirmed by Nicklin et al. , ASCT2 and LAT1 are functionally coupled in cancer cells: their cooperation maintains mTOR signaling, thereby supporting proliferation while preventing autophagy and apoptosis. In this model, glutamine imported by ASCT2 is exchanged for essential amino acids (EAAs) such as leucine by LAT1, and EAAs ensure sustained mTORC1 kinase activity. Pharmacological blockade of ASCT2 by γ-L-glutamyl-p-nitroanilide (GPNA) suppressed glutamine uptake, mTORC1 signaling, and cell growth in human triple-negative basal-like breast cancer cell lines .
Such successes in vitro have motivated the development of more potent and specific inhibitors of ASCT2. Aided by a homology model built on the atomic structure of the archaean aspartate transporter GltPh, a family of 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid (AABA) inhibitors was discovered , the most potent of which, later named V-9302, exhibited >100-fold improved potency over GPNA (IC50 of V-9302 vs. GPNA, 9.6 μM vs. ~1 mM). Further preclinical development of V-9302 seemed to justify high expectations as treatment with the drug decreased viability of lung, breast, and colorectal cancer cell lines, and arrested the growth of human cell line xenografts in nude mice .
The ASCT2-centered interpretation of these results, however, became strongly challenged by subsequent studies that demonstrated an inhibitory effect of AABA compounds independent of ASCT2, and identified SNAT2 and LAT1 as the true molecular targets of V-9302 . These data favor an alternative model of glutamine flow in cancer cells in which SNAT1 (the “loader”) is primarily responsible for glutamine import, ASCT2 and LAT1 (the “harmonizers”) jointly equilibrate intracellular and extracellular neutral amino acid pools, and SNAT2 (the “rescue” transporter) is induced upon disruption of amino acid balance . Further corroboration that ASCT2 and LAT1 are harmonizers rather than drivers of amino acid accumulation came from experiments where abrogation of either ASCT2 or LAT1 in human liver cancer cell lines failed to suppress mTOR signaling or proliferation, and amino acid levels, albeit initially reduced, recovered over time in the knockdown cells . Knockout of ASCT2 from colon and lung adenocarcinoma cell lines significantly reduced glutamine import without affecting leucine uptake or mTORC1 activity, which speaks against functional coupling between ASCT2 and LAT1 in this system .
Nevertheless, ASCT2-KO tumors exhibited reduced growth as xenografts in vivo ; also, in a very recent study using human head and neck squamous cell carcinoma cell cultures and xenografts, silencing of ASCT2 alone was able to increase oxidative stress, suppress the mTORC1 pathway, and attenuate tumor growth, albeit the effect was enhanced by simultaneous blockade of SNAT2 with V-9302 . While the relative contribution of various transporters to glutamine supply may be context-dependent, ASCT2 is still regarded as an attractive anticancer target, and the development of novel inhibitors – now using homology models based on the crystal structure of a closer relative, the human glutamate transporter EAAT1 (SCL1A3) – continues [35, 36]. The latest family of inhibitors built around sulfonamide and sulfonic acid ester scaffolds explore previously unvisited regions of the chemical design space .
No currently approved drug is known to interfere with ASCT2 function, and the FDA or EMA guidelines contain no recommendations on testing interactions of NCEs with ASCT2.2.
Table: Summary information for ASCT2
In vitro substrates used experimentally
ubiquitous; known physiological roles in T cells, brain, placenta; overexpressed in cancer
neutral amino acids; preferred uptake substrate: glutamine
glutamine (mostly), alanine
GPNA, benzylserine, benzylcysteine, O-(4-phenylbenzoyl)-L-serine, (R)-γ-(4-biphenylmethyl)-L-proline, AABA compounds (?), sulfonamide/sulfonic acid ester inhibitors
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