Characterizing unexpected interactions of a glutamine transporter inhibitor with members of the SLC1A transporter family.

The solute carrier 1A family comprises a group of membrane proteins that act as dual-function amino acid transporters and chloride (Cl-) channels and includes the alanine serine cysteine transporters (ASCTs) as well as the excitatory amino acid transporters. ASCT2 is regarded as a promising target for cancer therapy, as it can transport glutamine and other neutral amino acids into cells and is upregulated in a range of solid tumors. The compound L-γ-glutamyl-p-nitroanilide (GPNA) is widely used in studies probing the role of ASCT2 in cancer biology; however, the mechanism by which GPNA inhibits ASCT2 is not entirely clear. Here, we used electrophysiology and radiolabelled flux assays to demonstrate that GPNA activates the Cl- conductance of ASCT2 to the same extent as a transported substrate, whilst not undergoing the full transport cycle. This is a previously unreported phenomenon for inhibitors of the solute carrier 1A family but corroborates a body of literature suggesting that the structural requirements for transport are distinct from those for Cl- channel formation. We also show that in addition to its currently known targets, GPNA inhibits several of the excitatory amino acid transporters. Together, these findings raise questions about the true mechanisms of its anticancer effects.

Inhibition of guanosine monophosphate synthetase (GMPS) blocks glutamine metabolism and prostate cancer growth.

Glutamine is a critical nutrient in cancer; however, its contribution to purine metabolism in prostate cancer has not previously been determined. Guanosine monophosphate synthetase (GMPS) acts in the de novo purine biosynthesis pathway, utilizing a glutamine amide to synthesize the guanine nucleotide. This study demonstrates that GMPS mRNA expression correlates with Gleason score in prostate cancer samples, while high GMPS expression was associated with decreased rates of overall and disease/progression-free survival. Pharmacological inhibition or knockdown of GMPS significantly decreased cell growth in both LNCaP and PC-3 prostate cancer cells. We utilized [15 N-(amide)]glutamine and [U-13 C5 ]glutamine metabolomics to dissect the pathways involved and despite similar growth inhibition by GMPS knockdown, we show unique metabolic effects across each cell line. Using a PC-3 xenograft mouse model, tumor growth was also significantly decreased after GMPS knockdown, highlighting the importance of glutamine metabolism and providing support for GMPS as a therapeutic target in prostate cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure, Mechanism and Roles in Physiology and Cancer.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems-the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues GltPh and GltTk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

Benzylserine inhibits breast cancer cell growth by disrupting intracellular amino acid homeostasis and triggering amino acid response pathways.

BACKGROUND: Cancer cells require increased levels of nutrients such as amino acids to sustain their rapid growth. In particular, leucine and glutamine have been shown to be important for growth and proliferation of some breast cancers, and therefore targeting the primary cell-surface transporters that mediate their uptake, L-type amino acid transporter 1 (LAT1) and alanine, serine, cysteine-preferring transporter 2 (ASCT2), is a potential therapeutic strategy. METHODS: The ASCT2 inhibitor, benzylserine (BenSer), is also able to block LAT1 activity, thus inhibiting both leucine and glutamine uptake. We therefore aimed to investigate the effects of BenSer in breast cancer cell lines to determine whether combined LAT1 and ASCT2 inhibition could inhibit cell growth and proliferation. RESULTS: BenSer treatment significantly inhibited both leucine and glutamine uptake in MCF-7, HCC1806 and MDA-MB-231 breast cancer cells, causing decreased cell viability and cell cycle progression. These effects were not primarily leucine-mediated, as BenSer was more cytostatic than the LAT family inhibitor, BCH. Oocyte uptake assays with ectopically expressed amino acid transporters identified four additional targets of BenSer, and gas chromatography-mass spectrometry (GCMS) analysis of intracellular amino acid concentrations revealed that this BenSer-mediated inhibition of amino acid uptake was sufficient to disrupt multiple pathways of amino acid metabolism, causing reduced lactate production and activation of an amino acid response (AAR) through activating transcription factor 4 (ATF4). CONCLUSIONS: Together these data showed that BenSer blockade inhibited breast cancer cell growth and viability through disruption of intracellular amino acid homeostasis and inhibition of downstream metabolic and growth pathways.