Multiple Glutamine Transporters Sustain the Growth of Glutamine Synthetase‐Negative Human Oligodedroglioma Cells
Authors: Chiu Martina , Sabino Cosimo , Allegri Manfredi , Bianchi Massimiliano G , Bussolati Ovidio 
Published: November 2023

In a growing group of human cancers, glutamine (Gln) transport is considered a possible therapeutic target to achieve tumor control. However, Gln transport is mediated by several carriers in human cells, thus complicating the delineation of a strategy to obtain an effective intracellular shortage of the amino acid. To evaluate the feasibility of this approach, we have investigated the effects of Gln depletion on two cell lines (Hs683 and HOG) of human oligodendroglioma (OD), a rare brain tumors characterized by lack of expression of Glutamine Synthetase (GS). We have hypothesized that if OD cells do not express GS, they should exhibit a strict dependence upon extracellular Gln and, hence, on the activity of Gln transporters. When cultured under control conditions (DMEM supplemented with 4 mM Gln), OD cells did not express detectable GS (Western Blot) and, at variance with other cell models, GS expression was not enhanced by Gln starvation. Gln‐free incubation or the glutaminolytic enzyme L‐asparaginase, which converts Gln into glutamate and ammonium, completely suppressed cell viability, demonstrating the absolute requirement of OD cells for extracellular Gln. Radiolabeled 3H‐Gln uptake was characterized under control conditions with or without Na+ in the presence of specific transporter inhibitors: threonine (Thr) for ASCT2; MeAIB for SNAT1/SNAT2, histidine (His) for SNAT3 and leucine (Leu) for LAT1. All these transporters were found expressed in OD cells at mRNA and protein level. While Gln uptake in HOG cells was mainly Na+‐dependent and mostly inhibited by Thr, and thus attributable to ASCT2 activity, Hs683 cells transported Gln mainly through a Na+‐independent, Leu‐sensitive transporter, identifiable with LAT1. In both cell lines, GPNA, an amino acid analogue used to inhibit the ASCT2 transporter in several other tumor cell models not only suppressed the Na+‐dependent influx, but also affected the Na+‐independent uptake, thus behaving as an inhibitor of both ASCT2 and LAT1. In both cell lines, the Na+‐dependent influx was completely inhibited combining Thr with His, thus suggesting that SNAT3 also contributes to Gln uptake. Under conditions of amino acid deprivation, a MeAIB‐sensitive Gln transport also emerged, suggesting that during nutritional stress OD cells are able to concentrate Gln through the SNAT1/SNAT2 transporters. The effects of transporter inhibitors on cell viability were also tested. Viability was markedly hindered by GPNA in both OD cell lines, while it was only partially affected by high doses of Thr, His and MeAIB. In conclusion, we found that (1) OD cells are GS‐negative and strictly depend upon extracellular Gln; (2) OD cells display a complex transportome for Gln uptake; (3) the impressive effects of GPNA on OD cell viability should not be attributed only to a specific inhibition of the ASCT2 transporter but also to a shortage of essential amino acids due to LAT1 inhibition. On the basis of these results, depletion of extracellular Gln caused by L‐asparaginase seems a more straightforward approach than transport inhibition for the control of olidendrogliomas and, possibly, other GS‐negative tumors.