ASCT2 regulates glutamine uptake and cell growth in endometrial carcinoma.

Glutamine commonly becomes a conditionally essential amino acid in cancer. Glutamine is supplied to the cell by transporters such as ASCT2 (SLC1A5), which is frequently upregulated in multiple cancers. Here we investigated the expression of ASCT2 in endometrial carcinoma, and evaluated the contribution of ASCT2 to glutamine uptake and endometrial cancer cell growth. Analysis of human gene expression data showed that ASCT2 was significantly upregulated in both endometrioid and serous subtypes of endometrial carcinoma, compared to normal, age-matched endometrium. Furthermore, immunohistochemical staining of primary human endometrioid adenocarcinomas showed that tumours stain positive for ASCT2 in either a uniform or mosaic expression pattern, while normal adjacent glands appeared predominantly negative for ASCT2 staining. Chemical inhibition of glutamine transport by benzylserine or GPNA led to a significant decrease in endometrial cancer cell growth and spheroid cross-sectional area. ASCT2 knockdown recapitulated the decrease of cell growth and spheroid cross-sectional area in HEC1A cells, suggesting a reliance on ASCT2-mediated glutamine uptake. ASCT2 knockdown in Ishikawa cells led to lower glutamine uptake and cell growth, but did not affect spheroid area. Ishikawa cells express higher levels of the glutamine transporter SNAT1 compared to HEC1A cells, suggesting these cells may rely on both ASCT2 and SNAT1 for glutamine uptake. Since SNAT1 is also significantly upregulated in the endometrioid and serous subtypes, these data indicate that ASCT2 and SNAT1 could be used as markers of malignancy, and/or potential therapeutic targets in patients with endometrial carcinoma.

ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer.

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.

Glutamate potentiates lipopolysaccharide-stimulated interleukin-10 release from neonatal rat spinal cord astrocytes.

Interleukin-10 (IL-10) has important anti-inflammatory effects and can be protective in inflammatory conditions, such as chronic pain and infection. Exploring factors that modulate IL-10 levels may provide insight into pathomechanisms of inflammatory conditions and may provide a method of neuroprotection during these conditions. Lipopolysaccharide (LPS) stimulation of astrocytes is a source of IL-10; hence, it is of interest to investigate factors that modulate this process. Glutamate is present in increased concentrations in inflammatory conditions, and astrocytes also express glutamate receptors. The present study, therefore, investigated whether glutamate modulates LPS stimulation of IL-10 release from neonatal spinal cord astrocytes. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify IL-10 release from cultured neonatal spinal cord astrocytes, and reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure IL-10 mRNA expression. Glutamate (1 mM) significantly increased LPS (1 µg/ml)-stimulated IL-10 release from astrocytes by 166% and significantly upregulated IL-10 mRNA levels. Glutamate synergistically signaled through metabotropic glutamate receptor subgroups and the phospholipase C signaling pathway. Spinal cord astrocytes may, therefore, play a larger anti-inflammatory role than first thought in situations where glutamate and a high concentration of Toll-like receptor 4 (TLR4) agonists are present.

Lipopolysaccharide-stimulated interleukin-10 release from neonatal spinal cord microglia is potentiated by glutamate.

Interleukin-10 (IL-10) is a cytokine with important endogenous and therapeutic anti-inflammatory effects. Given this, it is of interest to investigate factors that modulate IL-10 levels in the central nervous system. IL-10 is released after lipopolysaccharide (LPS) stimulation of microglia. Microglia also express functional glutamate receptors and in inflammatory conditions are exposed to increased levels of glutamate. The aim of this research, then, is to investigate whether glutamate can modulate lipopolysaccharide stimulation of IL-10 release from neonatal rat spinal cord microglia. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify IL-10 release from cultured neonatal spinal cord microglia and reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure IL-10 mRNA expression. Glutamate (1 mM) significantly increased LPS (1 µg/ml)-stimulated IL-10 release from microglia by 172% (EC(50) of 103 µM) and significantly upregulated IL-10 mRNA levels. Glutamate potentiated LPS-stimulated IL-10 release by binding all subtypes of glutamate receptor. These results show that glutamate substantially increases the release of an anti-inflammatory cytokine from neonatal spinal cord microglia activated by a high concentration of LPS.

Antipsychotic drugs dose-dependently suppress the spontaneous hyperactivity of the chakragati mouse.

The chakragati (ckr) mouse has been proposed as a model of aspects of schizophrenia. The mice, created serendipitously as a result of a transgenic insertional mutation, exhibit spontaneous circling, hyperactivity, hypertone of the dopamine system, reduced social interactions, enlarged lateral ventricles, deficits in pre-pulse inhibition of acoustic startle and deficits in latent inhibition of conditioned learning. In this study, the dose-dependent effects of antipsychotic drugs (haloperidol, pimozide, risperidone, clozapine, olanzapine, ziprasidone, quetiapine and aripiprazole) on the spontaneous hyperactivity of the mice were investigated. All the antipsychotic drugs tested dose-dependently suppressed spontaneous hyperactivity. Aripriprazole, which is known to be a dopamine D2 receptor partial agonist, exhibited a tri-phasic dose-response, initially suppressing hyperactivity at low doses, having little effect on hyperactivity at intermediate doses, and suppressing activity again at high doses. These data suggest that the spontaneous circling and hyperactivity of the ckr mouse may allow screening of candidate antipsychotic compounds, distinguishing compounds with aripriprazole-like profiles.