Transient kinetics reveal the mechanism of competitive inhibition of the neutral amino acid transporter ASCT2.

ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L-cis hydroxyproline biphenyl ester (Lc-BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

lncRNA SYTL5-OT4 promotes vessel co-option by inhibiting the autophagic degradation of ASCT2.

AIMS: Vessel co-option is responsible for tumor resistance to antiangiogenic therapies (AATs) in patients with colorectal cancer liver metastasis (CRCLM). However, the mechanisms underlying vessel co-option remain largely unknown. Herein, we investigated the roles of a novel lncRNA SYTL5-OT4 and Alanine-Serine-Cysteine Transporter 2 (ASCT2) in vessel co-option-mediated AAT resistance. METHODS: SYTL5-OT4 was identified by RNA-sequencing and verified by RT-qPCR and RNA fluorescence in situ hybridization assays. The effects of SYTL5-OT4 and ASCT2 on tumor cells were investigated by gain- and loss-of-function experiments, and those of SYTL5-OT4 on ASCT2 expression were analyzed by RNA immunoprecipitation and co-immunoprecipitation assays. The roles of SYTL5-OT4 and ASCT2 in vessel co-option were detected by histological, immunohistochemical, and immunofluorescence analyses. RESULTS: The expression of SYTL5-OT4 and ASCT2 was higher in patients with AAT-resistant CRCLM. SYTL5-OT4 enhanced the expression of ASCT2 by inhibiting its autophagic degradation. SYTL5-OT4 and ASCT2 promoted vessel co-option by increasing the proliferation and epithelial-mesenchymal transition of tumor cells. Combination therapy of ASCT2 inhibitor and antiangiogenic agents overcame vessel co-option-mediated AAT resistance in CRCLM. CONCLUSION: This study highlights the crucial roles of lncRNA and glutamine metabolism in vessel co-option and provides a potential therapeutic strategy for patients with AAT-resistant CRCLM.

How Much Do You Fuse? A Comparison of Cell Fusion Assays in a Breast Cancer Model.

Cell fusion is a biological process that is crucial for the development and homeostasis of different tissues, but it is also pathophysiologically associated with tumor progression and malignancy. The investigation of cell fusion processes is difficult because there is no standardized marker. Many studies therefore use different systems to observe and quantify cell fusion in vitro and in vivo. The comparability of the results must be critically questioned, because both the experimental procedure and the assays differ between studies. The comparability of the fluorescence-based fluorescence double reporter (FDR) and dual split protein (DSP) assay was investigated as part of this study, in which general conditions were kept largely constant. In order to be able to induce both a high and a low cell fusion rate, M13SV1 breast epithelial cells were modified with regard to the expression level of the fusogenic protein Syncytin-1 and its receptor ASCT2 and were co-cultivated for 72 h with different breast cancer cell lines. A high number of fused cells was found in co-cultures with Syncytin-1-overexpressing M13SV1 cells, but differences between the assays were also observed. This shows that the quantification of cell fusion events in particular is highly dependent on the assay selected, but the influence of fusogenic proteins can be visualized very well.

Novel Withanolides from Tubocapsicum anomalum Suppress Triple-Negative Breast Cancer by Triggering Apoptosis and p53-ASCT2-SLC7A11-Mediated Ferroptosis.

Triple-negative breast cancer (TNBC) is a malignant breast cancer. There is an urgent need for effective drugs to be developed for TNBC. Tubocapsicum anomalum (T. anomalum) has been reported to have an anti-tumor effect, and six novel withanolides were isolated from it and designated as TAMEWs. However, its anti-TNBC effect is still unknown. The results of an MTT assay indicated a higher sensitivity of TNBC cells to TAMEWs compared to other cells. TAMEWs induced apoptosis via mitochondrial dysfunction. They caused increased levels of lipid ROS and Fe2+, with downregulation of GSH and cystine uptake, and it has been confirmed that TAMEWs induced ferroptosis. Additionally, the results of Western blotting indicate that TAMEWs significantly decrease the expressions of ferroptosis-related proteins. Through further investigation, it was found that the knockdown of the p53 gene resulted in a significant reversal of ferroptosis and the expressions of its associated proteins SLC7A11, ASCT2, and GPX4. In vivo, TAMEWs suppressed TNBC growth with no obvious damage. The IHC results also showed that TAMEWs induced apoptosis and ferroptosis in vivo. Our findings provide the first evidence that TAMEWs suppress TNBC growth through apoptosis and ferroptosis.

Discovery and Synthesis of Hydroxy-l-Proline Blockers of the Neutral Amino Acid Transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2).

As a conformationally restricted amino acid, hydroxy-l-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened synthetic hydroxy-l-proline derivatives using electrophysiological and radiolabeled uptake methods against amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We have discovered a novel class of alkoxy hydroxy-pyrrolidine carboxylic acids (AHPCs) that act as selective high-affinity inhibitors of the SLC1 family neutral amino acid transporters SLC1A4 and SLC1A5. AHPCs were computationally docked into a homology model and assessed with respect to predicted molecular orientation and functional activity. The series of hydroxyproline analogs identified here represent promising new agents to pharmacologically modulate SLC1A4 and SLC1A5 amino acid exchangers which are implicated in numerous pathophysiological processes such as cancer and neurological diseases.

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.

CircMAT2B facilitates the progression of head and neck squamous cell carcinoma via sponging miR-491-5p to trigger ASCT2-mediated glutaminolysis.

Circular RNAs (circRNAs) are well-known to exert significant roles in regulating the pathological processes, including human carcinogenesis. Currently, less is known about their exact roles in head and neck squamous cell carcinoma (HNSCC). Herein, we aimed to investigate and validate the role of a novel circRNA, circMAT2B, as well as its potential molecular mechanism in HNSCC progression. A cohort of 41 paired of HNSCC tumor tissues and adjacent normal tissues from HNSCC patients were collected. Further, we characterized circMAT2B expression patterns in HNSCC tissues and cell lines, as well as exploring its association with the prognosis of HNSCC patients. Biological functions on cell proliferation, apoptosis, migration, and invasion were assessed using Cell Counting Kit-8, EdU incorporation, TUNEL, wound healing, and transwell assays. Glutaminolysis was evaluated by measuring glutamine, glutamate, and α-ketoglutarate (α-KG) levels. The regulatory network of circMAT2B/miR-491-5p/ASCT2 axis was verified by RNA immunoprecipitation and luciferase reporter assays. Western blot was conducted to detect the level of ASCT2 and GLS1. Remarkably overexpressed circMAT2B was observed in HNSCC tissues and cell lines, of which high abundance was positively correlated with patients' poor prognosis. Silencing of circMAT2B inhibited cell proliferation, migration, and invasion, as well as glutaminolysis. miR-491-5p, interacted with ASCT2, was identified to be a downstream target of circMAT2B, thereby involving in circMAT2B-mediated biological effects. In summary, we draw a conclusion that circMAT2B could modulate the processes of cell proliferation, migration, invasion, and glutaminolysis of HNSCC cells partly via the miR-491-5p/ASCT2 axis by a molecular mechanism of competing endogenous RNA (ceRNA), implying an underlying circRNA-targeted therapy for HNSCC treatment.

ß-Escin reduces cancer progression in aggressive MDA-MB-231 cells by inhibiting glutamine metabolism through downregulation of c-myc oncogene.

BACKGROUND: The c-myc oncogene, which causes glutamine dependence in triple negative breast cancers (TNBC), is also the target of one of the signaling pathways affected by ß-Escin. METHODS AND RESULTS: We sought to determine how c-myc protein affects glutamine metabolism and the proteins, glutamine transporter alanine-serine-cysteine 2 (ASCT2) and glutaminase (GLS1), in ß-Escin-treated MDA-MB-231 cells using glutamine uptake and western blot analysis. Cell viability, colony formation, migration and apoptosis were also evaluated in MDA-MB-231 cells in response to ß-Escin treatment using MTS, colony forming, wound healing, and Annexin-V assay. We determined that ß-Escin decreased glutamine uptake and reduced c-myc and GLS1 protein expressions and increased the expression of ASCT2. In addition, this inhibition of glutamine metabolism decreased cell proliferation, colony formation and migration, and induced apoptosis. CONCLUSIONS: In this study, it was suggested that ß-Escin inhibits glutamine metabolism via c-myc in MDA-MB-231 cells, and it is thought that as a result of interrupting the energy supply in these cells via c-myc, it results in a decrease in the carcinogenic properties of the cells. Consequently, ß-Escin may be promising as a therapeutic agent for glutamine-dependent cancers.

Inhibition of Glutamine Uptake Resensitizes Paclitaxel Resistance in SKOV3-TR Ovarian Cancer Cell via mTORC1/S6K Signaling Pathway.

Ovarian cancer is a carcinoma that affects women and that has a high mortality rate. Overcoming paclitaxel resistance is important for clinical application. However, the effect of amino acid metabolism regulation on paclitaxel-resistant ovarian cancer is still unknown. In this study, the effect of an amino acid-deprived condition on paclitaxel resistance in paclitaxel-resistant SKOV3-TR cells was analyzed. We analyzed the cell viability of SKOV3-TR in culture conditions in which each of the 20 amino acids were deprived. As a result, the cell viability of the SKOV3-TR was significantly reduced in cultures deprived of arginine, glutamine, and lysine. Furthermore, we showed that the glutamine-deprived condition inhibited mTORC1/S6K signaling. The decreased cell viability and mTORC1/S6K signaling under glutamine-deprived conditions could be restored by glutamine and α-KG supplementation. Treatment with PF-4708671, a selective S6K inhibitor, and the selective glutamine transporter ASCT2 inhibitor V-9302 downregulated mTOR/S6K signaling and resensitized SKOV3-TR to paclitaxel. Immunoblotting showed the upregulation of Bcl-2 phosphorylation and a decrease in Mcl-1 expression in SKOV3-TR via the cotreatment of paclitaxel with PF-4708671 and V-9302. Collectively, this study demonstrates that the inhibition of glutamine uptake can resensitize SKOV3-TR to paclitaxel and represents a promising therapeutic target for overcoming paclitaxel resistance in ovarian cancer.

Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor.

BACKGROUND: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. RESULTS: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. CONCLUSIONS: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

Interaction of the neutral amino acid transporter ASCT2 with basic amino acids.

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative ß-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

Topotecan induces hepatocellular injury via ASCT2 mediated oxidative stress.

BACKGROUND: Topotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. MATERIALS AND METHODS: Methyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. RESULTS: MTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. CONCLUSION: Topotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production.

Lobetyolin induces apoptosis of colon cancer cells by inhibiting glutamine metabolism.

The purpose of the present study was to evaluate the anti-cancer property of Lobetyolin on colorectal cancer and explore its potential mechanism. Lobetyolin was incubated with HCT-116 cells in the absence or presence of ASCT2 inhibitor Benser or p53 inhibitor Pifithrin-α. The levels of glutamine, glutamic acid, α-ketoglutarate, ATP and GSH were determined to measure the glutamine metabolism. Annexin V-FITC/PI staining and TUNEL assay were applied to estimate the apoptotic condition. The levels of ASCT2 were examined by RT-qPCR, Western blot and immunofluorescence staining. The expressions of cleaved-caspase-3, caspase-3, cleaved-caspase-7, caspase-7, cleaved-PARP, PARP, p53, p21, bax and survivin were detected using Western blot analysis. As a result, the treatment with Lobetyolin effectively induced apoptosis and glutamine metabolism in HCT-116 cells through ASCT2 signalling. The inhibition of ASCT2 reduced the glutamine-related biomarkers and augmented the apoptotic process. We further found that the effect of Lobetyolin on HCT-116 was related to the expressions of p21 and bax, and transportation of p53 to nucleus. The inhibition of p53 by Pifithrin-α promoted the inhibitory effect of Lobetyolin on ASCT2-mediated apoptosis. Lobetyolin also exerted anti-cancer property in nude mice. In conclusion, the present work suggested that Lobetyolin could induce the apoptosis via the inhibition of ASCT2-mediated glutamine metabolism, which was possibly governed by p53.

Capture of a Hyena-Specific Retroviral Envelope Gene with Placental Expression Associated in Evolution with the Unique Emergence among Carnivorans of Hemochorial Placentation in Hyaenidae.

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the maternofetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial), with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. In this study, we performed RNA sequencing on hyena placental transcripts and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the maternofetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 million years ago (Mya), being found at the same genomic locus in all 4 extant hyena species. Its coding sequence has further been maintained during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans-both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in ex vivo assays, at variance with the syncytin-Car1 gene, which is found in all carnivorans, including the hyena, in which it is still present, transcriptionally active in the placenta, and fusogenic. Together, the present results illustrate the permanent renewal of placenta-specific genes by retroviral capture and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.IMPORTANCE The placenta is the most diverse organ among mammals, due in part to stochastic capture of retroviral envelope genes. In carnivorans, capture of syncytin-Car1 took place 80 Mya. It is fusogenic, expressed at the syncytialized placental maternofetal interface, and conserved among all carnivorans, consistent with their shared endotheliochorial placenta. Hyenas are a remarkable exception, with a highly invasive hemochorial placenta, as found in humans, where disruption of maternal blood vessels results in maternal blood bathing the syncytial maternofetal interface. In this study, we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

Target the human Alanine/Serine/Cysteine Transporter 2(ASCT2): Achievement and Future for Novel Cancer Therapy.

Glutamine metabolism, described as major energy and building blocks supply to cell growth, has gained great attention. Alanine-Serine-Cysteine Transporter (ASCT2), which belongs to solute carried (SLC) family transporters and is encoded by the SLC1A5 gene serves as a significant role for glutamine transport. Indeed, ASCT2 is often overexpressed in highly proliferative cancer cells to fulfill enhanced glutamine demand. So far, ASCT2 has been proved to be a significant target during the carcinogenesis process, and emerging evidence reveals that ASCT2 inhibitors can provide a benefit strategy for cancer therapy. Herein, we describe the structure of ASCT2, and summarize its related regulatory factors which are associated with antitumor activity. Moreover, this review article highlights the remarkable reform of discovery and development for ASCT2 inhibitors. On the basis of case studies, our perspectives for targeting ASCT2 and development of ASCT2 antagonist are discussed in the final part.

Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1).

It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.

Amino Acid Transporters Are a Vital Focal Point in the Control of mTORC1 Signaling and Cancer.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets.

Amino Acid Transporters as Targets for Cancer Therapy: Why, Where, When, and How.

Amino acids are indispensable for the growth of cancer cells. This includes essential amino acids, the carbon skeleton of which cannot be synthesized, and conditionally essential amino acids, for which the metabolic demands exceed the capacity to synthesize them. Moreover, amino acids are important signaling molecules regulating metabolic pathways, protein translation, autophagy, defense against reactive oxygen species, and many other functions. Blocking uptake of amino acids into cancer cells is therefore a viable strategy to reduce growth. A number of studies have used genome-wide silencing or knock-out approaches, which cover all known amino acid transporters in a large variety of cancer cell lines. In this review, these studies are interrogated together with other databases to identify vulnerabilities with regard to amino acid transport. Several themes emerge, such as synthetic lethality, reduced redundancy, and selective vulnerability, which can be exploited to stop cancer cell growth.

The glutamine transporter ASCT2 (SLC1A5) promotes tumor growth independently of the amino acid transporter LAT1 (SLC7A5).

The transporters for glutamine and essential amino acids, ASCT2 (solute carrier family 1 member 5, SLC1A5) and LAT1 (solute carrier family 7 member 5, SLC7A5), respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry via the LAT1 exchanger, thus activating mechanistic target of rapamycin complex 1 (mTORC1) and stimulating growth. Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549-ASCT2KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1KO cells, ASCT2KO cells displayed no amino acid (AA) stress response (GCN2/EIF2a/ATF4) or altered mTORC1 activity (S6K1/S6). We therefore conclude that ASCT2KO reduces tumor growth by limiting AA import, but that this effect is independent of LAT1 activity. These data were further supported by in vitro cell proliferation experiments performed in the absence of glutamine. Together these results confirm and extend ASCT2's pro-tumoral role and indicate that the proposed functional coupling model of ASCT2 and LAT1 is not universal across different cancer types.

Glutaminolysis-related genes determine sensitivity to xCT-targeted therapy in head and neck squamous cell carcinoma.

Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.