Cancer cells need to generate large amounts of glutathione (GSH) to buffer oxidative stress during tumor development. A rate-limiting step for GSH biosynthesis is cystine uptake via a cystine/glutamate antiporter Xc−. Xc− is a sodium-independent antiporter passively driven by concentration gradients from extracellular cystine and intracellular glutamate across the cell membrane. Increased uptake of cystine via Xc− in cancer cells increases the level of extracellular glutamate, which would subsequently restrain cystine uptake via Xc−. Cancer cells must therefore evolve a mechanism to overcome this negative feedback regulation. In this study, we report that glutamate transporters, in particular SLC1A1, are tightly intertwined with cystine uptake and GSH biosynthesis in lung cancer cells. Dysregulated SLC1A1, a sodium-dependent glutamate carrier, actively recycled extracellular glutamate into cells, which enhanced the efficiency of cystine uptake via Xc− and GSH biosynthesis as measured by stable isotope-assisted metabolomics. Conversely, depletion of glutamate transporter SLC1A1 increased extracellular glutamate, which inhibited cystine uptake, blocked GSH synthesis, and induced oxidative stress-mediated cell death or growth inhibition. Moreover, glutamate transporters were frequently upregulated in tissue samples of patients with non–small cell lung cancer. Taken together, active uptake of glutamate via SLC1A1 propels cystine uptake via Xc− for GSH biosynthesis in lung tumorigenesis.
Cellular GSH in cancer cells is not only determined by upregulated Xc− but also by dysregulated glutamate transporters, which provide additional targets for therapeutic intervention.
Cancer cells are facing two emergent needs, an increased demand of metabolic building blocks for growing biomass, and an increased demand of protection against reactive oxygen species (ROS). Compared with normal cells, cancer cells experience increased environmental stresses, including oxygen or nutrient deficiency, low pH, mediators of inflammation, and oxidative stress from ROS (1). ROS can be generated from increased metabolism within tumor cells, and induced by pro-inflammatory stimuli and TNFα in the microenvironment (2). High ROS levels in the tumor microenvironment can be detrimental, since ROS exerts oxidative stress that can trigger senescence or programmed cell death (1, 3). Thus, the metabolic profile of tumor cells, especially cancer stem cells, must be reprogrammed to counteract the toxicity of ROS.
Glutathione (GSH) is the most abundant antioxidant within all cells. GSH is synthesized in a two-step process. First, glutamate-cysteine ligase (GCL) catalyzes the formation of γ-glutamylcysteine from glutamate and cysteine, which is the rate limiting step. Second, GSH synthetase couples glycine to γ-glutamylcysteine to form GSH. The availability of cysteine, from a cystine transporter, is considered rate limiting for GSH synthesis. System Xc−, a Na+-independent cystine/glutamate antiporter composed of a light-chain subunit (xCT or SLC7A11) and a heavy-chain subunit (CD98hc or SLC3A2), mediates the extracellular cystine influx for exchange of intracellular glutamate efflux. Recent studies showed that CD44v could interact with and stabilize xCT at the plasma membrane, which emphasizes the importance of GSH synthesis in maintaining the stemness of cancer stem cells (4). Thus, the efficiency of cystine uptake via system Xc− is crucial for the GSH-dependent antioxidant system (5). Xc− antiporter is driven by the concentration gradients of glutamate and cystine across plasma membrane, which transports one cystine into the cell in the expense of exporting one glutamate out. Thus, extracellular glutamate acts as a competitive inhibitor for cystine uptake via system Xc− (6). Because high concentration of extracellular glutamate inhibit cystine uptake via Xc−, it remains a paradox how cancer cells enhance cystine uptake whereas expel glutamate in exchange for cystine. Presumably, cancer cells should evolve a mechanism to import extracellular glutamate for maintaining a glutamate gradient across membrane, which can facilitate cystine uptake via Xc− system for GSH biosynthesis.
Excitatory amino acid carrier 1 (EAAC1 or EAAT3) is a member of the EAAT family of high-affinity, sodium-dependent glutamate carriers encoded by solute carrier family 1 member 1 (SLC1A1; ref. 7). It is generally accepted that three Na+ ions and one H+ are cotransported whereas one K+ is counter-transported with each glutamate molecule (8). EAAT family has five members, EAAT1 to EAAT5. These proteins are “high-affinity” glutamate transporters. SLC1A1 (EAAT3) is mainly expressed in the brain, but also in the kidney and the intestinal mucosa. Within the brain, SLC1A1 serves as the predominant glutamate transporter. SLC1A1 uptakes synaptic released glutamate, the excitatory neurotransmitter, in the inter-neuronal cleft. Deficiency in uptake of extracellular glutamate leads to oxidative stress and exocitotoxicity (9). Slc1a1-deficient mice show age-dependent brain atrophy, learning/memory dysfunction, and reduced brain GSH levels (10). Thus, SLC1A1 is important for neuronal GSH synthesis. SLC1A1 is also present at low level in muscle and lung. However, the role of glutamate transporters in cancer cells is largely unknown.
Lung cancer is the leading cause of cancer death worldwide (11). Revealing the mechanism of lung tumorigenesis requires an animal model that resembles the pathological features of human lung cancer. G protein-coupled receptor family C group 5 type A (GPRC5A; also known as RAIG1), a newly identified lung tumor suppressor gene, is preferentially expressed in lung tissues (12–14). Gprc5a gene knockout (ko) in mice leads to development of spontaneous lung adenocarcinoma (13, 14), which is associated with chronic lung inflammation, persistent activation of NF-κB, EGFR, and STAT3 signaling (15–17). Importantly, GPRC5A is repressed in most of non–small cell lung cancer (NSCLC) and all of chronic obstructive pulmonary disease (COPD) (13, 18). Thus, Gprc5a-ko mice provide a unique mouse model that resembles lung cancer development in human.
In this study, we investigated the metabolic reprogramming of lung tumors in Gprc5a-ko mouse model. Unexpectedly, we found that glutamate transporter SLC1A1, dysregulated in lung cancer cells, is tightly intertwined with cystine uptake and GSH biosynthesis. Our study demonstrates that upregulation of glutamate transporter SLC1A1, via increasing glutamate influx, facilitates cystine uptake and GSH biosynthesis for lung tumor development.
Materials and Methods
Mice and tumorigenicity
Gprc5a-ko mice, from Dr. R. Lotan (University of Texas M.D. Anderson Cancer Center), were generated in a mixed background of 129sv × C57BL/6 as described previously (13). Mice were maintained according to a protocol approved by Shanghai Jiao Tong University School of Medicine Animal Care and Use Committee [experimental animal use permission No. SYXK (Shanghai) 2008-0050] in the specific pathogen-free animal facility in the university. Eight-week-old wild-type (WT; Gprc5a+/+) and Gprc5a−/− mice were received 2 weekly intraperitoneal injections of NNK (100 mg/kg of body weight; Midwest Research Institute, Kansas City, MO) dissolved in saline solution (0.9% NaCl) or saline alone. Ten to twelve months later, mice were sacrificed, one lube of lung was fixed in paraffin for hematoxylin and eosin staining analysis, the rest lung tissues were homogenized in liquid nitrogen for extraction of protein and RNA (17).
Derivation of primary mouse cells
Primary mouse lung cancer cells (SJT-1601) were isolated from NNK-induced lung tumor of Gprc5a-ko mouse. Cells were digested into single cells and seed the cells onto the palate over 3 days, then passing on the living cells for passaging. Unless otherwise stated, SJT-1601 cell were cultured in a humidified 37°C atmosphere of 21% oxygen, 5% carbon dioxide, in high glucose DMEM (Invitrogen) supplemented with 10% FBS (Gibco), penicillin, and streptomycin.
Cell lines and cell culture
Mouse tracheal epithelia cells (MTEC) were obtained from normal tracheal tissue of 3-week-old WT and Gprc5a-KO mice (C57 BL/6 × 129sv) as described previously (15, 16). The MTEC cells were cultured in K-SFM supplemented with epithelia growth factor (EGF, 5 ng/mL) and bovine pituitary extract (50 mg/mL, Invitrogen). The established mouse lung tumor cell line, named SJT-1601, was derived from the NNK-induced lung tumor of a Gprc5a-ko mouse. Human embryonic kidney cells, HEK293T, and NSCLC cells (H292G, Calu-1, and HCC827) were tested and authenticated by DNA typing at Shanghai Jiao Tong University Analysis Core. The 16HBE (Human bronchial epithelial cells) cells were as described previously (19). HBEC cells were cultured in serum-free medium, and all other cells were cultured in DMEM essential medium with 10% fetal calf serum, at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO2.
Tumorigenicity in nude mice
Eight-week-old nude mice were injected with A549-shNC/SLC1A1 (1.5 × 106 cells) combined with Matrigel. Two weeks later, the volume of tumors were measured and further analyzed.
Recombinant mouse FGF-basic (FGFβ, PMG0034) was from Gibco by life technologies. d-Luciferin (S7763) was from Selleck.cn. EGF (E9644), insulin (I3536), BSA (V900933), DMEM-high glucose (D0422-100ML), GSH-MEE (#G1404), vitamin E analog (α-tocopherol; #T3251) and l-buthionine-sulfoximine (BSO; B2515) were from Sigma-Aldrich. Matrigel Matrix (354262) was from Corning. B27(1639356) was from Gibico. l-Glutamic acid (D316BA0011) was from BBI Life Sciences. l-Glutamine (13C5, 99%; CLM-1822-H-0.5), l-Glutamic acid (13C5, 99%; CLM-1800-H-1), [13C6,15N2]-cystine (CNLM-4244-H-PK) and d-glucose (13C6, 99%; CLM-1396-5) were from Cambridge Isotope Laboratories. Annexin V-FITC reagent (C1062S), ROS Kit (S0033) was from Beyotime.
The following antibodies were used: anti-SLC1A1 antibody (12686-1-AP) for IHC was from Proteintech. Anti-SLC1A1 antibody (#14501), anti-cleaved-PARP (#5625), anti-phospho-p38 MAPK (#4511), and phospho-p38 (#8690T) were from Cell Signaling Technology. Anti-RAI3 (Gprc5a; sc-98884) was from Santa Cruz Technology. Anti-β-ACTIN-HRP (PM053-7) was from MBL. Anti-SLC1A1 antibody (#14501) was from Abcam. The antibodies were diluted according to manufacturers' instructions.
A 2% agarose solution was combined with DMEM-10%FBS (1:3 v/v; final concentration, 0.7%) and added to 24-well plates (0.5 mL per well), and allowed to solidify for 10 minutes at 4°C. MTEC or NSCLC cells (500 cells in 50 μL medium) were added over the solidified agarose. A top 0.5 mL agarose layer was added over the cells, consisting of the 2% agarose solution combined with DMEM—10%FBS (1:6 v/v; final concentration, 0.35%) along with Matrigel (1:30 v/v). The plates were incubated in 37°C, 5% CO2 incubator. After 2 weeks, established colonies were counted and photographed.
Sphere culture assay
Mouse tracheal epithelial cells (MTEC) and mouse lung cancer cell were cultured in sphere assay medium: DMEM/F12 medium with 0.4% BSA, 20 ng/mL EGF, 20 ng/mL FGFβ, 50 μg/mL insulin and B27. Low-adherent plates were used to optimize sphere formation with 1,000 cells in 24-well plates or 2,000 cells in 6-well plates. The media was changed every 3 days with sphere formation complete in 2 to 3 weeks. To avoid necrosis, care was taken to limit sphere size.
Cells were lysed with RIPA buffer or SDS buffer, and equal amounts of the protein samples were mixed with loading buffer and boiled for 5 minutes. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose (NC) membranes. Nonspecific binding sites were blocked with 5% (w/v) nonfat dry milk in TBST, and the membranes were incubated overnight on a shaking platform at 4°C with the specific primary antibodies diluted in 5% BSA with 0.05% sodium azide. HRP-conjugated secondary antibody was incubated at room temperature for 1 hour. Finally, the proteins were visualized by exposure with Immobilon Western Reagents. All antibodies were diluted for use according to manufacturers' instructions.
A549-shNC/SLC1A1 or Calu-1-shNC/SLC1A1 cells (600 cells per 6 cm dish) were seed in a culture plate. The plates were incubated for 2 weeks in 37°C, 5% CO2 incubator. After washing the plate twice with PBS, incubate with crystal violet for 15 minutes. Rinse with running water and take pictures.
The FACS detection assay
Living cell count
A549-shNC/SLC1A1 or Calu-1-shNC/SLC1A1 cells (2 × 105 cells) were seed in 6 cm dish, using different media according to experimental conditions, regular DMEM (C), glutamine-depleted medium (Gln−), and replenish of glutamate (Glu) in glutamine-depleted medium (Gln−Glu+). The plates were incubated for 48 hours in 37°C, 5% CO2 incubator. Digesting cells and counting the living cells using the Invitrogen Countess II FL (Thermo Fisher Scientific).
RNA extraction from cells and qRT-PCR analysis
Total RNA was isolated from cultured cells using an RNA simple Total Kit (TIANGEN). One microgram of RNA was reverse-transcribed using a FastQuant RT Kit (TIANGEN). Resulting cDNA was then diluted 1:20, and amplified with qPCR using 2XSYBR Green qPCR Master Kit (Bimake.cn) reagents in an ABI StepOne Plus detection system. Cycling conditions: heat ramp 95°C × 10 minutes, extension (95°C × 30 seconds, 60°C × 30 seconds, 72°C × 30 seconds) × 40 cycles, melt curve 95°C × 15 seconds, 60°C × 1 minutes, 95°C × 15 seconds with 0.3°C increments. Fold change gene expression was calculated by normalization to GAPDH using formula of 2 − ΔΔCT.
A tissue microarray composed of tumor and adjacent normal tissue was stained to identify SLC1A1 and GPRC5A proteins. The IHC protocol and score method were performed as described previously (22). All antibodies were diluted for use according to manufacturers' instructions.
Transfection and stably transfected cells
HEK293T cells were transfected with lenti-shRNA-SLC1A1, luciferase reporter plasmids, and lenti-SLC1A1 through PEI (plasmid: PEI 1:4) transfection reagents, and the media replaced 4 to 6 hours posttransfection. The lentivirus medium collected after 48 hours was used to infect MTEC cells and NSCLC cells. Stably transfected cells were selected by puromycin (2 μg/mL) until all cells fluoresced green (GFP-fusion protein in the vector).
The level of glutamate in the MTEC cell line and in the lung tumor cell line pellets was detected using the Glutamate Colorimetric Assay Kit, and performed according to the methods provided in the kit.
Human lung cancer tissues samples were obtained from Shanghai Chest Hospital, Shanghai Jiao Tong University (Shanghai, China). Cells or tissues were lysed with TRIzol, then total RNA was extracted with RNA Extract Kit and cDNA were prepared from 1.5 μg of total RNA using Fast Quant Kit. All mRNA were detected by ABI 7300 real-time PCR machine; the PCR primers are presented in Supplementary Table S1.
Aldefluor assay and analyzing the ALDH positive cell by FACS
The Aldefluor Assay Kit was used to analyze the population of high ALDH enzyme positive cells. A549, Calu-1, and SJT-1601 cells (4 × 105 cells/mL) were suspended in Aldefluor assay buffer with the activated reagent, then dividing into two aliquots containing ALDH substrate (BAAA, 5 μL/2 × 105 cells). Negative control aliquots were treated with a specific ALDH inhibitor (DEAB, 5 μL/2 × 105). Aliquots were incubated for 45 minutes at 37°C. The gates were established using the negative controls cells stained with DEAB. Finally, the data were analyzed in the software.
Stable isotope-assisted metabolomics in vitro and in vivo
For the cell metabolic flux analysis, A549 shNC/SLC1A1 cells and Calu1 shNC/SLC1A1 cells were cultured to 90% density, then changed to glutamine-deficient medium with 2 mmol/L [U-13C5]-glutamine or 2 mmol/L [U-13C5]-glutamate or 0.26 mmol/L [13C6,15N2]-cystine. After 12 hours, the labeled cells from each sample were harvested in 1.6 mL 80% (v/v) methanol solution. For comparison, cells were also cultured in glucose-deficient medium with 15 mmol/L [U-13C6]-glucose and were harvested in 1.6 mL 80% (v/v) methanol solution as outlined above.
For the metabolic flux analysis in vivo, on the first day, mice were surgically intubated through the jugular veins and allowed to recover for 2 to 3 days. Infusions were performed using [U-13C5]-glutamine as the tracer into mice using a microfluidic pump connected to the cannula for 6 hours (2 mg/kg/min). Lung tumor and para-cancer normal tissue were isolated, and then quenched in liquid nitrogen and store at −80°C for mass spectrometric analysis.
Data were analyzed using the software SPSS Statistics (IBM, Version 19). Data are presented as the mean ± SD. The differences of results were compared using two-tailed paired t test assuming unequal distribution. Multiple group comparisons used one-way ANOVA. P value <0.05 was considered to be statistically significant.
Other methods, including GC-MS analysis, UHPLC-qTOF-MS analysis, quantification of cystine in condition medium (CM), untargeted metabolomics by LC/MS-MS, in vivo biodistribution study in mouse model, in vivo FITC-Glutamate distribution study in mouse models, Pathway analysis and tissues metabolomics, refer to Supplementary Materials and Methods.
Dysregulated GSH synthesis and glutamate transporter Slc1a1 are associated with lung tumorigenesis in Gprc5a-ko mice
To determine the mechanism of lung tumorigenesis, we applied carcinogen-induced lung tumor model in Gprc5a-ko mice for characterization. WT and Gprc5a-ko (KO) mice were intraperitoneally injected with NNK (100 mg/kg body weight, once a week for twice) at two months of age; lung tissues were harvested 12 months later. All of Gprc5a-ko (KO-NNK-14m) mice (12 of 12, 100%) developed lung cancer, whereas none of wild-type (WT-NNK-14m mice) mice (0 of 12) did (Fig. 1A; ref. 13). Next, comprehensive metabolomics analysis was performed to characterize the metabolic profile in the lungs of all groups (Fig. 1B). Notably, the GSH level is dramatically increased in the Gprc5a-ko-NNK14m group, the only group with lung tumor, compared with the other groups (Fig. 1C). In addition, glutamate is moderately increased in lung tissues from Gprc5a-ko mice compared with those from WT mice (Fig. 1D). Among other intermediate metabolites of GSH synthesis, glutathione-oxidized (GSSG), S-methylglutathione, glycine, cysteine, and cystine were slightly increased in lung tissues of Gprc5a-ko-NNK14m mice compared with those of WT-NNK14m mice (Supplementary Figs. S1A–S1E), whereas other metabolites, such as cysteine-glutathione disulfide, glutamine, 5-oxoproline, and ophthalmate, were of no significant difference (Supplementary Figs. S1F–S1I). Taken together, increased level of GSH is highly associated with lung tumorigenesis in Gprc5a-ko mice.
To determine the mechanisms involved, we performed RNA-seq analysis using MTEC from WT and Gprc5a-ko (KO) mice. Pathway analysis showed that the most dramatically changed pathways are the metabolic pathways (Supplementary Fig. S2A). Among the differentially expressed genes in amino acid metabolism, we noticed that Slc1a1 was significantly upregulated in MTEC-KO compared with MTEC-WT (Fig. 1E). Upregulated Slc1a1 was validated by Western blot analysis (Fig. 1F) and qRT-PCR analysis, respectively (Fig. 1G and H). Slc1a1 encodes EAAC1, a member of the high-affinity glutamate transporters, playing an essential role in transporting glutamate across plasma membranes in neurons. To validate the biochemical role of upregulated Slc1a1, we examined the glutamate level in MTEC cells using a glutamate colorimetric assay. Indeed, glutamate concentration was significantly higher in MTEC-KO cells than in MTEC-WT (Fig. 1I; Supplementary Fig. S2B). Consistently, the glutamate level in CM of MTEC-KO cells is significantly lower than that of MTEC-WT (Fig. 1J), suggesting that MTEC-KO cells uptake more glutamate from medium than MTEC-WT cells do. Thus, upregulated Slc1a1 enhances glutamate uptake in Gprc5a-ko lung epithelial cells. Notably, upregulated Slc1a1 was also found in mouse lung tumor cell line SJT-1601 that was derived from Gprc5a-ko mouse lung tumor (Fig. 1K), supporting that lung tumorigenesis is beneficial from upregulated Slc1a1. Re-expression of Gprc5a in SJT-1601 cells inhibits Slc1a1 (Fig. 1K), suggesting that upregulated Slc1a1 is resulted from Gprc5a-deficiency. Upregulated SLC1A1 was also observed in human lung cancer cell line, whereas overexpression of GPRC5A inhibited SLC1A1 expression in A549 cells (Supplementary Fig. S2C). To determine Slc1a1 expression in vivo, we examined Slc1a1 levels in lung tissues via IHC staining. We found that, SLC1A1 IHC scores in S/TB, but not the alveolar region, was significantly upregulated in Gprc5a-ko (KO) mouse lungs compared with those of WT ones, both in 2m and 14m-NNK groups (Fig. 1L and M). Taken together, these results suggest that increased GSH and upregulated glutamate transporter Slc1a1 are associated with lung tumorigenesis in Gprc5a-ko mice.
Upregulated SLC1A1 is essential for increased glutamate uptake, GSH synthesis, and the malignant phenotype of lung cancer cells
To correlate the expression profile of SLC1A1 in human lung cancer cells, we examined the levels of SLC1A1 and xCT in a panel of human NSCLC cell lines. Western blot analysis showed that xCT was upregulated in all NSCLC cell lines, whereas SLC1A1 was upregulated in a subset of NSCLC cell lines (H292G, Calu-1, HCC827, A549), compared with that in normal human lung epithelial cell lines, HBEC (Fig. 2A). To determine the biological roles of SLC1A1, we silenced SLC1A1 using short hairpin (sh) RNA (shSLC1A1) in A549 cells (Fig. 2B) and Calu-1 cells (Supplementary Fig. S2D). SLC1A1-knockdown (KD) significantly reduced the colony formation (Fig. 2C), anchorage-independent growth (Fig. 2D), and 3D-sphere formation (Fig. 2E) in A549 and Calu-1 cells. These results suggest that upregulated SLC1A1 is essential for the malignant phenotypes of human lung cancer cells.
To determine the biochemical role of SLC1A1, we examined glutamate transportation and GSH synthesis in these cell lines. LC/MS-MS analysis showed that glutamate and GSH levels were significantly reduced in A549-shSLC1A1 cells compared with control cells, A549-nonspecific control (NC; Fig. 2F and G). GSH is known to buffer intracellular ROS for maintaining redox homeostasis in cells. To determine if GSH synthesis is required for maintaining the malignant features of the lung cancer cells, we examined the roles of GSH via BSO, an inhibitor of GSH synthesis, on A549 cells. The results showed that BSO treatment significantly suppressed stem cell-like subpopulation, in A549 cells (Fig. 2H), and SJT-1601 cells (Supplementary Fig. S3A). BSO treatment also reduced the number and size of colonies of A549 (Supplementary Fig. S3B). These findings suggest that GSH is required for maintaining the malignant features of lung tumor cells, similar to previous reports (27, 28). Next, we examined the effects of SLC1A1-KD on Aldh+ cells, a stem cell-enriched subpopulation. FACS analysis showed that Aldh+ population was significantly reduced in A549-siSLC1A1 cells (1.26%) compared with that in A549-siNC cells (3.11%; Fig. 2I and J). These observations suggest that SLC1A1 is required for maintaining the stemness of these cancer cells. Moreover, tumorigenicity of A549-shSLC1A1 cells was significantly reduced compared that of A549-shNC cells (Fig. 2K). Taken together, these results suggest that SLC1A1 overexpression is essential for maintaining the malignant phenotypes of lung cancer cells both in vitro and in vivo.
SLC1A1-mediated glutamate uptake alleviates glutamine deficiency-induced ROS and cell growth inhibition
GSH is an anti-ROS molecule. Generally, GSH level is inversely correlated with oxidative stress within cells. By examining ROS level, we found that ROS was significantly increased in A549-si-SLC1A1 cells compared with that in A549 si-NC cells (Fig. 3A). Consistently, silenced SLC1A1 by siRNA resulted in increased ROS targets (cleaved-PARP, p-P38; ref. 9) as compared with NC in both A549 and Calu-1 lung cancer cells (Fig. 3B). These suggest that SLC1A1-KD reduces GSH and increases oxidative stress, leading to reduced stem cell subpopulation and increased programmed cell death.
Intracellular glutamate is largely converted from extracellular glutamine, imported via glutamine transporters. Upregulated glutamate transporter in tumor cells suggests that there is a functional link between SLC1A1 upregulation and the malignant phenotypes. To determine the role of extracellular glutamate on tumor cells, we examined the growth of tumor cells by culturing them in glutamine-depleted medium with or without exogenous glutamate. Depletion of glutamine in medium (Gln−) inhibited the growth in A549-shNC cells (Fig. 3C). However, addition of exogenous glutamate in medium (Gln−Glu+) restored the growth in A549-shNC cells (Fig. 3C), but failed to do so in A549-sh-SLC1A1 cells (Fig. 3C). These findings suggest that glutamate transporter SLC1A1 is essential for cell growth in the glutamine-depleted medium but supplied with glutamate (Gln−Glu+). Consistently, depletion of glutamine in the medium (Gln−) reduced ALDH+ cell, whereas addition of exogenous glutamate in the medium (Gln−Glu+) largely restored ALDH+ cell population in A549-siNC and Calu-1-siNC cells; however, this effect was lost in A549-siSLC1A1 and Calu-1-siSLC1A1 cells (Fig. 3D; Supplementary Figs. S3C and S3D). These findings suggest that SLC1A1 is essential for restoration of ALDH+ cell population when extracellular glutamate is available. To determine the role of SLC1A1 on programmed cell death, we examined Annexin V, an apoptotic marker, in cells. The results showed that, following culture in the medium (Gln−Glu+), Annexin V levels were significantly increased in A549-siSLC1A1 and Calu-1-siSLC1A1 cells, in comparison to those of si-NC control cells (Fig. 3E and F; Supplementary Figs. S4A and S4B). These observations suggest that SLC1A1-mediated uptake of glutamate inhibits apoptotic subpopulation, a type of programmed cell death, in the medium (Gln−Glu+).
Increased ROS is implicated to induce various forms of programmed cell death and growth inhibition in cells (1, 9). Next, we asked if glutamine depletion-induced programmed cell death and proliferation inhibition were resulted from increased ROS. Indeed, ROS was increased in si-SLC1A1-C cells compared with si-NC-C A549 cells in medium (Gln−Glu+; Fig. 3G and H); whereas addition of reducing agent GSH-MEE or α-tocopherol significantly reduced ROS in A549-si-SLC1A1, but did not in A549-si-NC (Fig. 3G and H). These findings suggest that SLC1A1-mediated glutamate uptake is essential for buffering ROS induced via glutamine depletion. In addition, we also examined the effects of SLC1A1 overexpression in 16HBE, an immortalized normal lung epithelial cell line. The results showed that the ROS level was significantly reduced in 16HBE-SLC1A1 cells in comparison with that in 16HBE-V cells when cultured in medium (Gln−Glu+; Fig. 3I; Supplementary Fig. S4C). These results suggest that SLC1A1 overexpression confers 16HBE cells the extra-ability to buffer ROS.
To determine the biological impact of SLC1A1 deficiency-induced ROS, we examined the total living cell number, the net outcome of all forms of cell death and growth inhibition, in medium with or without glutamine (Gln) or glutamate (Glu). Depletion of glutamine in medium (Gln−) in 48 hours leads to significantly reduced cell numbers, compared with those in normal medium (C), in both A549-shNC and A549-shSLC1A1 cells (Fig. 3J); importantly, replenish of glutamate (Glu) in glutamine-depleted medium (Gln−Glu+) largely restored the proliferation of A549-shNC cells, but not that of A549-shSLC1A1 cells (Fig. 3J). Consistently, clonogenic assay (culture for 2 weeks) showed that glutamine-depletion in medium (Glu−) completely eliminated the colonies forming activity of A549 cells; whereas replenish of glutamate in Gln-depleted medium (Gln−Glu+) largely restored the colony forming activity in A549-shNC cells, but not that of A549-shSLC1A1 cells (Fig. 3K). These findings suggest that glutamine depletion-induced cell death and/or growth inhibition can be largely restored by exogenous glutamate, which is dependent on SLC1A1.
To determine if SLC1A1-mediated biological functions were resulted from the impact of ROS, we examined the role of reducing agents on cell growth at various conditions. Culture of A549-shSLC1A1 cells in Gln−Glu+ medium resulted in significantly inhibited cell growth as compared with that of A549-shNC cells. Importantly, treatment of A549-shSLC1A1 cells with reducing agents, GSH-MEE and α-tocopherol, largely restored the cell growth, whereas the cell growth of A549-shNC cells were not further increased (Fig. 3L). These findings suggest that SLC1A1-KD confers the susceptibility to ROS-mediated cell death and/or proliferation inhibition in Gln−Glu+ medium.
Active glutamate uptake via SLC1A1 propels passive cystine uptake and GSH synthesis in lung cancer cells
Xc− is a Na+-independent antiporter, which is driven by the concentration gradients of cystine and glutamate across membrane. If extracellular glutamate were high, the glutamate gradient across membrane would be reduced, leading to decreased cystine import. We hypothesized that, glutamate uptake via SLC1A1, which reduce extracellular glutamate, is to maintain a high concentration gradient of glutamate across membrane, which propels passive cystine uptake via antiporter Xc−. To determine if the glutamate imported via SLC1A1 is indeed used for GSH synthesis, we performed a stable isotope tracing analysis and assessed the fate of [U-13C5]-glutamine-derived GSH (Fig. 4A; refs. 29–31). Of note, the labeling of GSH (M5 isotopologue; five-labeled carbons), glutamate (M5 isotopologue), and metabolic intermediate γ-glutamylcysteine (M5 isotopologue) from the glutamine-carbon was significantly reduced in A549-shSLC1A1 cells compared with those from control A549-shNC cells (Fig. 4B–D). In addition, metabolic intermediates involved in the synthesis of GSH, such as GSSG and pyroglutamate were also reduced in A549-shSLC1A1 cells compared with the metabolites from A549-shNC cells (Supplementary Figs. S5A and S5B). These findings suggest that the glutamate uptake via SLC1A1 facilitates GSH synthesis. To rule out the possibility that altered uptake of labeled glutamine was due to reduced cell proliferation, we also examined the incorporation of the metabolites by using a tracer [U-13C6]-glucose in the same experimental condition. The results showed that labeled glutamate (M5 isotopologue), GSH (M5 isotopologue), and intermediate γ-glutamylcysteine (M5 isotopologue) derived from labeled glucose carbon exhibited no significant change between two cell lines (Supplementary Figs. S5C–S5E). Thus, SLC1A1 facilitates GSH synthesis via glutamine uptake.
SLC1A1 is a glutamate transporter, and its impact on glutamine uptake must be indirect. To determine the role of SLC1A1 on glutamate uptake, we examined GSH synthesis using [U-13C5]-labeled glutamate in Calu-1-shSLC1A1 and -shNC cells (Supplementary Fig. S2D). 13C-labeled GSH (M5 isotopologue) metabolites were measured 6 hours after treatment with [U-13C5]-glutamate in medium (Gln−). GSH (M5 isotopologue) from 13C-labeled glutamate carbons were significantly reduced in Calu-1-shSLC1A1 cells compared with vector control, Calu-1-shNC (Fig. 4E). Consistently, labeled glutamate (M5 isotopologue) from [U-13C5]-glutamate was also significantly reduced in Calu-1-shSLC1A1 compared with control cells (Fig. 4F), and 13C-labeled glutamate carbons in the CM of Calu-1-shSLC1A1 cells was higher than that in the CM of Calu-1-shNC cells (Fig. 4G). These results suggest that glutamate uptake by Calu-1-shSLC1A1 cells was reduced as compared with that by Calu-1-shNC cells. In comparison, the 13C incorporation in the intermediate metabolites, aspartate, malate and succinate of TCA cycle were not changed in Calu-1-shSLC1A1 cells (Fig. 4H and I; Supplementary Fig. S5F). Glutamine depletion in medium did not induce significant apoptotic cells in 6 hours in A549 and Calu-1 cells (Fig. 3E and F), suggesting that reduced GSH and Glu are not due to cell death. Taken together, SLC1A1 promotes glutamate uptake for GSH synthesis rather than feeding the TCA cycle anaplerosis. For further characterization, we also examined GSH metabolites in 16HBE and 16HBE-SLC1A1 cells. 13C-labeled GSH (M5 isotopologue) from labeled glutamate carbons was significantly increased in 16HBE-SLC1A1 cells compared with the parental cell line (Fig. 4J). These results suggest that SLC1A1 overexpression enhances GSH synthesis in 16HBE cells. Taken together, SLC1A1 enhances GSH synthesis via glutamate uptake.
To determine the role of SLC1A1 on cystine uptake for GSH synthesis, we performed stable isotope-assisted metabolomics using [13C6–15N2]-labeled cystine as the tracer in Calu-1 and A549 cells (Supplementary Fig. S5H). 13C-labeled GSH (M4 isotopologue) was significantly decreased in Calu-1-siSLC1A1 cells compared with that in control Calu-1-siNC cells (Fig. 4K), suggesting that SLC1A1 is essential for efficient cystine uptake. The M8 isotopologue of GSSG were also decreased in Calu-1-siSLC1A1 cells (Supplementary Fig. S5G), supporting the notion. Similarly, [13C3–15N1]-labeled GSH was also reduced in A549-siSLC1A1 cells compared with A549-siNC cells (Fig. 4L). Consistently, [13C6–15N2]-labeled cystine in CM of Calu-1-siSLC1A1 and A549-siSLC1A1 cells were significantly higher than those in control si-NC cells (Fig. 4M), suggesting that SLC1A1-KD reduces cystine uptake from medium (Supplementary Fig. S5H). Thus, glutamate transporter SLC1A1 facilitates cystine uptake for GSH synthesis in lung cancer cells.
Lung tumors preferentially uptake glutamate for GSH synthesis in vivo
Cellular metabolic processes can be greatly influenced by tissue microenvironment in vivo (31). To determine the roles of glutamate uptake and GSH synthesis in vivo, we performed an experiment tracing [U-13C5]-glutamine in lung tumors from Gprc5a-ko mice. Gprc5a-ko mice develop lung tumors by 14 months of age (5a-ko-NNK14m; refs. 13, 14). Before sacrifice, [U-13C5]-glutamine (2 mg/kg/min, 6 hours) was injected by jugular vein catheterization to these mice (31). The lung tissues were harvested after 6 hours infusion, and metabolites from lung tumors and normal lung tissues were measured (Fig. 5A). The results showed that M5 isotopologues of GSH and glutamate derived from labeled-glutamine carbon were significantly increased in lung tumors compared with adjacent normal lung tissues (Fig. 5B and C). On contrary, the M5 isotopologues of GSSG and glutamine from the labeled glutamine carbon were similar in tumors and adjacent lung normal tissues (Supplementary Figs. S6A and S6B). Taken together, these results suggest that GSH and glutamate are preferentially synthesized and used for the growth and survival of lung tumors in vivo.
To determine the fate of glutamate, we designed FITC-labeled glutamate [5-FITC-(Acp)-Glu] to track the location of lung tumors in mouse lung tumor model. Gprc5a-ko (KO-NNK-14m) mice developed lung tumors, which were detectable by MRI imaging (red circle, Fig. 5D). Importantly, glutamate-FITC via intraperitoneal injection was much brighter in tumor regions than in adjacent normal tissues (Fig. 5E). Light images (red arrow, Fig. 5F) and HE staining (Fig. 5G) confirmed the location of tumors. These suggest that lung tumor tissues prefer glutamate uptake compared with normal lung tissues. In experimental metastasis model, mouse lung tumor cells, SJT-1601-luc, were intravenously injected in nude mice. Three weeks later, lung tumors were developed, as imaged by luciferase activity via intraperitoneal injection luciferin (Fig. 5H) or FITC-Glutamate via tail vain injection. Luminescence imaging showed that metastatic tumors were formed in mouse lungs (Fig. 5I, bottom). Importantly, FITC-glutamate also showed strong imaging in tumor tissues in lungs, which is largely overlapped with luciferase images (Fig. 5I, top), both in mice injected with SJT-1601-luc at 106 (Fig. 5I, right) or 105 (Fig. 5I, left). To determine if SLC1A1 expression is essential for glutamate uptake in tumor tissue in vivo, we examined the uptake of FITC-glutamate in tumors derived from A549-shNC and A549-shSLC1A1 cells. To compare glutamate uptake by tumors with similar size, we subcutaneously inoculated 1.5 × 106 A549-shNC cells and 3 × 106 A549-shSLC1A1 cells in nude mice. When similar size of tumors were formed (in 2 weeks), FITC-Glutamate was injected via tail vein. Two hours later, tumors were isolated for fluorescence photography. The results showed that the tumor mass from A549-shNC cells exhibited about two-fold higher FITC intensity than that from A549-shSLC1A1 cells, suggesting that SLC1A1-KD significantly reduces uptake of FITC-glutamate in lung tumor (Supplementary Figs. S6C and S6D). Thus, SLC1A1 is essential for efficient FITC-glutamate uptake in tumor tissues.
Dysregulated glutamate transporters are prevalent in NSCLC tissues
To determine if SLC1A1 is upregulated in human lung cancer, we measured mRNA from 16 paired lung NSCLC tissues and normal tissues by using qRT-PCR. The result showed that SLC1A1 expression was upregulated in a subset (7/16) of lung tumor tissues compared with adjacent normal tissues, but not in other samples (Fig. 6A). We reasoned that other glutamate transporters might perform similar function as SLC1A1. Then, we examined the mRNA levels of other glutamate transporters, SLC1A2, SLC1A3. The results showed that most of lung tumors (12/16) expressed either one or more types of the glutamate transporters tested (Fig. 6A–C). To further characterize the expression of SLC1A1 and GPRC5A in human lung tumors, we performed IHC staining using a tissue microarray of human lung tumor and adjacent normal tissue (n = 76 pairs). GPRC5A was highly expressed in adjacent-normal tissue compared with lung tumor tissue; whereas SLC1A1 was highly expressed in most of lung tumor tissues compared with adjacent-tumor tissues (Fig. 6D). The IHC score showed that there is an inverse correlation between GPRC5A and SLC1A1 on average (Fig. 6E and F), supporting that GPRC5A deficiency contributes SLC1A1 upregulation in NSCLCs. In addition, we measured the GSH and glutamine levels in 30 pairs of freshly isolated NSCLC and adjacent normal lung tissues. The results showed that GSH and glutamine were significantly higher in lung tumor tissues than that in normal lung tissues (Fig. 6G and H), supporting that GSH synthesis is greatly enhanced in tumor tissues.
To extend this observation further, we collected data on GPRC5A and SLC1A1 expression from The Human Protein Atlas at www.proteinatlas.org, and found that there is an inverse correlation between two genes, which is consistent with our original data (Supplementary Figs. S7A–S7D). We also analyzed published data from The Cancer Genome Atlas; patients with lung cancer who were divided into two groups according to the level of SLC1A1 expression (32–34). The analysis showed that patients with a high expression of SLC1A1 showed poor survival compared with those with low expression (Fig. 6I), supporting the promotion role of SLC1A1 in lung tumor progression. Taken together, these data suggest that glutamate transporters, in particular SLC1A1, are frequently upregulated in lung cancer tissues, which predicts poor survival.
In this study, we show that upregulated glutamate transporter SLC1A1 is essential for increased uptake of glutamate and cystine for GSH synthesis, which contributes to the malignant phenotypes of lung cancer cells. Consistently, carcinogen-induced lung tumors and metastatic tumors exhibited increased uptake of glutamate compared with adjacent normal lung tissues, which can be used as a potential diagnosis marker for lung cancer. Finally, upregulated glutamate transporters, in particular SLC1A1, as well as GSH synthesis, are found in NSCLC samples, supporting the role in tumorigenesis.
Lung tumorigenesis is associated with chronic inflammation (35, 36) and COPD (37–40). Inflammation has been linked to a high level of ROS production (41). To buffer the high level of ROS, malignant cells must gain the extra ability to generate a high level of GSH since the regular program of GSH synthesis under physiological conditions is inadequate for the need in lung tumorigenesis (42). In this study, we found that glutamate transporters, in particular SLC1A1, are overexpressed in NSCLC tissues. Glutamate transporters SLC1A1-5 are mostly found in neuronal cells, to uptake extracellular glutamate released in cleft, which prevents excitotoxic damage. SLC1A1 is also critical in GSH synthesis in neurons (43). System Xc− imports cystine while exporting glutamate in a 1:1 ratio. Thus, extracellular glutamate acts as a competitive inhibitor for cystine uptake via system Xc−. Because SLC1A1-mediated glutamate uptake reduces extracellular glutamate, it would facilitate cystine uptake. Consistent with this model, it was reported that transient over-expression of SLC1A1 (EAAT3) in hippocampal HT22 cells increases intracellular GSH in the presence of high glutamate concentrations, and protected host cells from oxidative glutamate toxicity (44–46). Thus, although cystine is considered as the rate-limiting amino acid (47), the glutamate gradient across membrane is crucial for efficient cystine uptake and GSH synthesis, especially in cancer cells that need high level of GSH. Consistent with concept, other members of glutamate transporters were found to be upregulated in those cancer cells when SLC1A1 was not upregulated. It remains to determine the role of other SLC1A family members that are also implicated in human tumors although the mechanisms remain unclear (48). Previously, we showed that chronic inflammation was associated with lung tumorigenesis in Gprc5a-ko mouse mice; and Gprc5a-deletion leads to aberrant activation of NF-κB and STAT3 signaling in lung epithelial cells (15, 49, 50). It remains to be determined what mechanisms are involved in upregulation of glutamate transporters, such as SLC1A1, in tumor cells. Nevertheless, it appears that upregulated glutamate transporters, in particular SLC1A1, a program used in neuron, have been hijacked by lung tumor cells for cancer progression. GSH synthesis in normal cells is sufficient for homeostasis, which is independent of SLC1A1. However, metabolism in cancer cells is dysregulated, which generates extra high level of ROS. Our assumption is that SLC1A1-mediated glutamate uptake confers to synthesize an extra-level of GSH for buffering increased ROS in cancer cells.
Analogues of glutamate, such as 4-[18F]fluoro L-glutamate (BAY 85–8050), have been reported to be used for tumor imaging (51). PET imaging with excellent tumor visualization and high tumor to background ratios was achieved in preclinical ectopia tumor models via subcutaneous inoculation. However, the hypothetic explanation theory proposed is very different from ours, in which the authors assumed that xCT is responsible for the preference of glutamate uptake in cancer cells. In this study, we applied FITC-Glutamate tracing assay in orthotropic tumor model, in which lung tumors preferentially uptake glutamate compared with normal lung tissues. Moreover, we showed that SLC1A1 is essential for preferential glutamate uptake in tumor tissues in vivo. Thus, we conclude that it is glutamate transporter, such as SLC1A1, rather than xCT, that is mainly responsible for preferential glutamate uptake.
In general, cancer stem cells, metastatic cancer cells, and cancer cells associated with relapse are more resistant to oxidative stress provoked by chemo- or radiotherapy than non-stem-like cancer cells, whereas increased GSH has been linked to stem-like features (47, 52, 53). xCT overexpression and high GSH synthesis have been identified in cancer stem cells (4, 54). Here, we report that the glutamate transporters, in particular SLC1A1, are highly expressed to maintain a high level of GSH synthesis. The efficacy of cancer therapies, including radiotherapy and anticancer drugs, is attributable in part to the production of ROS and the consequent induction of oxidative stress in cancer cells, it is therefore expected that targeting of antioxidant systems in cancer stem cells, such as glutamate transporter SLC1A1 and xCT, in combination of chemo- or radiotherapy, would improve the efficacy of cancer treatment. Taken together, dysregulated glutamate transporter SLC1A1 is essential for increased glutamate uptake, which facilitates cystine uptake and GSH synthesis for lung tumorigenesis (Fig. 7).
No disclosures were reported.
W. Guo: Resources, data curation, formal analysis, funding acquisition, investigation, methodology, writing—original draft. K. Li: Investigation, methodology. B. Sun: Investigation, methodology. D. Xu: Investigation, methodology. L. Tong: Investigation, methodology. H. Yin: Investigation, methodology. Y. Liao: Investigation, methodology. H. Song: Investigation, methodology. T. Wang: Investigation, methodology. B. Jing: Investigation, methodology. M. Hu: Investigation, methodology. S. Liu: Investigation, methodology. Y. Kuang: Investigation, methodology. J. Ling: Investigation, methodology. Q. Li: Investigation, methodology. Y. Wu: Data curation, formal analysis. Q. Wang: Data curation, formal analysis. F. Yao: Resources, data curation, formal analysis. B.P. Zhou: Conceptualization, formal analysis, supervision, writing—original draft. S.-H. Lin: Conceptualization, formal analysis, supervision, funding acquisition, methodology, writing—review and editing. J. Deng: Conceptualization, formal analysis, supervision, funding acquisition, investigation, writing—original draft, writing—review and editing.
This work was supported by grants from the National Nature Science Foundation of China 91957120, 81672911 (to S.-H. Lin), 81902338 (to W. Guo), 81620108022, 91129303, 91729302, 81572759, and Shenzhen Municipal Government of China (KQTD20170810160226082 to J. Deng).
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