Elevated SLC1A5 associated with poor prognosis and therapeutic resistance to transarterial chemoembolization in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant tumor, and glutamine is vital for tumor cells. The role of glutamine transporter SLC1A5 in tumor progression and transarterial chemoembolization (TACE) efficacy is under study. This research seeks to determine the impact of SLC1A5 expression on the prognosis and TACE efficacy of HCC and elucidate its mechanisms. METHODS: SLC1A5 expression in HCC, correlation with patient outcomes, and response to TACE were studied in an open access liver cancer dataset and confirmed in our cohort. Additionally, the correlation between SLC1A5 expression and hypoxia, angiogenesis and immune infiltration was analyzed and verified by immunohistochemistry, immunofluorescence and transcriptome sequencing. Liver cancer cell lines with SLC1A5 expression knockdown or overexpression were constructed, and cell proliferation, colony formation, apoptosis, migration and drug sensitivity as well as in vivo xenograft tumor were measured. A gene set enrichment analysis was conducted to determine the signaling pathway influenced by SLC1A5, and a western blot analysis was performed to detect protein expression alterations. RESULTS: SLC1A5 expression was higher in HCC tissue and associated with poor survival and TACE resistance. Hypoxia could stimulate the upregulation of glutamine transport, angiogenesis and SLC1A5 expression. The SLC1A5 expression was positively correlated with hypoxia and angiogenesis-related genes, immune checkpoint pathways, macrophage, Tregs, and other immunosuppressive cells infiltration. Knockdown of SLC1A5 decreased proliferation, colony formation, and migration, but increased apoptosis and increased sensitivity to chemotherapy drugs. Downregulation of SLC1A5 resulted in a decrease in Vimentin and N-cadherin expression, yet an increase in E-cadherin expression. Upregulation of SLC1A5 increased Vimentin and N-cadherin expression, while decreasing E-cadherin. Overexpression of ß-catenin in SLC1A5-knockdown HCC cell lines could augment Vimentin and N-cadherin expression, suppress E-cadherin expression, and increase the migration and drug resistance. CONCLUSIONS: Elevated SLC1A5 was linked to TACE resistance and survival shortening in HCC patients. SLC1A5 was positively correlated with hypoxia, angiogenesis, and immunosuppression. SLC1A5 may mediate HCC cell migration and drug resistance via Epithelial-mesenchymal transition (EMT) pathway.

Particulate matter facilitates amphiregulin-dependent lung cancer proliferation through glutamine metabolism.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) causes lung cancer, the molecular mechanisms underlying the PM-induced increases in lung cancer progression remain unclear. We applied the lung cancer cell line A549 (Parental; A549.Par) to PM for an extended period to establish a mimic PM-exposed lung cancer cell line, A549.PM. Our results indicate that A549.PM exhibits higher cell growth and proliferation abilities compared to A549.Par cells in vitro and in vivo. The RNA sequencing analysis found amphiregulin (AREG) plays a critical role in PM-induced cell proliferation. We observed that PM increases AREG-dependent lung cancer proliferation through glutamine metabolism. In addition, the EGFR/PI3K/AKT/mTOR signaling pathway is involved in PM-induced solute carrier family A1 member 5 (SLC1A5) expression and glutamine metabolism. Our findings offer important insights into how lung cancer proliferation develops upon exposure to PM.

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.

Differential metabolic secretion between muscular dystrophy mouse-derived spindle cell sarcomas and rhabdomyosarcomas drives tumor type development.

The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.

DRP1 inhibition-mediated mitochondrial elongation abolishes cancer stemness, enhances glutaminolysis, and drives ferroptosis in oral squamous cell carcinoma.

BACKGROUND: Mitochondrial dynamics play a fundamental role in determining stem cell fate. However, the underlying mechanisms of mitochondrial dynamics in the stemness acquisition of cancer cells are incompletely understood. METHODS: Metabolomic profiling of cells were analyzed by MS/MS. The genomic distribution of H3K27me3 was measured by CUT&Tag. Oral squamous cell carcinoma (OSCC) cells depended on glucose or glutamine fueling TCA cycle were monitored by 13C-isotope tracing. Organoids and tumors from patients and mice were treated with DRP1 inhibitors mdivi-1, ferroptosis inducer erastin, or combination with mdivi-1 and erastin to evaluate treatment effects. RESULTS: Mitochondria of OSCC stem cells own fragment mitochondrial network and DRP1 is required for maintenance of their globular morphology. Imbalanced mitochondrial dynamics induced by DRP1 knockdown suppressed stemness of OSCC cells. Elongated mitochondria increased α-ketoglutarate levels and enhanced glutaminolysis to fuel the TCA cycle by increasing glutamine transporter ASCT2 expression. α-KG promoted the demethylation of histone H3K27me3, resulting in downregulation of SNAI2 associated with stemness and EMT. Significantly, suppressing DRP1 enhanced the anticancer effects of ferroptosis. CONCLUSION: Our study reveals a novel mechanism underlying mitochondrial dynamics mediated cancer stemness acquisition and highlights the therapeutic potential of mitochondria elongation to increase the susceptibility of cancer cells to ferroptosis.

Deficiency in SLC25A15, a hypoxia-responsive gene, promotes hepatocellular carcinoma by reprogramming glutamine metabolism.

BACKGROUND & AIMS: The role of solute carrier family 25 member 15 (SLC25A15), a critical component of the urea cycle, in hepatocellular carcinoma (HCC) progression remains poorly understood. This study investigated the impact of SLC25A15 on HCC progression and its mechanisms. METHODS: We systematically investigated the function of SLC25A15 in HCC progression using large-scale data mining and cell, animal, and organoid models. Furthermore, we analyzed its involvement in reprogramming glutamine metabolism. RESULTS: SLC25A15 expression was significantly decreased in HCC tissues, and patients with low SLC25A15 levels had a poorer prognosis. Hypoxia-exposed HCC cells or tissues had lower SLC25A15 expression. A positive correlation between HNF4A, a transcription factor suppressed by hypoxia, and SLC25A15 was observed in both HCC tissues and cells. Modulating HNF4A levels altered SLC25A15 mRNA levels. SLC25A15 upregulated SLC1A5, increasing glutamine uptake. The reactive metabolic pathway of glutamine was increased in SLC25A15-deficient HCC cells, providing energy for HCC progression through additional lipid synthesis. Ammonia accumulation due to low SLC25A15 levels suppressed the expression of OGDHL (oxoglutarate dehydrogenase L), a switch gene that mediates SLC25A15 deficiency-induced reprogramming of glutamine metabolism. SLC25A15-deficient HCC cells were more susceptible to glutamine deprivation and glutaminase inhibitors. Intervening in glutamine metabolism increased SLC25A15-deficient HCC cells' response to anti-PD-L1 treatment. CONCLUSION: SLC25A15 is hypoxia-responsive in HCC, and low SLC25A15 levels result in glutamine reprogramming through SLC1A5 and OGDHL regulation, promoting HCC progression and regulating cell sensitivity to anti-PD-L1. Interrupting the glutamine-derived energy supply is a potential therapeutic strategy for treating SLC25A15-deficient HCC. IMPACT AND IMPLICATIONS: We first demonstrated the tumor suppressor role of solute carrier family 25 member 15 (SLC25A15) in hepatocellular carcinoma (HCC) and showed that its deficiency leads to reprogramming of glutamine metabolism to promote HCC development. SLC25A15 can serve as a potential biomarker to guide the development of precision therapeutic strategies aimed at targeting glutamine deprivation. Furthermore, we highlight that the use of an inhibitor of glutamine utilization can enhance the sensitivity of low SLC25A15 HCC to anti-PD-L1 therapy.

tRF-29-79 regulates lung adenocarcinoma progression through mediating glutamine transporter SLC1A5.

In recent decades, considerable evidence has emerged indicating the involvement of tRNA-derived fragments (tRFs) in cancer progression through various mechanisms. However, the biological effects and mechanisms of tRFs in lung adenocarcinoma (LUAD) remain unclear. In this study, we screen out tRF-29-79, a 5'-tRF derived from tRNAGlyGCC, through profiling the tRF expressions in three pairs of LUAD tissues. We show that tRF-29-79 is downregulated in LUAD and downregulation of tRF-29-79 is associated with poorer prognosis. In vivo and in vitro assay reveal that tRF-29-79 inhibits proliferation, migration and invasion of LUAD cells. Mechanistically, we discovered that tRF-29-79 interacts with the RNA-binding protein PTBP1 and facilitates the transportation of PTBP1 from nucleus to cytoplasm, which regulates alternative splicing in the 3' untranslated region (UTR) of SLC1A5 pre-mRNA. Given that SLC1A5 is a core transporter of glutamine, we proved that tRF-29-79 mediate glutamine metabolism of LUAD through affecting the stability of SLC1A5 mRNA, thus exerts its anticancer function. In summary, our findings uncover the novel mechanism that tRF-29-79 participates in glutamine metabolism through interacting with PTBP1 and regulating alternative splicing in the 3' UTR of SLC1A5 pre-mRNA.

LncRNA SLC1A5-AS/MZF1/ASCT2 Axis Contributes to Malignant Progression of Hepatocellular Carcinoma.

BACKGROUND: Hypoxia is a pivotal factor influencing cellular gene expression and contributing to the malignant progression of tumors. Metabolic anomalies under hypoxic conditions are predominantly mediated by mitochondria. Nonetheless, the exploration of hypoxia-induced long noncoding RNAs (lncRNAs) associated with mitochondria remains largely uncharted. METHODS: We established hypoxia cell models using primary human hepatocytes (PHH) and hepatocellular carcinoma (HCC) cell lines. We isolated mitochondria for high-throughput sequencing to investigate the roles of candidate lncRNAs in HCC progression. We employed in vitro and in vivo assays to evaluate the functions of solute carrier family 1 member 5 antisense lncRNA (SLC1A5-AS). RNA-seq was utilized to scrutinize the comprehensive genome profile regulated by SLC1A5-AS in HCC. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were utilized to validate the expression of alanine-serine-cysteine transporter 2 (ASCT2, encoded by the SLC1A5 gene), and a glutamine uptake assay was employed to estimate the glutamine uptake capacity of Huh-7 cells after SLC1A5-AS overexpression. To delve into the mechanisms governing the regulation of SLC1A5 expression by SLC1A5-AS, we employed a biotin-labeled SLC1A5-AS probe in conjunction with a western blot assay to confirm the interactions between SLC1A5-AS and candidate transcription factors. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were utilized to authenticate the effects of the predicted transcription factors on SLC1A5 promoter activity. RESULTS: Following the screening, we identified CTB-147N14.6, derived from the antisense strand of the SLC1A5 gene, which we have named SLC1A5-AS. SLC1A5-AS exhibited significantly elevated expression levels in HCC tissue and was associated with poor prognosis in HCC patients. In vitro and in vivo assays revealed that the overexpression of SLC1A5-AS significantly heightened cell invasion and metastasis. RNA-seq data unveiled SLC1A5-AS involvement in glutamine metabolism, left-handed amino (L-amino) acid transmembrane transporter activity, and the nuclear factor kappa-B (NF-κB) signaling pathway. Overexpression of SLC1A5-AS markedly increased ASCT2 mRNA/protein levels, thereby enhancing glutamine uptake and promoting the growth and metastasis of HCC cells. Mechanistically, higher RNA levels of SLC1A5-AS directly bound with myeloid zinc finger 1 (MZF1), acting as a transcriptional repressor, thus diminishing its binding to the SLC1A5 promoter region. CONCLUSIONS: Our findings unveil a novel role for the lncRNA SLC1A5-AS in glutamine metabolism, suggesting that targeting SLC1A5-AS/MZF1, in conjunction with ASCT2 inhibitor treatment, could be a potential therapeutic strategy for this disease.

SLC1A5 is a novel biomarker associated with ferroptosis and the tumor microenvironment: a pancancer analysis.

Solute carrier family 1 member 5 (SLC1A5) is a member of the solute carrier (SLC) superfamily of transporters and plays an important role in tumors as a key transporter of glutamine into cells. However, the relationship between SLC1A5, which is involved in immune regulation, and immune cell infiltration in the tumor microenvironment has not been elucidated, and the relationship between SLC1A5 and ferroptosis is rarely reported. Therefore, we comprehensively analyzed the expression level of SLC1A5 across cancers and compared it with that in normal tissues. Then, the relationship between SLC1A5 expression and the tumor immune microenvironment was analyzed by single-cell analysis, gene set enrichment analysis (GSEA), and Tumor Immune Estimation Resource (TIMER). Next, the correlations of the SLC1A5 expression level with immunotherapy response, immunomodulator expression, tumor mutation burden (TMB) and microsatellite instability (MSI) were evaluated. Finally, in vitro experiments verified that SLC1A5 participates in ferroptosis of glioma cells to regulate tumor progression. Our results indicated that SLC1A5 is aberrantly expressed in most cancer types and closely associated with prognosis. The GSEA results showed that SLC1A5 is involved in immune activation processes and closely related to the infiltration levels of different immune cells in different cancer types. Upon further investigation, we found that SLC1A5 is a suppressor of ferroptosis in glioma, and SLC1A5 knockdown inhibited the proliferation and migration of glioma cells in vitro. In conclusion, we conducted a pancancer analysis of SLC1A5, demonstrated its role as a prognostic biomarker in cancer patients and explored its potential biological functions.

Targeting Pulmonary Fibrosis by SLC1A5-Dependent Glutamine Transport Blockade.

The neutral amino acid glutamine plays a central role in TGF-ß (transforming growth factor-ß)-induced myofibroblast activation and differentiation. Cells take up glutamine mainly through a transporter expressed on the cell surface known as solute carrier SLC1A5 (solute carrier transporter 1A5). In the present work, we demonstrated that profibrotic actions of TGF-ß are mediated, at least in part, through a metabolic maladaptation of SLC1A5 and that targeting SLC1A5 abrogates multiple facets of fibroblast activation. This approach could thus represent a novel therapeutic strategy to treat patients with fibroproliferative diseases. We found that SLC1A5 was highly expressed in fibrotic lung fibroblasts and fibroblasts isolated from idiopathic pulmonary fibrosis lungs. The expression of profibrotic targets, cell migration, and anchorage-independent growth by TGF-ß required the activity of SLC1A5. Loss or inhibition of SLC1A5 function enhanced fibroblast susceptibility to autophagy; suppressed mTOR, HIF (hypoxia-inducible factor), and Myc signaling; and impaired mitochondrial function, ATP production, and glycolysis. Pharmacological inhibition of SLC1A5 by the small-molecule inhibitor V-9302 shifted fibroblast transcriptional profiles from profibrotic to fibrosis resolving and attenuated fibrosis in a bleomycin-treated mouse model of lung fibrosis. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in fibrosis, providing a framework for new paradigm-shifting therapies targeting cellular metabolism for this devastating disease.

A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells.

The ability of tumor cells to alter their metabolism to support survival and growth presents a challenge to effectively treat cancers. Carbonic anhydrase IX (CAIX) is a hypoxia-induced, metabolic enzyme that plays a crucial role in pH regulation in tumor cells. Recently, through a synthetic lethal screen, we identified CAIX to play an important role in redox homeostasis. In this study, we show that CAIX interacts with the glutamine (Gln) transporter, solute carrier family 1 member 5 (SLC1A5), and coordinately functions to maintain redox homeostasis through the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis. Inhibition of CAIX increases Gln uptake by SLC1A5 and concomitantly increases GSH levels. The combined inhibition of CAIX activity and Gln metabolism or the GSH/GPX4 axis results in an increase in lipid peroxidation and induces ferroptosis, both in vitro and in vivo. Thus, this study demonstrates cotargeting of CAIX and Gln metabolism as a potential strategy to induce ferroptosis in tumor cells.

CircRNA OXCT1 promotes the malignant progression and glutamine metabolism of non-small cell lung cancer by absorbing miR-516b-5p and upregulating SLC1A5.

Previous study has demonstrated the high expression of circular RNA 3-oxoacid CoA-transferase 1 (circ-OXCT1) in lung adenocarcinoma tumor tissues. However, the role and possible mechanism of circ-OXCT1 in non-small cell lung cancer (NSCLC) progression was unclear.Quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) staining assay were performed to detect the expression of circ-OXCT1, microRNA-516b-5p (miR-516b-5p), solute carrier family 1 member 5 (SLC1A5) and other indicated protein markers. Cell proliferation was measured by Cell counting kit 8 (CCK8), colony formation and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Flow cytometry was employed to detect the rate of apoptotic cells. Cell migration and invasion were measured using transwell assay. The relative glutamine uptake and α-ketoglutarate (α-KG) production was determined using commercial kits. Interaction between miR-516b-5p and circ-OXCT1 or SLC1A5 was predicted by bioinformatics analysis and confirmed via luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assay was implemented to demonstrate the effect of circ-OXCT1 in tumor growth.Circ-OXCT1 and SLC1A5 were upregulated and miR-516b-5p was downregulated in NSCLC tissues and cells. Functional experiments revealed that circ-OXCT1 silencing suppressed cell proliferation, migration and invasion, but promoted cell apoptosis in vitro. Circ-OXCT1 knockdown repressed tumor formation in vivo. Besides, miR-516b-5p was a target of circ-OXCT1, and miR-516b-5p inhibitor could relieve circ-OXCT1 absence-mediated effects in NSCLC cells. SLC1A5 was identified as a target of miR-516b-5p. Circ-OXCT1 promoted SLC1A5 expression by target binding with miR-516b-5p.Circ-OXCT1 facilitated NSCLC progression via miR-516b-5p-dependent regulation of SLC1A5, which provided a possible circRNA-targeted therapy for NSCLC.

Circ_0000069 contributes to the growth, metastasis and glutamine metabolism in renal cell carcinoma (RCC) via regulating miR-125a-5p-dependent SLC1A5 expression.

BACKGROUND: Circular RNAs (circRNAs) have emerged as critical mediators in various cancers, including renal cell carcinoma (RCC). In the present research, the functions of circ_0000069 in RCC were explored. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay, western blot assay and immunohistochemistry (IHC) assay were performed for the expression of circ_0000069, microRNA-125a-5p (miR-125a-5p) and solute carrier family 1 member 5 (SLC1A5). Cell Counting Kit-8 (CCK-8) assay and 5'-ethynyl-2'-deoxyuridine (EdU) assay were performed for cell proliferation. Flow cytometry assay was manipulated for cell apoptosis. Transwell assay and wound-healing assay were utilized for cell invasion and migration. Glutamine metabolism level was evaluated by examining glutamine consumption, α-ketoglutarate production and glutamate production. Dual-luciferase reporter assay was used to analyze the relationships of circ_0000069, miR-125a-5p and SLC1A5. Murine xenograft model assay was conducted to analyze the function of circ_0000069 in vivo. RESULTS: Circ_0000069 level was abnormally upregulated in RCC tissues and cells. Knockdown of circ_0000069 inhibited the proliferation, invasion, migration and glutamine metabolism and promoted the apoptosis in RCC cells in vitro and restrained tumor growth in vivo. Circ_0000069 served as the sponge for miR-125a-5p. MiR-125a-5p inhibition ameliorated the effects of circ_0000069 knockdown on RCC cell malignant behaviors. SLC1A5 was identified as the target gene of miR-125a-5p. Moreover, miR-125a-5p overexpression repressed the progression of RCC cells, while SLC1A5 elevation abrogated the effect. CONCLUSION: Circ_0000069 knockdown inhibited the carcinogenesis of RCC by regulating miR-125a-5p/SLC1A5 axis.

Mesothelioma cancer cells are glutamine addicted and glutamine restriction reduces YAP1 signaling to attenuate tumor formation.

Glutamine addiction is an important phenotype displayed in some types of cancer. In these cells, glutamine depletion results in a marked reduction in the aggressive cancer phenotype. Mesothelioma is an extremely aggressive disease that lacks effective therapy. In this study, we show that mesothelioma tumors are glutamine addicted suggesting that glutamine depletion may be a potential therapeutic strategy. We show that glutamine restriction, by removing glutamine from the medium or treatment with inhibitors that attenuate glutamine uptake (V-9302) or conversion to glutamate (CB-839), markedly reduces mesothelioma cell proliferation, spheroid formation, invasion, and migration. Inhibition of the SLC1A5 glutamine importer, by knockout or treatment with V-9302, an SLC1A5 inhibitor, also markedly reduces mesothelioma cell tumor growth. A relationship between glutamine utilization and YAP1/TEAD signaling has been demonstrated in other tumor types, and the YAP1/TEAD signaling cascade is active in mesothelioma cells and drives cell survival and proliferation. We therefore assessed the impact of glutamine depletion on YAP1/TEAD signaling. We show that glutamine restriction, SLC1A5 knockdown/knockout, or treatment with V-9302 or CB-839, reduces YAP1 level, YAP1/TEAD-dependent transcription, and YAP1/TEAD target protein (e.g., CTGF, cyclin D1, COL1A2, COL3A1, etc.) levels. These changes are observed in both cells and tumors. These findings indicate that mesothelioma is a glutamine addicted cancer, show that glutamine depletion attenuates YAP1/TEAD signaling and tumor growth, and suggest that glutamine restriction may be useful as a mesothelioma treatment strategy.

Procyanidin B2 3,3″-di-O-gallate suppresses IFN-γ production in murine CD4+ T cells through the regulation of glutamine influx via direct interaction with ASCT2.

Excessive activation of CD4+ T cells increases cytokine production substantially and induces immune-mediated diseases. Procyanidins are polyphenols with anti-inflammatory properties. Procyanidin B2 (PCB2) gallate [specifically, PCB2 3,3''-di-O-gallate (PCB2DG)] inhibits cytokine production through the suppression of glycolysis via mammalian target of rapamycin (mTOR) in T cells. Several amino acids play critical roles in T cell activation, especially glutamine, which is important in mTOR signaling and interferon-γ (IFN-γ) production in CD4+ T cells. However, the mechanisms underlying the effects of PCB2DG, including its interaction partners, have yet to be clarified. In the present study, the mechanisms underlying the inhibitory effect of PCB2DG on IFN-γ through glutamine metabolism regulation were investigated. We found that PCB2DG treatment reduced intracellular glutamine levels in CD4+ T cells, whereas the addition of glutamine abrogated the inhibitory effects of PCB2DG on IFN-γ production. The PCB2DG-induced reduction in intracellular glutamine accumulation led to the upregulated expression of activating transcription factor 4, which was induced by the cytoprotective signaling pathway in the amino acid response. In addition, the mRNA and protein expression levels of alanine serine cysteine transporter 2 (ASCT2), a major glutamine transporter in CD4+ T cells, were not altered by PCB2DG treatment. Further analysis using a target identification strategy revealed that PCB2DG binds to ASCT2, suggesting that PCB2DG interacts directly with this major glutamine transporter to inhibit glutamine influx. Overall, this study indicates that ASCT2 is a novel target protein of a dietary polyphenol and provides new insights into the mechanism underlying the immunomodulatory effects of polyphenols.

SLC1A5 enhances malignant phenotypes through modulating ferroptosis status and immune microenvironment in glioma.

Glioma is the most common type of primary malignant tumor in the central nervous system with limited treatment satisfaction. Finding new therapeutic targets has remained a major challenge. Ferroptosis is a novel and distinct type of programmed cell death, playing a regulatory role in the progression of tumors. However, the role of ferroptosis or ferroptosis-related genes (FRGs) in glioma progression has not been extensively studied. In our study, a novel ferroptosis-related prognostic model, including 7 genes, was established, in which patients classified into the high-risk group had more immuno-suppressive status and worse prognosis. Among these 7 genes, we screened solute carrier family 1 member 5 (SLC1A5), an FRG, as a possible new target for glioma treatment. Our results showed that the expression of SLC1A5 was significantly upregulated in glioblastoma tissues compared with the low-grade gliomas. In addition, SLC1A5 knockdown could significantly inhibit glioma cell proliferation and invasion, and reduce the sensitivity of ferroptosis via the GPX4-dependent pathway. Furthermore, SLC1A5 was found to be related to immune response and SLC1A5 knockdown decreased the infiltration and M2 polarization of tumor-associated macrophages. Pharmacological inhibition of SLC1A5 by V9302 was confirmed to promote the efficacy of anti-PD-1 therapy. Overall, we developed a novel prognostic model for glioma based on the seven-FRGs signature, which could apply to glioma prognostic and immune status prediction. Besides, SLC1A5 in the model could regulate the proliferation, invasion, ferroptosis and immune state in glioma, and be applied as a prognostic biomarker and potential therapeutic target for glioma.

circ_0025033 promotes ovarian cancer development via regulating the hsa_miR-370-3p/SLC1A5 axis.

BACKGROUND: Circular RNAs (circRNAs) appear to be important modulators in ovarian cancer. We aimed to explore the role and mechanism of circ_0025033 in ovarian cancer. METHODS: qRT-PCR was conducted to determine circ_0025033, hsa_miR-370-3p, and SLC1A5 mRNA expression. Functional experiments were conducted, including Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, tube formation, xenograft tumor model assay, western blot analysis of protein levels, and analysis of glutamine metabolism using commercial kits. Their predicted interaction was confirmed using dual-luciferase reporter and RNA pull-down. RESULTS: circ_0025033 was upregulated in ovarian cancer; its knockdown induced proliferation, invasion, angiogenesis, glutamine metabolism, and apoptosis in vitro, and blocked tumor growth in vivo. circ_0025033 regulated ovarian cancer cellular behaviors via sponging hsa_miR-370-3p. In parallel, SLC1A5 might abolish the anti-ovarian cancer role of hsa_miR-370-3p. Furthermore, circ_0025033 affected SLC1A5 via regulating hsa_miR-370-3p. CONCLUSION: circ_0025033 might promote ovarian cancer progression via hsa_miR-370-3p/SLC1A5, providing an interesting insight into ovarian cancer tumorigenesis.

Circ_0000808 promotes the development of non-small cell lung cancer by regulating glutamine metabolism via the miR-1827/SLC1A5 axis.

BACKGROUND: Circular RNA (circRNA) has been proved to be an important molecular target for cancer treatment. However, the function and molecular mechanism of circ_0000808 in non-small cell lung cancer (NSCLC) are still unclear. METHODS: Quantitative real-time PCR was used to detect the expression of circ_0000808, miR-1827, and solute carrier family 1 member 5 (SLC1A5). Cell proliferation, apoptosis, migration, and invasion were measured by cell counting kit 8 assay, colony formation assay, EdU staining, flow cytometry, wound healing assay, and transwell assay. The protein expression was measured by Western blot analysis. Dual-luciferase reporter assay and RIP assay were used to investigate the interactions between miR-1827 and circ_0000808 or SLC1A5. Cell glutamine metabolism was assessed by determining glutamine uptake, glutamate production, and α-ketoglutarate production. Xenograft mouse model was used to assess the in vivo effects of circ_0000808. RESULTS: Circ_0000808 expression was upregulated in NSCLC tissues and cancer cells, and its silencing inhibited NSCLC cell proliferation, migration, and invasion and led to apoptosis. Further results confirmed that circ_0000808 interacted with miR-1827 to positively regulate SLC1A5. The rescue experiments showed that miR-1827 inhibitor reversed the suppressive effect of circ_0000808 knockdown on the malignant behaviors of NSCLC cells. Also, SLC1A5 overexpression abolished the inhibition effect of miR-1827 on NSCLC cell progression. In addition, circ_0000808/miR-1827/SLC1A5 axis positively regulated the glutamine metabolism process in NSCLC cells. Moreover, circ_0000808 knockdown reduced the NSCLC tumor growth in vivo. CONCLUSION: In summary, our data showed that circ_0000808 enhanced the progression of NSCLC by promoting glutamine metabolism through the miR-1827/SLC1A5 axis.

Glutamine Transporter SLC1A5 Regulates Ionizing Radiation-Derived Oxidative Damage and Ferroptosis.

Ionizing radiation-derived oxidative stress and ferroptosis are one of the most important biological effects on destroying the liver tumor, whereas radioresistance of liver tumor remains a leading cause of radiotherapy (RT) failure mainly because of the protective antiferroptosis, in which oxidative stress and subsequent lipid peroxidation are the key initiators. Thus, it is of great importance to overcome ferroptosis resistance to improve the therapeutic efficacy of RT in liver tumor patients. Irradiation-resistant HepG2 cells (HepG2-IRR) were established by long-term exposure to X-ray (2 to 8 Gy), and targeted metabolomics analysis revealed an obvious increase in intracellular amino acids in HepG2-IRR cells upon ferroptosis stress. Among these amino acids with obvious changes, N-acetylglutamine, a derivative of glutamine, is essential for the redox homeostasis and progression of tumor cells. Interestingly, the treatment of glutamine starvation could promote the ferroptosis effect significantly, whereas glutamine supplementation reversed the ferroptosis effect completely. Consistent with the changes in amino acids pattern, the glutamine transporter SLC1A5 was verified in liver tumor samples from TCGA training and validation cohorts as an independent prognostic amino acid-ferroptosis gene (AFG). A risk score for screening prognosis based on the SLC1A5, SLC7A11, ASNS, and TXNRD1 demonstrated that a high-risk score was correlated with poor survival. In vitro studies had shown that the knockdown of SLC1A5 resulted in a significant decrease in cell viability and promoted lipid peroxidation and oxidative damage introduced by irradiation (10 Gy). Collectively, our findings indicated that SLC1A5 may act as a suppressor gene against ferroptosis and can be a potential target for ionizing radiation mediated effects.

The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.

N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects in vitro and in vivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.