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BACKGROUND: Breast cancer (BC) remains heterogeneous in terms of prognosis and response to treatment. Metabolic reprogramming is a critical part of oncogenesis and a potential therapeutic target. Glutaminase (GLS), which generates glutamate from glutamine, plays a role in triple-negative breast cancer (TNBC). However, targeting GLS directly may be difficult, as it is essential for normal cell function. This study aimed to determine potential targets in BC associated with glutamine metabolism and evaluate their prognostic value in BC. METHODS: The iNET model was used to identify genes in BC that are associated with GLS using RNA-sequencing data. The prognostic significance of tripartite motif-containing 2 (TRIM2) mRNA was assessed in BC transcriptomic data (n = 16,575), and TRIM2 protein expression was evaluated using immunohistochemistry (n = 749) in patients with early-stage invasive breast cancer with long-term follow-up. The associations between TRIM2 expression and clinicopathological features and patient outcomes were evaluated. RESULTS: Pathway analysis identified TRIM2 expression as an important gene co-expressed with high GLS expression in BC. High TRIM2 mRNA and TRIM2 protein expression were associated with TNBC (p < 0.01). TRIM2 was a predictor of poor distant metastasis-free survival (DMFS) in TNBC (p < 0.01), and this was independent of established prognostic factors (p < 0.05), particularly in those who received chemotherapy (p < 0.05). In addition, TRIM2 was a predictor of shorter DMFS in TNBC treated with chemotherapy (p < 0.01). CONCLUSIONS: This study provides evidence of an association between TRIM2 and poor patient outcomes in TNBC, especially those treated with chemotherapy. The molecular mechanisms and functional behaviour of TRIM2 and the functional link with GLS in BC warrant further exploration using in vitro models.
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BACKGROUND: T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1. METHODS: Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively. RESULTS: The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001). CONCLUSION: Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.
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Glutamina , Leucemia Mieloide Aguda , Humanos , Ácido Glutámico , Receptor 2 Celular del Virus de la Hepatitis A , Células HL-60RESUMEN
@#Objective To investigate the effect of glutaminase 1(GLS1)specific inhibitor BPTES[bis-2-（5-phenylacetamido-1,3,4-thiadiazol-2-yl）ethyl sulfide]on the liver fibrosis in the mouse model of liver fibrosis induced by carbon tetrachloride(CCl4).Methods Male C57BL/6J mice were intraperitoneally injected with olive oil(control group),10%CCl4(10 μL/g,model group)or 10% CCl4(10 μL/g)+ BPTES(10 mg/kg,treatment group),with 10 mice in each group,two doses a week for four weeks to establish liver fibrosis model. Collagen deposition in mouse liver tissue was observed by Sirius red staining. The expression levels of actin alpha 2(Acta2),collagen typeⅠalpha 1(Col1a1)GLS1 and GLS1 protein were detected by qRT-PCR and immunohistochemical staining.Results Compared with the control group,the liver tissue of mice in the model group was generally enlarged,the surface was not smooth and granular,and the ratio of liver mass to tibia length significantly increased(t = 2. 979,P < 0. 05);The Sirius red positive area of collagen deposition increased signifi-cantly in the liver tissue of mice in the model group(t = 7. 661,P < 0. 01),the relative expression levels of Acta2 and Col1a1 significantly increased(t = 4. 335 and 5. 319,respectively,each P < 0. 01),and the mRNA and protein levels of GLS1 significantly increased(t = 5. 319 and 9. 725,respectively,each P < 0. 01). However,compared with the model group,the BPTES treatment group had a reduction in liver mass,a significant reduction in the Sirius red positive area of collagen deposition in liver tissue(t = 7. 427,P < 0. 01),and a significant reduction in the relative expressions of Atca2 and Col1a1(t = 3. 713 and 2. 628,respectively,each P < 0. 05).Conclusion Inhibition of GLS1activity can significantly improve the degree of liver fibrosis induced by CCl4,providing a new idea for the treatment of liver fibrosis.
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In rapidly proliferating cancer cells, glutamine is a major source of energy and building blocks. Increased glutamine uptake and enhanced glutaminolysis are key metabolic features of many cancers. Glutamine is metabolized by glutaminase (GA), which is encoded by two genes: GLS and GLS2. In contrast to isoforms arising from the GLS gene, which clearly act as oncoproteins, the role of GLS2 products in tumorigenesis is far from well understood. While in some cancer types GLS2 is overexpressed and drives cancer development, in some other types it is downregulated and behaves as a tumor suppressor gene. In this review, we describe the essential functions and regulatory mechanisms of human GLS2 and the cellular compartments in which GLS2 has been localized. Furthermore, we present the context-dependent oncogenic and tumor-suppressor properties of GLS2, and delve into the mechanisms underlying these phenomena.
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Aberrant glutamatergic signaling has been implicated in altered metabolic activity and the demand to synthesize biomass in several types of cancer including melanoma. In the last decade, there has been a significant contribution to our understanding of metabolic pathways. An increasing number of studies are now emphasizing the importance of glutamate functioning as a signaling molecule and a building block for cancer progression. To that end, our group has previously illustrated the role of glutamatergic signaling mediated by metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous development of metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1, is one of the most abundant amino acids in humans and the predominant excitatory neurotransmitter in the central nervous system. Elevated levels of glutaminolytic mitochondrial tricarboxylic acid (TCA) cycle intermediates, especially glutamate, have been reported in numerous cancer cells. Herein, we highlight and critically review metabolic bottlenecks that are prevalent during tumor evolution along with therapeutic implications of limiting glutamate bioavailability in tumors.
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Metabolic programs are known to be altered in cancers arising from various tissues. Malignant transformation can alter signaling pathways related to metabolism and increase the demand for both energy and biomass for the proliferating cancerous cells. This scenario is further complexed by the crosstalk between transformed cells and the microenvironment. One of the most common metabolic alterations, which occurs in many tissues and in the context of multiple oncogenic drivers, is the increased demand for the amino acid glutamine. Many studies have attributed this increased demand for glutamine to the carbon backbone and its role in the tricarboxylic acid (TCA) cycle anaplerosis. However, an increasing number of studies are now emphasizing the importance of glutamine functioning as a proteogenic building block, a nitrogen donor and carrier, an exchanger for import of other amino acids, and a signaling molecule. Herein, we highlight the recent literature on glutamine's versatile role in cancer, with a focus on nitrogen metabolism, and therapeutic implications of glutamine metabolism in cancer.
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Osteoblast-mediated bone formation is a complex process involving various pathways and regulatory factors, including cytokines, growth factors, and hormones. Investigating the regulatory mechanisms behind osteoblast differentiation is important for bone regeneration therapy. miRNAs are known as important regulators, not only in a variety of cellular processes, but also in the pathogenesis of bone diseases. In the present study, we investigated the potential roles of miR-206 during osteoblast differentiation. We report that miR-206 expression was significantly down-regulated in human bone marrow mesenchymal stem cells (BMSCs) at days 7 and 14 during osteogenic induction. Furthermore, miR-206 overexpressing BMSCs showed attenuated alkaline phosphatase (ALP) activity, Alizarin Red staining, and osteocalcin secretion. The mRNA levels of osteogenic markers, Runx2 and Osteopontin (OPN), were significantly down-regulated in miR-206 overexpressing BMSCs. We observed that significantly increased glutamine uptake at days 7 and 14 during the osteogenic induction and inhibition of glutamine metabolism by knocking down glutaminase (GLS)-suppressed osteogenic differentiation of BMSCs. Here, we discover that miR-206 could directly bind to the 3'-UTR region of GLS mRNA, resulting in suppressed GLS expression and glutamine metabolism. Finally, restoration of GLS in miR-206 overexpressing BMSCs led to recovery of glutamine metabolism and osteogenic differentiation. In summary, these results reveal a new insight into the mechanisms of the miR-206-mediated osteogenesis through regulating glutamine metabolism. Our study may contribute to the development of therapeutic agents against bone diseases.
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Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Glutaminasa/genética , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , Osteogénesis/genética , Regiones no Traducidas 3'/genética , Células Cultivadas , Regulación de la Expresión Génica , Glutaminasa/metabolismo , Glutamina/metabolismo , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismoRESUMEN
INTRODUCTION: The kidney-type glutaminase (GLS) controlling the first step of glutamine metabolism is overexpressed in many cancer cells. Targeting inhibition of GLS shows obvious inhibitory effects on cancer cell proliferation. Therefore, extensive research and development of GLS inhibitors have been carried out in industrial and academic institutions over the past decade to address this unmet medical need. AREAS COVERED: This review covers researches and patent literatures in the field of discovery and development of small molecule inhibitors of GLS for cancer therapy over the past 16 years. EXPERT OPINION: The detailed ligand-receptor interaction information from their complex structure not only guides the rational drug design, but also facilitates in silico structure-based virtual ligand screening of novel GLS inhibitors. Multi-drug combination administration is of great significance both in terms of safety and efficacy.
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Antineoplásicos/farmacología , Glutaminasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/farmacología , Glutaminasa/metabolismo , Humanos , Ligandos , Neoplasias/enzimología , Neoplasias/patología , Patentes como AsuntoRESUMEN
In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.
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Vesículas Extracelulares/enzimología , Glutaminasa/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Quinona Reductasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Caveolina 1/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Vesículas Extracelulares/metabolismo , Glutaminasa/biosíntesis , Glutamina/metabolismo , Humanos , Immunoblotting , Masculino , Modelos Moleculares , NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Metástasis de la Neoplasia , Estrés Oxidativo , Neoplasias de la Próstata/metabolismo , Quinona Reductasas/biosíntesis , Quinona Reductasas/químicaRESUMEN
Clinical treatment for colorectal cancer (CRC) thus far encounters a huge challenge due to oxaliplatin-resistance. As crucial rate-limiting enzymes in aerobic glycolysis and glutaminolysis, pyruvate kinase M2 type (PKM2) and kidney-type glutaminase (GLS1) are proposed to carry important implications in colorectal carcinogenesis and drug-resistance. This study aimed to explore the possible association of oxaliplatin-resistance with aerobic glycolysis/glutaminolysis indexed by PKM2/GLS1 expression. PKM2 and GLS1 expression was quantified by polymerase chain reaction (PCR) and Western blot techniques in CRC cell lines. The abilities of cell formation, kinetics, migration, invasion, survival and apoptosis, as well as permeability glycoprotein (Pgp) expression were inspected before and after knocking-down PKM2/GLS1 expression. In addition, the influence of knocking-down PKM2/GLS1 expression was evaluated in vivo. Differentiated PKM2 and GLS1 expression in both THC8307 and THC8307/Oxa cell lines was identified. In the THC8307 cell line, PKM2 and GLS1 can accelerate malignant behaviors, increase oxaliplatin-resistance, upregulate Pgp expression, and inhibit cell apoptosis. Contrastingly in the THC8307/Oxa cell line, knockdown of PKM2/GLS1 expression can restrain malignant behaviors, reestablish oxaliplatin-sensitivity, downregulate Pgp expression, and induce cell apoptosis. In xenograft, knockdown of PKM2/GLS1 expression can significantly inhibit tumor growth, reduce Pgp expression, and increase tumor apoptosis. Taken together, the present findings enriched our knowledge by demonstrating a significant association of PKM2 and GLS1 with oxaliplatin-resistance in CRC. We further propose that knockdown of PKM2/GLS1 expression may constitute a novel therapeutic strategy toward effective treatment for CRC.
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Antineoplásicos/farmacología , Proteínas Portadoras/genética , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Expresión Génica , Glutaminasa/genética , Proteínas de la Membrana/genética , Compuestos Organoplatinos/farmacología , Hormonas Tiroideas/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Ratones , Oxaliplatino , Fenotipo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión a Hormona TiroideRESUMEN
The lack of sensitive and specific biomarkers hinders pathological diagnosis and prognosis for hepatocellular carcinoma (HCC). Since glutaminolysis plays a crucial role in carcinogenesis and progression, we sought to determine if the expression of kidney-type and liver-type glutaminases (GLS1 and GLS2) were informative for pathological diagnosis and prognosis of HCC. We compared the expression of GLS1 and GLS2 in a large set of clinical samples including HCC, normal liver, and other liver diseases. We found that GLS1 was highly expressed in HCC; whereas, expression of GLS2 was mainly confined to non-tumor hepatocytes. The sensitivity and specificity of GLS1 for HCC were 96.51% and 75.21%, respectively. A metabolic switch from GLS2 to GLS1 was observed in a series of tissues representing progressive pathologic states mimicking HCC oncogenic transformation, including normal liver, fibrotic liver, dysplasia nodule, and HCC. We found that high expression of GLS1 and low expression of GLS2 in HCC correlated with survival time of HCC patients. Expression of GLS1 and GLS2 were independent indexes for survival time; however, prognosis was predominantly determined by the level of GLS1 expression. These findings indicate that GLS1 expression is a sensitive and specific biomarker for pathological diagnosis and prognosis of HCC.