GLS1 Expression Research Articles

Glutaminolysis is associated with mitochondrial pathway activation and can be therapeutically targeted in glioblastoma

BackgroundGlioblastoma is an aggressive cancer that originates from abnormal cell growth in the brain and requires metabolic reprogramming to support tumor growth. Metabolic reprogramming involves the upregulation of various metabolic pathways. Although the activation of specific metabolic pathways in glioblastoma cell lines has been documented, the comprehensive profile of metabolic reprogramming and the role of each pathway in glioblastoma tissues in patients remain elusive.MethodsWe analyzed 38 glioblastoma tissues. As a test set, we examined 20 tissues from Kyushu University Hospital, focusing on proteins related to several metabolic pathways, including glycolysis, the one-carbon cycle, glutaminolysis, and the mitochondrial tricarboxylic acid cycle. Subsequently, we analyzed an additional 18 glioblastoma tissues from Kagoshima University Hospital as a validation set. We also validated our findings using six cell lines, including U87, LN229, U373, T98G, and two patient-derived cells.ResultsThe levels of mitochondria-related proteins (COX1, COX2, and DRP1) were correlated with each other and with glutaminolysis-related proteins (GLDH and GLS1). Conversely, their expression was inversely correlated with that of glycolytic proteins. Notably, inhibiting the glutaminolysis pathway in cell lines with high GLDH and GLS1 expression proved effective in suppressing tumor growth.ConclusionsOur findings confirm that glioblastoma tissues can be categorized into glycolytic-dominant and mitochondrial-dominant types, as previously reported. The mitochondrial-dominant type is also glutaminolysis-dominant. Therefore, inhibiting the glutaminolysis pathway may be an effective treatment for mitochondrial-dominant glioblastoma.

Buyang Huanwu Decoction restores the balance of mitochondrial dynamics after cerebral ischemia-reperfusion through calcium overload reduction by the PKCε-Nampt-Sirt5 axis

Ethnopharmacological relevanceStroke is a common condition that poses a significant threat to human health. Buyang Huanwu Decoction (BYHWD) is a traditional treatment used for stroke management. However, the exact mechanism by which BYHWD mitigates cerebral ischemia-reperfusion by regulating calcium overload and restoring mitochondrial function is not yet fully understood. AimThe objective of this research was to examine the neuroprotective properties of BYHWD in reducing the damage produced by cerebral ischemia/reperfusion (I/R) injury via the modulation of calcium overload and mitochondrial dynamics (MD). MethodsMCAO/R model success was evaluated via PSI laser scatter flowmetry. The neurological function scores were assessed. The cerebral infarct (CI) volume was detected via TTC staining. NeuN expression was detected via immunohistochemistry, and degenerated neurons were observed via FJC staining. The mitochondrial permeability transition pore (mPTP), the mitochondrial membrane potential (MMP), and ATP were detected. The reactive oxygen species (ROS) content and the NAD+/NADH ratio were determined. The glutamate (Glu) and glutamine (Gln) contents as well as the Ca2+ concentration were determined. The expression of PKCε, p-PKCε, namely, Sirt5, GLS, Drp1, p-Drp1 616, Fis1, Opa1, and Mfn2 was determined via Western blotting. Immunohistochemistry was used to detect p-PKCε, which is expressed at high levels. Immunofluorescence was used to detect p-Drp1 616, Opa1 and Sirt5 fluorescence intensity. ResultsBYHWD treatment enhanced neurological function, decreased the amount of CI, mitigated neuronal damage, decreased mPTP opening, restored the MMP, increased ATP synthesis, and decreased the ROS content after brain I/R. It also increased PKCε, p-PKCε, Sirt5, GLS, Opa1 and Mfn2 expression; downregulated p-Drp1 616, Drp1 and Fis1 expression; elevated the NAD+/NADH ratio and Gln content; and decreased the Glu content and Ca2+ concentration. The effects of BYHWD were reversed by the administration of the PKCε inhibitor εV1-2. BYHWD administration led to increased PKCε mRNA expression. ConclusionsBYHWD modulates MD by diminishing calcium overload through the PKCε-Nampt-Sirt5 axis, which restores mitochondrial function and mitigates brain I/R damage.

Comprehensive analysis of the expression level, prognostic value, and immune infiltration of cuproptosis-related genes in human breast cancer.

As a novel cell death form, cuproptosis results from copper combining with lipidated proteins in the tricarboxylic acid cycle. To the best of our knowledge no study has yet comprehensively analyzed the relationship between cuproptosis-related genes and breast cancer. The expression, prognostic value, mutations, chemosensitivity, and immune infiltration of cuproptosis-related genes in breast carcinoma patients were analyzed, PPI networks were constructed, and enrichment analyses were performed based on these genes. TIMER, UALCAN, Kaplan-Meier plotter, Human Protein Atlas, cBioPortal, STRING, GeneMANIA, DAVID, and R program v4.0.3 were used to accomplish the analyses above. Compared to normal breast tissues, FDX1, LIAS, LIPT1, DLD, DLAT, PDHA1, MTF1, and GLS were down-regulated in breast cancer tissues, while CDKN2A was up-regulated. High expression of FDX1, LIAS, DLD, DLAT, MTF1, GLS, and CDKN2A were associated with favorable overall survival. Cuproptosis-related genes showed a high alteration rate (51.3%) in breast cancer, contributing to worse clinical outcomes. The expression levels of FDX1, LIPT1, DLD, DLAT, PDHA1, PDHB, MTF1, GLS, and CDKN2A were associated positively with 1 or more immune cell infiltrations in breast cancer. Patients with high levels of B cell, CD4+ T cell, CD8+ T cell, and dendritic cell infiltration had a higher survival rate at 10 years. This study comprehensively investigated relationships between cuproptosis and breast cancer by bioinformatic analyses. We found that cuproptosis-related genes were generally lowly expressed in breast carcinoma tissue. As the critical gene of cuproptosis, high expression of FDX1 was related to favorable prognoses in breast cancer patients; thus, it might be a potential prognostic marker. Moreover, genes associated with cuproptosis were linked to immune infiltration in breast cancer and this relationship affected the prognosis of breast cancer.

GLS2 reduces the occurrence of epilepsy by affecting mitophagy function in mouse hippocampal neurons.

Altered mitophagy has been observed in various neurological disorders, such as epilepsy. The role of mitophagy in causing neuronal damage during epileptic episodes is significant, and recent research has indicated that GLS2 plays a crucial role in regulating autophagy. However, exactly how GLS2 affects epilepsy is still unclear. To investigate the expression and distribution characteristics of GLS2 in epilepsy, and then observed the changes in behavior and electrophysiology caused by overexpression of GLS2 in epileptic mice, and determined whether GLS2 regulated seizure-like changes in the mouse model through the protective mechanism of mitophagy. The expression of GLS2 in a kainic acid (KA)-induced epileptic mouse model and aglutamate-inducedneuronal excitatory damage in HT22 cells model was downregulation. In brief, overexpression of GLS2 can alleviate epileptic activity. Subsequently, we demonstrated that GLS2 interacts with mitophagy-related proteins in a KA-induced epilepsy mouse model. Mechanistically, overexpression of GLS2 inhibited mitophagy in epileptic mice, downregulating the expression of LC3 and reducing ROS production. This study proves the GLS2 expression pattern is abnormal in epileptic mice. The function of mitophagy in hippocampal neurons is affected by GLS2, and overexpression of GLS2 can reduce the occurrence of seizure-like events (SLEs) by altering mitophagy function. Thus, GLS2 might control seizures, and our findings provide a fresh avenue for antiepileptic treatment and offer novel insights into treating and preventing epilepsy.

Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells.

Myelodysplastic syndrome (MDS) features bone marrow failure and a heightened risk of evolving into acute myeloid leukemia (AML), increasing with age and reducing overall survival. Given the unfavorable outcomes of MDS, alternative treatments are necessary. Glutamine, the most abundant amino acid in the blood, is metabolized first by the enzyme glutaminase (GLS). To investigate whether GLS is involved in the progression of MDS. The efficacy of GLS inhibitors (CB839 or IPN60090) and BCL2 inhibitor venetoclax was also examined. We employed GLS inhibitors (CB839, IPN60090) and the BCL2 inhibitor venetoclax, prepared as detailed. MDS and AML cell lines were cultured under standard and modified (hypoxic, glutamine-free) conditions. Viability, proliferation, and caspase activity were assessed with commercial kits. RT-PCR quantified gene expression post-shRNA transfection. Mitochondrial potential, ATP levels, proteasome activity, and metabolic functions were evaluated using specific assays. Statistical analyses (t-tests, ANOVA) validated the findings. The glutamine-free medium inhibited the growth of MDS cells. GLS1 expression was higher in AML cells than in normal control samples (GSE15061), whereas GLS2 expression was not. Treatment of MDS and AML cells for 72 h was inhibited in a dose-dependent manner by GLS inhibitors. Co-treatment with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax and GLS inhibitors increased potency. Cells transfected with GLS1 short hairpin RNA showed suppressed proliferation under hypoxic conditions and increased sensitivity to venetoclax. Targeting glutaminolysis and BCL2 inhibition enhances the therapeutic efficacy and has been proposed as a novel strategy for treating high-risk MDS and AML.

Mitochondrial reprogramming by activating OXPHOS via glutamine metabolism in African American patients with bladder cancer.

Bladder cancer (BLCA) mortality is higher in African American (AA) patients compared with European American (EA) patients, but the molecular mechanism underlying race-specific differences are unknown. To address this gap, we conducted comprehensive RNA-Seq, proteomics, and metabolomics analysis of BLCA tumors from AA and EA. Our findings reveal a distinct metabolic phenotype in AA BLCA characterized by elevated mitochondrial oxidative phosphorylation (OXPHOS), particularly through the activation of complex I. The results provide insight into the complex I activation-driven higher OXPHOS activity resulting in glutamine-mediated metabolic rewiring and increased disease progression, which was also confirmed by [U]13C-glutamine tracing. Mechanistic studies further demonstrate that knockdown of NDUFB8, one of the components of complex I in AA BLCA cells, resulted in reduced basal respiration, ATP production, GLS1 expression, and proliferation. Moreover, preclinical studies demonstrate the therapeutic potential of targeting complex I, as evidenced by decreased tumor growth in NDUFB8-depleted AA BLCA tumors. Additionally, genetic and pharmacological inhibition of GLS1 attenuated mitochondrial respiration rates and tumor growth potential in AA BLCA. Taken together, these findings provide insight into BLCA disparity for targeting GLS1-Complex I for future therapy.

Prostate Cancer's Silent Partners: Fibroblasts and Their Influence on Glutamine Metabolism Manipulation.

Cancer-associated fibroblast (CAF)s in the tumour microenvironment (TME) modulate the extracellular matrix, interact with cancer cells, and facilitate communication with infiltrating leukocytes, significantly contributing to cancer progression and therapeutic response. In prostate cancer (PCa), CAFs promote malignancy through metabolic rewiring, cancer stem cell regulation, and therapy resistance. Pre-clinical studies indicate that targeting amino acid metabolism, particularly glutamine (Gln) metabolism, reduces cancer proliferation and stemness. However, most studies lack the context of CAF-cancer interaction, focusing on monocultures. This study assesses the influence of CAFs on PCa growth by manipulating Gln metabolism using colour-labelled PCa cell lines (red) and fibroblast (green) in a co-culture system to evaluate CAFs' effects on PCa cell proliferation and clonogenic potential. CAFs increased the proliferation of hormone-sensitive LNCaP cells, whereas the castration-resistant C4-2 cells were unaffected. However, clonogenic growth increased in both cell lines. Gln deprivation and GLS1 inhibition experiments revealed that the increased growth rate of LNCAP cells was associated with increased dependence on Gln, which was confirmed by proteomic analyses. Tissue analysis of PCa patients revealed elevated GLS1 levels in both the PCa epithelium and stroma, suggesting that GLS1 is a therapeutic target. Moreover, the median overall survival analysis of GLS1 expression in the PCa epithelium and stroma identified a "high-risk" patient group that may benefit from GLS1-targeted therapies. Therefore, GLS1 targeting appears promising in castration-resistant PCa patients with high GLS1 epithelium and low GLS1 stromal expression.

Risk factor prediction and immune correlation analysis of cuproptosis-related gene in osteoarthritis.

Osteoarthritis (OA) is a widespread inflammatory joint disease with significant global disability burden. Cuproptosis, a newly identified mode of cell death, has emerged as a crucial factor in various pathological conditions, including OA. In this context, our study aims to investigate the intrinsic relationship between cuproptosis-related genes (CRGs) and OA, and assess their potential as biomarkers for OA diagnosis and treatment. Datasets from the GEO databases were analysed the differential expression of CRGs, leading to the identification of 10 key CRGs (CDKN2A, DLD, FDX1, GLS, LIAS, LIPT1, MTF1, PDHA1, DLAT and PDHB). A logistic regression analysis and calibration curves were used to show excellent diagnostic accuracy. Consensus clustering revealed two CRG patterns, with Cluster 1 indicating a closer association with OA progression. RT-PCR confirmed a significant increase in the expression levels of these nine key genes in IL-1β-induced C28/i2 cells, and the expression of CDKN2A and FDX1 were also elevated in conditioned monocytes, while the expression of GLS and MTF1 were significantly decreased. Invitro experiments demonstrated that the expression levels of these 7/10 CRGs were significantly increased in chondrocytes induced by IL-1β, and upon stimulation with cuproptosis inducers, chondrocyte apoptosis was exacerbated, accompanied by an increase in the expression of cuproptosis-related proteins. These further substantiated our research findings and indicated that the nine selected cuproptosis genes have high potential for application in the diagnosis of OA.

TFAP2A Activates S100A2 to Mediate Glutamine Metabolism and Promote Lung Adenocarcinoma Metastasis.

Lung adenocarcinoma (LUAD) is a fatal disease with metabolic abnormalities. The dysregulation of S100 calcium-binding protein A2 (S100A2), a member of the S100 protein family, is connected to the development of various cancers. The impact of S100A2 on the LUAD occurrence and metastasis, however, has not yet been reported. The functional mechanism of S100A2 on LUAD cell metastasis was examined in this article. The expression of TFAP2A and S100A2 in LUAD tissues and cells was analyzed by bioinformatics and qRT-PCR, respectively. The enrichment pathway analysis was performed on S100A2. Bioinformatics analysis determined the binding relationship between TFAP2A and S100A2, and their interaction was validated through dual-luciferase and chromatin immunoprecipitation experiments. Cell viability was determined using cell counting kit-8 (CCK-8). A transwell assay was performed to analyze the invasion and migration of cells. Immunofluorescence was conducted to obtain vimentin and E-cadherin expression, and a western blot was used to detect the expression of MMP-2, MMP-9, GLS, and GLUD1. The kits measured the NADPH/NADP ratio, glutathione (GSH)/glutathione disulfide (GSSG) levels, and the contents of glutamine, α-KG, and glutamate. S100A2 was upregulated in LUAD tissues and cells, and S100A2 mediated glutamine metabolism to induce LUAD metastasis. Additionally, the transcriptional regulator TFAP2A was discovered upstream of S100A2, and TFAP2A expression was upregulated in LUAD, which indicated that TFAP2A promoted the S100A2 expression. The rescue experiment found that upregulation of S100A2 could reverse the inhibitory effects of silencing TFAP2A on glutamine metabolism and cell metastasis. In conclusion, by regulating glutamine metabolism, the TFAP2A/S100A2 axis facilitated LUAD metastasis. This suggested that targeting S100A2 could be beneficial for LUAD treatment.

The Anti-proliferative Effect of a Novel Glutaminase Inhibitor IN-3 on Prostate Cancer Cells.

This study aimed to evaluate anti-cancer potential of a novel glutaminase (GLS) inhibitor IN-3 in prostate cancer cells. The cell viability upon IN-3 treatment was examined using crystal violet staining and IC50 values were calculated for cancer cell lines PC-3 and LNCaP and normal fibroblasts CCD1072sk. The expression levels of GLS isoforms were determined by real-time polymerase chain reaction after IN-3 treatment. The metastatic prostate cancer dataset was downloaded from C-Bioportal and the expressions of GLS isoforms were analyzed. The IC50 values of IN-3 for LNCaP, PC-3 and CCD1072sk were 2.13, 6.14 and 15.39 μM respectively. The dose dependent effect of IN-3 was evident even in low concentration with 1 μM in LNCaP and 2 μM in PC-3 and these anti-proliferative effects of IN-3 were highly significant with p-values lower than 0.0001. The treatment of PC-3 cells with 10 μM IN-3 elevated the expression of kidney type GLS isoform of GLS1 but not GLS2. Comparison of metastatic and localized prostate cancer tissues showed that GLS1 was highly expressed not only in primary but also in metastatic prostate cancer tissues. GLS1 expression was significantly higher than GLS2 expression with p-values lower than 0.001. The GLS inhibitor IN-3 may be a potent anti-cancer agent in prostate cancer by demonstrating its differential effect between cancer and normal cells. Further studies are warranted to elucidate its drug potential in prostate cancer.

Therapeutic Targeting of the GLS1-c-Myc Positive Feedback Loop Suppresses Glutaminolysis and Inhibits Progression of Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is addicted to glutaminolysis. Targeting this metabolic dependency has emerged as a potential therapeutic approach for HNSCC. In this study, we conducted a bioinformatic analysis of The Cancer Genome Atlas HNSCC cohort that revealed a robust correlation between expression of MYC (encoding the protein c-Myc) and glutaminase 1 (GLS1), which catalyzes the first step in glutaminolysis. Intriguingly, disruption of GLS1 signaling in HNSCC cells by genetic depletion or CB-839 treatment resulted in a reduction in c-Myc protein stability via a ubiquitin-specific peptidase 1-dependent ubiquitin-proteasome pathway. On the other hand, c-Myc directly binds to the promoter region of GLS1 and upregulates its transcription. Notably, the GLS1-c-Myc pathway enhanced acetyl-coenzyme A carboxylase-dependent Slug acetylation, prompting cancer cell invasion and metastasis. Thus, the GLS1-c-Myc axis emerged as a positive feedback loop critical for driving the aggressiveness of HNSCC. Therapeutically, combining CB-839 with the c-Myc inhibitor MYCi975 strongly suppressed GLS1-c-Myc signaling, resulting in a superior antitumor effect compared with either single agent in an orthotopic mouse model of HNSCC. These findings hold promise for the development of effective therapies for patients with HNSCC, addressing an urgent need arising from the significant incidence and high metastatic rate of the disease. Significance: GLS1 and c-Myc form a positive feedback loop that promotes head and neck cancer metastasis and can be targeted as a promising therapeutic strategy for this disease.

Quercetin Attenuates Acute Kidney Injury Caused by Cisplatin by Inhibiting Ferroptosis and Cuproptosis.

Ferroptosis, an iron- and ROS-dependent form of regulated cell death. Cuproptosis is a novel form of cellular demise mode. Quercetin, a natural flavonoid, has demonstrated a range of pharmacological activities, including anti-cancer, anti-inflammatory, and antioxidant properties. In this research, we investigated the quercetin effect on cisplatin-induced acute kidney and its mechanism associated ferroptosis and cuproptosis. The HK-2 cells were used in this research. Cell viability was evaluated using the CCK-8 assay. Acute kidney injury (AKI) models were established to perform in vivo experiments. Renal tissue homogenate was used to determine ROS, LPO, MDA, PA, etc., to assess ferroptosis and cuproptosis. To perform bioinformatic analysis, microarray data from the GEO database was utilized. Real-time PCR analysis and ELISA was explored the mechanism of ferroptosis and cuproptosis. We found that ferroptosis and cuproptosis in AKI were abnormally activated caused by cisplatin, and that quercetin attenuated AKI by inhibiting ferroptosis and cuproptosis. QCT suppressed ferroptosis by reducing malondialdehyde (MDA) and ROS levels and increasing glutathione (GSH) levels and alleviated cuproptosis by reducing copper ion, pyruvate (PA) and HSP70 levels. Moreover, bioinformatic analysis revealed that the ferroptosis-related gene SLC7A11 and the cuproptosis-related genes ATP7B and GLS were the differential expression genes. And QCT significantly increased the expression or activity of SLC7A11, GPX4, ATP7B, and GLS in Cis-AKI mice. Our findings highlight the clinical importance of quercetin, which guards against cisplatin-induced acute kidney injury by suppressing ferroptosis and cuproptosis.

Inhibition of glutaminolysis alone and in combination with HDAC inhibitor has anti-myeloma therapeutic effects.

Aim: This study aimed to investigate drug candidates and their efficacy in treating refractory multiple myeloma (MM) despite significant therapeutic advances and the introduction of novel agents. Our study focused on how myeloma cells mediate the metabolic pathways essential for survival. Therefore, we examined the role of glutaminolysis in this process. Methods: We investigated the role of glutaminolysis in myeloma cell growth. In addition, we analyzed the ability of CB-839 (telaglenastat), a glutaminase (GLS) inhibitor, to suppress myeloma cell proliferation and enhance the sensitivity to histone deacetylase (HDAC) inhibitors. Results: Glutamate deprivation significantly reduced MM cell proliferation. We observed an upregulation of GLS1 expression in MM cell lines compared to that in normal controls. CB-839 inhibits MM cell proliferation in a dose-dependent manner, resulting in enhanced cytotoxicity. Additionally, intracellular α-ketoglutarate and nicotinamide adenine dinucleotide phosphate levels decreased after CB-839 administration. Combining panobinostat with CB-839 resulted in enhanced cytotoxicity and increased caspase 3/7 activity. Cells transfected with GLS shRNA exhibited reduced cell viability and elevated sub-G1 phase according to cell cycle analysis results. Compared to control cells, these cells also showed increased sensitivity to panobinostat. Conclusion: Glutaminolysis contributes to the viability of MM cells, and the GLS inhibitor CB-839 has been proven to be an effective treatment for enhancing the cytotoxic effect of HDAC inhibition. These results are clinically relevant and suggest that CB-839 is a potential therapeutic candidate for patients with MM.

PoRVA G9P[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Identification of subclusters and prognostic genes based on GLS-associated molecular signature in ulcerative colitis

Ulcerative colitis (UC) is a chronic and recurrent inflammatory disease that affects the colon and rectum. The response to treatment varies among individuals with UC. Therefore, the aim of this study was to identify and explore potential biomarkers for different subtypes of UC and examine their association with immune cell infiltration. We obtained UC RNA sequencing data from the GEO database, which included the training set GSE92415 and the validation set GSE87473 and GSE72514. UC patients were classified based on GLS and its associated genes using consensus clustering analysis. We identified differentially expressed genes (DEGs) in different UC subtypes through a differential expression analysis of the training cohort. Machine learning algorithms, including Weighted Gene Co-Expression Network Analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO), and Support Vector Machine Recursive Feature Elimination (SVM-RFE), were utilized to identify marker genes for UC. The CIBERSORT algorithm was used to determine the abundance of various immune cells in UC and their correlation with UC signature genes. Finally, we validated the expression of GLS through in vivo and ex vivo experiments. The expression of GLS was found to be elevated in patients with UC compared to normal patients. GLS and its related genes were able to classify UC patients into two subtypes, C1 and C2. The C1 subtype, as compared to the C2 subtype, showed a higher Mayo score and poorer treatment response. A total of 18 DEGs were identified in both subtypes, including 7 up-regulated and 11 down-regulated genes. Four UC signature genes (CWH43, HEPACAM2, IL24, and PCK1) were identified and their diagnostic value was validated in a separate cohort (AUC > 0.85). Furthermore, we found that UC signature biomarkers were linked to the immune cell infiltration. CWH43, HEPACAM2, IL24, and PCK1 may serve as potential biomarkers for diagnosing different subtypes of UC, which could contribute to the development of targeted molecular therapy and immunotherapy for UC.

Distinct cuproptosis patterns in hepatocellular carcinoma patients correlate with unique immune microenvironment characteristics and cell-cell communication, contributing to varied overall survival outcomes.

Hepatocellular carcinoma (HCC), a prevalent cancer, is linked to cuproptosis in tumor progression. However, cuproptosis's impact on HCC prognosis and its role in the tumor microenvironment remain unclear. We aimed to explore the correlation between cellular cuproptosis and the immune microenvironment in HCC, providing potential immunotherapeutic insights. Examining cuproptosis-related genes and the immune microenvironment through consensus clustering and WGCNA. Risk models were constructed using LASSO Cox analysis and validated in an independent cohort. Gene expression data from The Cancer Genome Atlas (TCGA) database and single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database were utilized. We scored cuproptosis expression and explored immunoinfiltration and cell-cell communication. Differential signals in T_memory cells were compared across different cuproptosis levels. Cuproptosis genes associated with fibroblast recruitment (GLS) and macrophage infiltration (FDX1). Liver cancer patients categorized into two subtypes based on cuproptosis gene expression. High expression of DLAT, GLS, and CDKN2A linked to immunosuppression (TGF-β), while high FDX1, MTF1, LIAS, and LIPT1 expression enhanced communication with non-immune cells. Developed reliable prognostic signature score and nomogram using cuproptosis-related genes. Single-cell analysis revealed differences in T_memory and TAM infiltration based on cuproptosis scores, with SPP1 and MIF as dominant signaling molecules. Finally, the results of in vitro experiments showed that when DLAT or CDKN2A was knocked down, the proliferation, migration, and invasion of HCC cells were significantly decreased. Our study demonstrates that cuproptosis affects the immune microenvironment and cell-cell communication. Identified 9 genetic markers predicting survival outcomes and immunotherapy responses. Evaluating cuproptosis signaling can optimize immunotherapeutic strategies for hepatocellular carcinoma.

Analysis of anti-tumor effect and mechanism of GLS1 inhibitor CB-839 in colorectal cancer using a stroma-abundant tumor model

BackgroundGlutaminase 1 (GLS1), a key enzyme in glutamine metabolism in cancer cells, acts as a tumor promoter and could be a potential therapeutic target. CB-839, a GLS1-specific inhibitor, was developed recently. Herein, we aimed to elucidate the anti-tumor effects and mechanism of action of CB-839 in colorectal cancer (CRC). MethodsUsing the UCSC Xena public database, we evaluated GLS1 expression in various cancers. Immunostaining for GLS1 was performed on 154 surgically resected human CRC specimens. Subsequently, we examined the GLS1 mRNA expression levels in eight CRC cell lines and evaluated the association between GLS1 expression and CB-839 efficacy. To create a reproducible CRC model with abundant stroma and an allogeneic immune response, we co-transplanted CT26 and stem cells into BALB/c mice and treated them with CB-839. Finally, RNA sequencing of mouse tumors was performed. ResultsDatabase analysis showed higher GLS1 expression in CRC tissues than in normal colon tissues. Clinical samples from 114 of the 154 patients with CRC showed positive GLS1 expression. GLS1 expression in clinical CRC tissues correlated with vascular invasion. CB-839 treatment inhibited cancer cell proliferation depending on GLS1 expression in vitro and inhibited tumor growth and metastasis in the CRC mouse model. RNA sequencing revealed that CB-839 treatment inhibited stromal activation, tumor growth, migration, and angiogenesis. These findings were validated through in vitro and in vivo experiments and clinical specimen analysis. ConclusionsGLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

Tanshinone IIA alleviates pulmonary fibrosis by modulating glutamine metabolic reprogramming based on [U-13C5]-glutamine metabolic flux analysis

IntroductionGlutamine metabolic reprogramming, mediated by glutaminase (GLS), is an important signal during pulmonary fibrosis (PF) progression. Tanshinone IIA (Tan IIA) is a naturally lipophilic diterpene with antioxidant and antifibrotic properties. However, the potential mechanisms of Tan IIA for regulating glutamine metabolic reprogramming are not yet clear. ObjectivesThis study aimed was to evaluate the role of Tan IIA in intervening in glutamine metabolic reprogramming to exert anti-PF and to explore the potential new mechanisms of metabolic regulation. MethodsFibrotic characteristics was detected via immunofluorescence and western blotting analysis. Cell proliferation was examined with EdU Assay. Cell metabolites were labeled by using stable isotope [U-13C5]-glutamine. By utilizing 100% 13C glutamine tracers and employing network analysis to investigate the activation of metabolic pathways in fibroblasts, as well as evaluating the impact of Tan IIA on these pathways, we accurately quantified the absolute flux of glutaminolysis, proline synthesis, and the TCA cycle pathway using isotopomer network compartmental analysis (INCA), a user-friendly software tool for 13C metabolic flux analysis (13C-MFA). Molecular docking was used for identifying the binding of Tan IIA with target protein. ResultsTan IIA ameliorate TGF-β1-induced myofibroblast proliferation, reduce collagen I and III and α-SMA protein expression in MRC-5 and NIH-3T3 cells. Furthermore, Tan IIA regulate mitochondrial energy metabolism by modulating TGF-β1-stimulated glutamine metabolic reprogramming in NIH-3T3 cells and inhibiting GLS1 expression, which reduced the metabolic flux of glutamine into mitochondria in myofibroblasts, and also targeted inhibited the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), P5C reductase 1 (PYCR1), and phosphoserine aminotransferase 1 (PSAT1), and reduced proline hydroxylation and blocked the collagen synthesis pathway. ConclusionTan IIA reverses glutamine metabolic reprogramming, reduces mitochondrial energy expenditure, and inhibits collagen matrix synthesis by modulating potential targets in glutamine metabolism. This novel perspective sheds light on the essential role of glutamine metabolic reprogramming in PF.

Clinical and histopathological factors for recurrence and metastasis in lacrimal gland adenoid cystic carcinoma in Chinese patients.

To investigate the association of metabolism-related proteins and clinicopathological features with poor prognosis in lacrimal gland adenoid cystic carcinoma (LGACC). Clinicopathological data for 39 Chinese patients with LGACC enrolled were retrospectively analysed. Disease progression included death, recurrence, further nodal metastasis, and distant metastasis. Expression of ASCT2 and GLS1 were evaluated by immunohistochemistry. Kaplan-Meier survival curves and Cox proportional hazards regression models were used for risk factor analyses. At the end of follow-up, 14 patients (35.9%) developed local recurrence, 13 patients (33.3%) developed distant metastasis, 3 patients (7.7%) developed lymph node metastasis, and 9 patients (23.1%) died. Among the 13 patients who developed distant metastasis, lung metastasis was observed in 8 patients (61.5%), the brain in 8 patients (61.5%), and bone in 1 patient (7.7%). ASCT2 was expressed in 16 (57.14%) cases, while GLS1 had high expression in 19 (67.9%) cases. Advanced T category (≥T3), bone erosion, basaloid subtype, and ASCT2 (-) were associated with disease progression. Basaloid subtype was an independent risk factor for local recurrence (P = 0.028; HR, 12.12; 95% CI, 1.3-111.5). ASCT2(-) was an independent risk factor for distant metastasis (P = 0.016; HR, 14.46; 95% CI, 1.6-127.5) and was associated with basaloid subtype (P = 0.019). For LGACC, ≥T3 category, basaloid subtype, and bone erosion were high-risk predictors. ASCT2(-) was an independent risk factor for distant metastasis, which suggested that it could be a potential biomarker for LGACC.

Abstract 3061: A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness

Abstract Both GLS1 and c-Myc are upregulated in head and neck squamous cell carcinoma (HNSCC) primary tumors and further increased in metastatic tumors. However, the regulation between these two molecules remains largely unknown. GLS1 is a critical enzyme that regulates glutamate, which plays an important role in cancer metabolism that supports cancer growth and survival. Our bioinformatic analysis of the TCGA HNSCC cohort revealed a strong correlation between c-Myc and GLS1 expression. We describe the importance of c-Myc in regulating GLS1 and vice versa. c-Myc protein directly binds to the promoter of the GLS1 gene and upregulates its expression at the transcriptional level. Interestingly, blocking GLS1 signaling in HNSCC cells by lentiviral shRNA knockdown or CB-839 treatment downregulates USP1, one of the best characterized human DUBs, which in turn reduces c-Myc protein stability via the ubiquitin-proteasome pathway. The GLS1-c-Myc axis thus represents a novel positive feedback loop that is critical for driving the aggressiveness of HNSCC. As the treatment of HNSCC remains a major challenge, these novel findings provide the molecular basis for combining GLS1-specific inhibitors with c-Myc-targeted therapy for the treatment of HNSCC patients. Citation Format: Jianqiang Yang, Fanghui Chen, Fan Yang, Yong Teng. A positive feedback loop between GLS1 and c-Myc drives tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3061.