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.

Synergistic Effects of Glutamine Deprivation and Metformin in Acute Myeloid Leukemia.

OBJECTIVE: The metabolic reprogramming of acute myeloid leukemia (AML) cells is a compensatory adaptation to meet energy requirements for rapid proliferation. This study aimed to examine the synergistic effects of glutamine deprivation and metformin exposure on AML cells. METHODS: SKM-1 cells (an AML cell line) were subjected to glutamine deprivation and/or treatment with metformin or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES, a glutaminase inhibitor) or cytarabine. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis and reactive oxygen species (ROS) by flow cytometry. Western blotting was conducted to examine the levels of apoptotic proteins, including cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, the human long noncoding RNA (lncRNA) microarray was used to analyze gene expression after glutamine deprivation, and results were confirmed with quantitative RT-PCR (qRT-PCR). The expression of metallothionein 2A (MT2A) was suppressed using siRNA. Cell growth and apoptosis were further detected by CCK-8 assay and flow cytometry, respectively, in cells with MT2A knockdown. RESULTS: Glutamine deprivation or treatment with BPTES inhibited cell growth and induced apoptosis in SKM-1 cells. The lncRNA microarray result showed that the expression of MT family genes was significantly upregulated after glutamine deprivation. MT2A knockdown increased apoptosis, while proliferation was not affected in SKM-1 cells. In addition, metformin inhibited cell growth and induced apoptosis in SKM-1 cells. Both glutamine deprivation and metformin enhanced the sensitivity of SKM-1 cells to cytarabine. Furthermore, the combination of glutamine deprivation with metformin exhibited synergistic antileukemia effects on SKM-1 cells. CONCLUSION: Targeting glutamine metabolism in combination with metformin is a promising new therapeutic strategy for AML.

Engineering of protein glutaminase for highly efficient modification of fish myofibrillar protein through structure-based and computational-aided strategy.

Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp31, Ser72, Asn121, Asp471, and Glu485 were crucial for maintaining PG5-myosin interaction, with Ser72 and Asn121 making significant energy contributions.

YTHDF1 regulates GID8-mediated glutamine metabolism to promote colorectal cancer progression in m6A-dependent manner.

Dysregulation of epigenetics is a hallmark of cancer development, and YTHDF1 stands out as a crucial epigenetic regulator with the highest DNA copy number variation among all N6-methyladenosine (m6A) regulators in colorectal cancer (CRC) patients. Here, we aimed to investigate the specific contribution of YTHDF1 overexpression to CRC progression and its consequences. Through multiple bioinformatic analyses of human cancer databases and clinical CRC samples, we identified GID8/Twa1 as a crucial downstream target of YTHDF1. YTHDF1 manipulates GID8 translation efficiency in an m6A-dependent manner, and high expression of GID8 is associated with more aggressive tumor progression and poor overall survival. Mechanistically, GID8 is intimately associated with glutamine metabolic demands by maintaining active glutamine uptake and metabolism through the regulation of excitatory amino acid transporter 1 (SLC1A3) and glutaminase (GLS), thereby facilitating the malignant progression of CRC. Inhibition of GID8 attenuated CRC proliferation and metastasis both in vitro and in vivo. In summary, we identified a previously unknown target pertaining to glutamine uptake and metabolism in tumor cells, underscoring the potential of GID8 in the treatment of CRC.

Glutamine metabolism improves left ventricular function but not macrophage-mediated inflammation following myocardial infarction.

Glutamine is a critical amino acid that serves as an energy source, building block, and signaling molecule for the heart tissue and the immune system. However, the role of glutamine metabolism in regulating cardiac remodeling following myocardial infarction (MI) is unknown. In this study, we show in adult male mice that glutamine metabolism is altered both in the remote (contractile) area and in infiltrating macrophages in the infarct area after permanent left anterior descending artery occlusion. We found that metabolites related to glutamine metabolism were differentially altered in macrophages at days 1, 3, and 7 after MI using untargeted metabolomics. Glutamine metabolism in live cells was increased after MI relative to no MI controls. Gene expression in the remote area of the heart indicated a loss of glutamine metabolism. Glutamine administration improved left ventricle (LV) function at days 1, 3, and 7 after MI, which was associated with improved contractile and metabolic gene expression. Conversely, administration of BPTES, a pharmacological inhibitor of glutaminase-1, worsened LV function after MI. Neither glutamine nor BPTES administration impacted gene expression or bioenergetics of macrophages isolated from the infarct area. Our results indicate that glutamine metabolism plays a critical role in maintaining LV contractile function following MI and that glutamine administration improves LV function. Glutamine metabolism may also play a role in regulating macrophage function, but macrophages are not responsive to exogenous pharmacological manipulation of glutamine metabolism.NEW & NOTEWORTHY Glutamine metabolism is altered in both infarct macrophages and the remote left ventricle (LV) following myocardial infarction (MI). Supplemental glutamine improves LV function following MI while inhibiting glutamine metabolism with BPTES worsens LV function. Supplemental glutamine or BPTES does not impact macrophage immunometabolic phenotypes after MI.

HMGA2-mediated glutamine metabolism is required for Cd-induced cell growth and cell migration.

Cadmium (Cd) exposure significantly increases the risk of lung cancer. The demand for glutamine is increasing in cancers, including lung cancer. In this study, we investigated the role of glutamine metabolism in Cd-induced cell growth and migration. Firstly, we found that 2 µM Cd-treatment up-regulated the expression of ASCT2 (alanine, serine, cysteine-preferring transporter 2) and ASNS (asparagine synthetase) while downregulating mitochondrial glutaminase GLS1 in A549 cells. The same results were obtained in male BALB/c mice treated with 0.5 and 1 mg Cd/kg body weight. Subsequently, both glutamine deprivation and transfection with siASCT2 revealed that glutamine played a role in Cd-induced cell growth and migration. Furthermore, using 4-PBA (5 mM), an inhibitor of endoplasmic reticulum (ER) stress, Tm (0.1 µg/ml), an inducer of ER stress, siHMGA2, and over-expressing HMGA2 plasmids we demonstrated that ER stress/HMGA2 axis was involved in inducing ASCT2 and ASNS, while inhibiting GLS1. Additionally, the chromatin immunoprecipitation assay using an HMGA2 antibody revealed the direct binding of the HMGA2 to the promoter sequences of the ASCT2, ASNS, and GLS1 genes. Finally, dual luciferase reporter assay determined that HMGA2 increased the transcription of ASCT2 and ASNS while inhibiting the transcription of GLS1. Overall, we found that ER stress-induced HMGA2 controls glutamine metabolism by transcriptional regulation of ASCT2, ASNS and GLS1 to accelerate cell growth and migration during exposure to Cd at low concentrations. This study innovatively revealed the mechanism of Cd-induced cell growth which offers a fresh perspective on preventing Cd toxicity through glutamine metabolism.

mTORC2-driven chromatin cGAS mediates chemoresistance through epigenetic reprogramming in colorectal cancer.

Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor that initiates a STING-dependent innate immune response, binds tightly to chromatin, where its catalytic activity is inhibited; however, mechanisms underlying cGAS recruitment to chromatin and functions of chromatin-bound cGAS (ccGAS) remain unclear. Here we show that mTORC2-mediated phosphorylation of human cGAS serine 37 promotes its chromatin localization in colorectal cancer cells, regulating cell growth and drug resistance independently of STING. We discovered that ccGAS recruits the SWI/SNF complex at specific chromatin regions, modifying expression of genes linked to glutaminolysis and DNA replication. Although ccGAS depletion inhibited cell growth, it induced chemoresistance to fluorouracil treatment in vitro and in vivo. Moreover, blocking kidney-type glutaminase, a downstream ccGAS target, overcame chemoresistance caused by ccGAS loss. Thus, ccGAS coordinates colorectal cancer plasticity and acquired chemoresistance through epigenetic patterning. Targeting both mTORC2-ccGAS and glutaminase provides a promising strategy to eliminate quiescent resistant cancer cells.

HuR controls glutaminase RNA metabolism.

Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.

The combined inhibition of SLC1A3 and glutaminase in osimertinib-resistant EGFR mutant cells.

BACKGROUND: We investigated the unknown mechanisms of osimertinib-resistant EGFR-mutant lung cancer. METHODS: An osimertinib-resistant cell line (PC-9/OsmR2) was established through continuous exposure to osimertinib using an EGFR exon 19 deletion (19Del) lung adenocarcinoma cell line (PC-9). EGFR 19Del (M1), L858R/T790M/C797S (M6), and L858R/C797S (M8) expression vectors were introduced into Ba/F3 cells. A second osimertinib-resistant line (M1/OsmR) was established through continuous exposure to osimertinib using M1 cells. RESULTS: SLC1A3 had the highest mRNA expression level in PC-9/OsmR2 compared to PC-9 cells by microarray analysis and SLC1A3 was increased by flow cytometry. In PC-9/OsmR2 cells, osimertinib sensitivity was significantly increased in combination with siSLC1A3. Because SLC1A3 functions in glutamic acid transport, osimertinib with a glutaminase inhibitor (CB-839) or an SLC1A3 inhibitor (TFB-TBOA) increased the sensitivity. Also, CB-839 plus TFB-TBOA without osimertinib resulted in greater susceptibility than did CB-839 or TFB-TBOA plus osimertinib. Comprehensive metabolome analysis showed that the M1/OsmR cells had significantly more glutamine and glutamic acid than M1 cells. CB-839 plus osimertinib exerted a synergistic effect on M6 cells and an additive effect on M8 cells. CONCLUSION: Targeting glutaminase and glutamic acid may overcome the osimertinib-resistant EGFR-mutant lung cancer.

Discovery and mechanistic analysis of a novel source protein glutaminase PG5 and its potential application.

In this study, we successfully obtained a novel source protein glutaminase PG5 with specific activity of 10.4 U/mg, good tolerance and broad substrate profile through big data retrieval. Structural analysis and site-directed mutagenesis revealed that the catalytic pocket of Mature-PG5 contained a large number of aromatic amino acids and hydrophobic amino acids, and that Ser72 greatly affects the properties of the catalytic pocket and the affinity of PG5 for the substrate. In addition, molecular dynamics analysis revealed that the opening and closing between amino acid residues Gly65 and Thr66 with Cys164 at the catalytic cleft could affect substrate binding and product release. In addition, PG5 effectively improved the solubility of fish myofibrillar proteins under low-salt conditions while enhancing their foaming and emulsification properties. This study offers valuable insights into the catalytic mechanism of PG5, which will contribute to its future directed evolution and application in the food industry.

All three MutL complexes are required for repeat expansion in a human stem cell model of CAG-repeat expansion mediated glutaminase deficiency.

The Repeat Expansion Diseases (REDs) arise from the expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion. Furthermore, whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase (GLS) gene. We show that alleles with as few as ~ 120 repeats show detectable expansions in culture despite relatively low levels of R-loops formed at this locus. Additionally, using a CRISPR-Cas9 knockout approach we show that PMS2 and MLH3, the constituents of MutLα and MutLγ, the 2 mammalian MutL complexes known to be involved in mismatch repair (MMR), are essential for expansion. Furthermore, PMS1, a component of a less well understood MutL complex, MutLß, is also important, if not essential, for repeat expansion in these cells. Our results provide insights into the factors important for expansion and lend weight to the idea that, despite some differences, the same mechanism is responsible for expansion in many, if not all, REDs.

SIRT4 Has an Anti-Cancer Role in Cervical Cancer by Inhibiting Glutamine Metabolism via the MEK/ERK/C-Myc Axis.

BACKGROUND/AIM: Glutamine metabolism is crucial in cell proliferation, aging, and apoptosis across various cancer types. Existing research indicates that Sirtuin 4 (SIRT4), primarily located in mitochondria, modulates this process. This study aimed to clarify the regulatory relationship between SIRT4 and glutamine metabolism in cervical cancer. MATERIALS AND METHODS: SIRT4 mRNA levels and their clinical correlation to cervical cancer were analyzed using the UALCAN database. Immunohistochemistry (IHC) was performed to assess SIRT4 protein expression in tissue samples from cervical cancer patients. Transient transfection was employed to create Hela and Siha cell lines with overexpressed SIRT4, mitogen-activated extracellular signal-regulated kinase (MEK), and glutaminase 1 (GLS1). The impact on cellular functions was studied using MTT, soft agar, transwell, and western blotting assays. Glutamate and ATP levels were also measured to evaluate metabolic changes. RESULTS: Low levels of SIRT4 mRNA in cervical cancer tissues correlated with tumor metastasis and poor survival rates. Overexpression of SIRT4 led to suppressed cell proliferation, colony growth, and motility, along with significant down-regulation of GLS expression, a key contributor to glutamine metabolism. Additionally, SIRT4 overexpression resulted in the inactivation of the MEK/ERK/c-myc signaling pathway, while overexpression of MEK reversed these effects. Notably, the inhibitory effects of SIRT4 on cell proliferation, colony formation, migration, and invasion in Hela and Siha cells were significantly attenuated following GLS1 overexpression. CONCLUSION: SIRT4 acts as an anti-cancer agent in cervical cancer by inhibiting glutamine metabolism through the MEK/ERK/c-myc signaling pathway, providing a novel sight for cervical cancer therapy.

IGF2BP3 promotes glutamine metabolism of endometriosis by interacting with UCA1 to enhances the mRNA stability of GLS1.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) has been implicated in numerous inflammatory and cancerous conditions. However, its precise molecular mechanisms in endometriosis (EMs) remains unclear. The aim of this study is to examine the influence of IGF2BP3 on the occurrence and progression of EMs and to elucidate its underlying molecular mechanism. METHODS: Efects of IGF2BP3 on endometriosis were confrmed in vitro and in vivo. Based on bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assays and Fluorescent in situ hybridization (FISH) were used to show the association between IGF2BP3 and UCA1. Single-cell spatial transcriptomics analysis shows the expression distribution of glutaminase 1 (GLS1) mRNA in EMs. Study the effect on glutamine metabolism after ectopic endometriotic stromal cells (eESCs) were transfected with Sh-IGF2BP3 and Sh-UCA1 lentivirus. RESULTS: Immunohistochemical staining have revealed that IGF2BP3 was upregulated in ectopic endometriotic lesions (EC) compared to normal endometrial tissues (EN). The proliferation and migration ability of eESCs were greatly reduced by downregulating IGF2BP3. Additionally, IGF2BP3 has been observed to interact with urothelial carcinoma associated 1 (UCA1), leading to increased stability of GLS1 mRNA and subsequently enhancing glutamine metabolism. Results also demonstrated that IGF2BP3 directly interacts with the 3' UTR region of GLS1 mRNA, influencing its expression and stability. Furthermore, UCA1 was able to bind with c-MYC protein, stabilizing c-MYC mRNA and consequently enhancing GLS1 expression through transcriptional promotion. CONCLUSION: These discoveries underscored the critical involvement of IGF2BP3 in the elevation and stability of GLS1 mRNA in the context of glutamine metabolism by interacting with UCA1 in EMs. The implications of our study extended to the identification of possible therapeutic targets for individuals with EMs.

Targeting glutamine metabolism improves sarcoma response to radiation therapy in vivo.

Diverse tumor metabolic phenotypes are influenced by the environment and genetic lesions. Whether these phenotypes extend to rhabdomyosarcoma (RMS) and how they might be leveraged to design new therapeutic approaches remains an open question. Thus, we utilized a Pax7Cre-ER-T2/+; NrasLSL-G12D/+; p53fl/fl (P7NP) murine model of sarcoma with mutations that most frequently occur in human embryonal RMS. To study metabolism, we infuse 13C-labeled glucose or glutamine into mice with sarcomas and show that sarcomas consume more glucose and glutamine than healthy muscle tissue. However, we reveal a marked shift from glucose consumption to glutamine metabolism after radiation therapy (RT). In addition, we show that inhibiting glutamine, either through genetic deletion of glutaminase (Gls1) or through pharmacological inhibition of glutaminase, leads to significant radiosensitization in vivo. This causes a significant increase in overall survival for mice with Gls1-deficient compared to Gls1-proficient sarcomas. Finally, Gls1-deficient sarcomas post-RT elevate levels of proteins involved in natural killer cell and interferon alpha/gamma responses, suggesting a possible role of innate immunity in the radiosensitization of Gls1-deficient sarcomas. Thus, our results indicate that glutamine contributes to radiation response in a mouse model of RMS.

[Exploration of mechanism of total triterpenoids from fruits of Chaenomeles speciosa against senescent GES-1 cells induced by D-galactose based on Gln/GLS1/α-KG metabolic axis and mitochondrial apoptosis signaling pathway].

Total triterpenoids from the fruits of Chaenomeles speciosa(TCS) are active components in the prevention and treatment of gastric mucosal damage, which have potential anti-aging effects. However, it is still unclear whether TCS can improve gastric aging, especially its molecular mechanism against gastric aging. On this basis, this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal) to provide scientific data for the clinical use of TCS to prevent gastric aging. GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE) plasmid of glutaminase 1(GLS1) were induced to aging by D-gal, and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide(BPTES) were given. Cell survival rate, positive rate of ß-galactosidase(SA-ß-gal) staining, mitochondrial membrane potential(MMP), and apoptosis were investigated. GLS1 activity, levels of glutamine(Gln), glutamate(Glu), α-ketoglutarate(α-KG), urea, and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA) and colorimetric methods. The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot. The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group, TCS significantly decreased the SA-ß-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells, inhibited the survival of senescent cells, and promoted their apoptosis(P<0.01). It decreased the activity of GLS1 and the content of Gln, Glu, α-KG, urea, and ammonia in supernatant and cell(P<0.01), reduced the concentration of cytochrome C(Cyto C) in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P<0.01). The mRNA expression of Bcl-2 and Bcl-xl, the protein expression of pro-caspase-9 and pro-caspase-3, and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells were decreased(P<0.01). Cyto C concentration in the cytoplasm, the mRNA expressions of Bax, Bad, apoptosis protease activating factor 1(Apaf-1), and protein expressions of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 were increased(P<0.01). The aforementioned results indicate that TCS can counteract the senescent GES-1 cells induced by D-gal, and its mechanism may be closely related to suppressing the Gln/GLS1/α-KG metabolic axis, activating the mitochondrial apoptosis pathway, and thereby accelerating the apoptosis of the senescent cells and eliminating senescent cells.

Targeting the glutamine metabolism to suppress cell proliferation in mesenchymal docetaxel-resistant prostate cancer.

Docetaxel (DX) serves as a palliative treatment option for metastatic prostate cancer (PCa). Despite initial remission, acquired DX resistance is inevitable. The mechanisms behind DX resistance have not yet been deciphered, but a mesenchymal phenotype is associated with DX resistance. Mesenchymal phenotypes have been linked to metabolic rewiring, obtaining most ATP production by oxidative phosphorylation (OXPHOS) powered substantially by glutamine (Gln). Likewise, Gln is known to play an essential role in modulating bioenergetic, redox homeostasis and autophagy. Herein, investigations of Gln deprivation on DX-sensitive and -resistant (DR) PCa cells revealed that the DR cell sub-lines were susceptible to Gln deprivation. Mechanistically, Gln deprivation reduced OXPHOS and ATP levels, causing a disturbance in cell cycle progression. Genetic and chemical inhibition of the Gln-metabolism key protein GLS1 could validate the Gln deprivation results, thereby representing a valid therapeutic target. Moreover, immunohistological investigation of GLS1 revealed a high-expressing GLS1 subgroup post-docetaxel failure, exhibiting low overall survival. This subgroup presents an intriguing opportunity for targeted therapy focusing on glutamine metabolism. Thus, these findings highlight a possible clinical rationale for the chemical inhibition of GLS1 as a therapeutic strategy to target mesenchymal DR PCa cells, thereby delaying accelerated tumour progression.

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

BACKGROUND: Glutaminase 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). METHODS: Using 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. RESULTS: Database 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. CONCLUSIONS: GLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

A paradigm shift in cancer research based on integrative multi-omics approaches: glutaminase serves as a pioneering cuproptosis-related gene in pan-cancer.

BACKGROUND: Cuproptosis is a newly identified form of unprogrammed cell death. As a pivotal metabolic regulator, glutaminase (GLS) has recently been discovered to be linked to cuproptosis. Despite this discovery, the oncogenic functions and mechanisms of GLS in various cancers are still not fully understood. METHODS: In this study, a comprehensive omics analysis was performed to investigate the differential expression levels, diagnostic and prognostic potential, correlation with tumor immune infiltration, genetic alterations, and drug sensitivity of GLS across multiple malignancies. RESULTS: Our findings revealed unique expression patterns of GLS across various cancer types and molecular subtypes of carcinomas, underscoring its pivotal role primarily in energy and nutrition metabolism. Additionally, GLS showed remarkable diagnostic and prognostic performance in specific cancers, suggesting its potential as a promising biomarker for cancer detection and prognosis. Furthermore, we focused on uterine corpus endometrial carcinoma (UCEC) and developed a novel prognostic model associated with GLS, indicating a close correlation between GLS and UCEC. Moreover, our exploration into immune infiltration, genetic heterogeneity, tumor stemness, and drug sensitivity provided novel insights and directions for future research and laid the foundation for high-quality verification. CONCLUSION: Collectively, our study is the first comprehensive investigation of the biological and clinical significance of GLS in pan-cancer. In our study, GLS was identified as a promising biomarker for UCEC, providing valuable evidence and a potential target for anti-tumor therapy. Overall, our findings shed light on the multifaceted functions of GLS in cancer and offer new avenues for further research.

CircCOL1A1 promotes proliferation, migration, and invasion of colorectal cancer (CRC) cells and glutamine metabolism through GLS1 up-regulation by sponging miR-214-3p.

BACKGROUND: Circular ribose nucleic acids (circRNAs), an abundant type of noncoding RNAs, are widely expressed in eukaryotic cells and exert a significant impact on the initiation and progression of various disorders, including different types of cancer. However, the specific role of various circRNAs in colorectal cancer (CRC) pathology is still not fully understood. METHODS: The initial step involved the use of quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of circRNAs and messenger RNA (mRNA) in CRC cell lines and tissues. Subsequently, functional analyses of circCOL1A1 knockdown were conducted in vitro and in vivo through cell counting kit (CCK)-8, colony formation and transwell assays, as well as xenograft mouse model of tumor formation. Molecular expression and interactions were investigated using luciferase reporter assays, Western blot analysis, RNA immunoprecipitation (RIP), and immunohistochemical staining. RESULTS: The RT-qPCR results revealed elevated levels of circCOL1A1 expressions in CRC tissues and cell lines as compared to the normal counterparts. In addition, circCOL1A1 expression level was found to be correlated with TNM stage, lymph node metastases, distant metastases, and invasion. Knockdown of circCOL1A1 resulted in impaired invasion, migration, and proliferation of CRC cells, and suppressed tumor generation in the animal model. We further demonstrated that circCOL1A1 could act as a sponge for miR-214-3p, suppressing miR-214-3p activity and leading to the upregulation of GLS1 protein to promote glutamine metabolism. CONCLUSION: These findings suggest that circCOL1A1 functions as an oncogenic molecule to promote CRC progression via miR-214-3p/GLS1 axis, hinting on the potential of circCOL1A1 as a therapeutic target for CRC.

Development of a Universal One-Step Purification and Activation Method to Engineer Protein-Glutaminase through Rational Design.

Cytotoxic enzymes often exist as zymogens containing prodomains to keep them in an inactive state. Protein-glutaminase (PG), which can enhance various functional characteristics of food proteins, is an enzyme containing pro-PG and mature-PG (mPG). However, poor activity and stability limit its application while tedious purification and activation steps limit its high-throughput engineering. Here, based on structural analysis, we replaced the linker sequence between pro-PG and mPG with the HRV3C protease recognition sequence and then coexpressed it with HRV3C protease in Escherichia coli to develop an efficient one-step purification and activation method for PG. We then used this method to obtain several mutants designed by a combination of computer-aided approach and beneficial point mutations. The specific activity (131.6 U/mg) of the best variant D1 was 4.14-fold that of the wild type, and t1/2 and T5010 increased by 13 min and 7 °C, respectively. D1 could effectively improve the solubility and emulsification of wheat proteins, more than twice the effect of the wild type. We also discussed the mechanism underlying the improved properties of D1. In summary, we not only provide a universal one-step purification and activation method to facilitate zymogen engineering but also obtain an excellent PG mutant.