glutaminase and Carcinoma--Non-Small-Cell-Lung

To evaluate the in vitro activity of polyethylene glycol-conjugated L-asparaginase (PEG-Lasparaginase) against fresh human tumor specimens, using the human tumor clonogenic assay (HTCA), and to perform a phase I dose-escalation clinical trial of PEG-L-asparaginase. The goal of the clinical study was to determine the toxicity and optimum biologic dose of PEG-L-asparaginase based on depletion of serum L-asparagine in patients with advanced solid tumors.. A modified method for determination of serum L-asparagine is described. PEG-L-asparaginase was administered by intramuscular injection every 2 weeks to 28 patients with various types of advanced solid tumor malignancies. At least 3 patients were evaluated at each dose level: 250 IU/m2, 500 IU/m2, 1,000 IU/m2, 1,500 IU/m2, 2,000 IU/m2.. The in vitro HTCA studies suggested good antitumor activity against malignant melanoma and multiple myeloma. Serum L-asparagine was most consistently and profoundly depleted (up to 4 weeks) in patients treated with 2,000 IU/m2. Patients receiving this dose level also showed more frequent grade 1, grade 2, and occasional grade 3 toxicities of fatigue/weakness, nausea/vomiting, and anorexia/ weight loss. Three patients developed hypersensitivity reactions, but these were not dose related. Two patients developed deep vein thromboses. We saw no episodes of clinical pancreatitis, but there were minor fluctuations of serum amylase and lipase. We saw no partial or complete responses in patients treated in this study, including 11 patients with malignant melanoma.. We conclude that PEG-L-asparaginase is generally well tolerated in patients with advanced solid tumors, and a dosage of 2,000 IU/m2 by intramuscular injection every 2 weeks results in significant depletion of serum L-asparagine.

Topics: Adult; Antineoplastic Agents; Asparaginase; Asparagine; Carcinoma, Non-Small-Cell Lung; Drug Screening Assays, Antitumor; Glutaminase; Humans; Lung Neoplasms; Melanoma; Neoplasm Proteins; Neoplasms; Polyethylene Glycols; Skin Neoplasms

Lung cancer, a malignant disease, is one of the leading causes of patient death. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Currently, chemotherapeutic agents such as cisplatin are widely used against lung cancer. However, development of chemoresistance, which led to poor prognosis and low survival rate greatly limited the clinical applications of cisplatin. Sinomenine (SIN) is a bioactive component of sinomenium acutum. Accumulating evidence revealed SIN exhibits potential anti-tumor activities in various types of cancers. However, the precise molecular mechanisms for the sinomenine-induced anti-cancer effects have not been fully elucidated. Here, we assessed the effects of sinomenine on cisplatin sensitivity in NSCLC cells. The combination of SIN with cisplatin showed synergistically inhibitory effects on lung cancer cells by calculating the combination index (CI value) using the Calcusyn 2.0 software. Moreover, we detected that the glutamine metabolism was significantly suppressed by sinomenine treatments in lung cancer cells. Under low glutamine supply, A549 cells showed less sensitivity to sinomenine treatments. Meanwhile, miR-200a-3p was found to be significantly induced by SIN treatments. We demonstrated a suppressive role of miR-200a-3p on glutamine metabolism. Furthermore, miR-200a-3p was downregulated but the glutamine metabolism was significantly hyperactive in A549 cisplatin resistant cells compared with parental cells. Bioinformatical analysis and luciferase assay demonstrated the glutaminase (GLS), a key enzyme of glutamine metabolism, is the direct target of miR-200a-3p in lung cancer cells. Finally, rescue experiments demonstrated that recovery of GLS in miR-200a-3p overexpressing-cisplatin resistant cells successfully overrode the sinomenine-mediated cisplatin sensitization. In summary, this study revealed a new molecular mechanism for the sinomenine-promoted cisplatin sensitization, contributing to investigating the sinomenine-based therapeutic agents against chemoresistant NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Glutaminase; Glutamine; Humans; Lung Neoplasms; MicroRNAs

Increased insight into the mutational landscape of squamous cell lung cancers (LUSCs) in the past decade has not translated into effective targeted therapies for patients with this disease. NRF2, encoded by NFE2L2, and its upstream regulator, KEAP1, control key aspects of redox balance and are frequently mutated in NSCLCs.. Here, we describe the specific potent activity of TAK-228, a TORC1/2 inhibitor, in NSCLC models harboring NRF2-activating alterations and results of a phase 2 clinical trial of TAK-228 in patients with advanced NSCLC harboring NRF2-activating alterations including three cohorts (NFE2L2-mutated LUSC, KEAP1-mutated LUSC, KRAS/NFE2L2- or KEAP1-mutated NSCLC).. TAK-228 was most efficacious in a LUSC cohort with NFE2L2 alterations; the overall response rate was 25% and median progression-free survival was 8.9 months. Additional data suggest that concurrent inhibition of glutaminase with the glutaminase inhibitor CB-839 might overcome metabolic resistance to therapy in these patients.. TAK-228 has single-agent activity in patients with NRF2-activated LUSC. This study reframes oncogenic alterations as biologically relevant based on their downstream effects on metabolism. This trial represents, to the best of our knowledge, the first successful attempt at metabolically targeting NSCLC and identifies a promising targeted therapy for patients with LUSC, who are bereft of genotype-directed therapies.

Topics: Carcinoma, Non-Small-Cell Lung; Glutaminase; Humans; Kelch-Like ECH-Associated Protein 1; Lung Neoplasms; Mutation; NF-E2-Related Factor 2

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Glutaminase; Histone Deacetylase 6; Humans; Lung Neoplasms; Mutation; Proto-Oncogene Proteins p21(ras)

Inhibiting cancer metabolism via glutaminase (GAC) is a promising strategy to disrupt tumor progression. However, mechanism regarding GAC acetylation remains mostly unknown. In this study, we demonstrate that lysine acetylation is a vital post-translational modification that inhibits GAC activity in non-small cell lung cancer (NSCLC). We identify that Lys311 is the key acetylation site on GAC, which is deacetylated by HDAC4, a class II deacetylase. Lys311 acetylation stimulates the interaction between GAC and TRIM21, an E3 ubiquitin ligase of the tripartite motif (TRIM) family, therefore promoting GAC K63-linked ubiquitination and inhibiting GAC activity. Furthermore, GAC

Topics: Acetylation; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Transformation, Neoplastic; Glutaminase; Histone Deacetylases; Humans; Lung Neoplasms; Repressor Proteins; Ubiquitination

Non-small-cell lung cancer (NSCLC) cell lines vary in their sensitivity to glutaminase inhibitors, so it is important to identify the metabolic assets underling their efficacy in cancer cells. Even though specific genetic lesions such as in KRAS and LKB1 have been associated with reliance on glutamine for their metabolic needs, we found no distinction between glutaminase inhibitor CB-839 sensitivity and resistant phenotypes in NSCLC cells with or without these genetic alterations. We demonstrated the close relationship between environmental alanine uptake and catabolism. This response depended on the individual cell's ability to employ alanine aminotransferase (GPT2) to compensate the reduced glutamate availability. It may, therefore, be useful to determine GPT2 levels to predict which NSCLC patients would benefit most from glutaminase inhibitor treatment.

Topics: Alanine; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Glutaminase; Humans; Lung Neoplasms

Phosphoserine aminotransferase 1 (PSAT1) is an enzyme implicated in serine biosynthesis, and its overexpression has been linked to cancer cell proliferation. Therefore, targeting PSAT1 is considered to be an anticancer strategy.. The viability of non-small cell lung cancer (NSCLC) cells was measured by MTT assay. Protein and mRNA expression were determined by western blot and reverse transcription polymerase chain reaction, respectively.. Glutamine-limiting conditions were generated through glutamine deprivation or CB-839 treatment, which induced PSAT1 expression in NSCLC cells. PSAT1 expression induced by glutamine-limiting conditions was regulated by activating transcription factor 4. Knock-down of PSAT1 enhanced the sensitivity of NSCLC cells to glutamine-limiting conditions. Interestingly, ionizing radiation induced PSAT1 expression, and knocking down PSAT1 increased cell sensitivity to ionizing radiation.. Inhibiting PSAT1 might aid in the treatment of lung cancer, and PSAT1 may be a therapeutic target for lung cancer.

Topics: Activating Transcription Factor 4; Benzeneacetamides; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Gene Knock-In Techniques; Glutaminase; Glutamine; Humans; Lung; Lung Neoplasms; Radiation Tolerance; RNA, Messenger; Thiadiazoles; Transaminases

Topics: Antineoplastic Agents; Apoptosis; Benzeneacetamides; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase-Activating Kinase; Cyclin-Dependent Kinases; Drug Synergism; G2 Phase Cell Cycle Checkpoints; Glutaminase; Glycolysis; Humans; Lung Neoplasms; Phenylenediamines; Protein Kinase Inhibitors; Pyrimidines; Thiadiazoles

The anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase, is responsible for the transition from metaphase to anaphase and the exit from mitosis. The anaphase promoting complex subunit 10 (APC10), a subunit of the APC/C, executes a vital function in substrate recognition. However, no research has reported the connection between APC10 and cancer until now. In this study, we uncovered a novel, unprecedented role of APC10 in tumor progression, which is independent of APC/C. First, aberrant increase of APC10 expression was validated in non-small cell lung cancer (NSCLC) cells and tissues, and the absence of APC10 repressed cell proliferation and migration. Of great interest, we found that APC10 inhibition induced cell cycle arrest at the G0/G1 phase and reduced the expression of the APC/C substrate, Cyclin B1; this finding is different from the conventional concept of the accumulation of Cyclin B1 and cell cycle arrest in metaphase. Further, APC10 was found to interact with glutaminase C (GAC), and the inhibition of APC10 weakened glutamine metabolism and induced excessive autophagy. Taken together, these findings identify a novel function of APC10 in the regulation of NSCLC tumorigenesis and point to the possibility of APC10 as a new target for cancer therapy.

Topics: A549 Cells; Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; Autophagy; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cytoplasm; Disease Progression; G1 Phase; Glutaminase; Glutamine; Humans; Lung Neoplasms; Resting Phase, Cell Cycle; RNA, Small Interfering; Signal Transduction; Transfection

Glutamine metabolism plays an important role in cancer development and progression. Glutaminase C (GAC), the first enzyme in glutaminolysis, has emerged as an important target for cancer therapy and many studies have focused on the mechanism of enhanced GAC expression in cancer cells. However, little is known about the post-translational modification of GAC. Here, we report that phosphorylation is a crucial post-translational modification of GAC, which is responsible for the higher glutaminase activity in lung tumor tissues and cancer cells. We identify the key Ser314 phosphorylation site on GAC that is regulated by the NF-κB-PKCε axis. Blocking Ser314 phosphorylation by the S314A mutation in lung cancer cells inhibits the glutaminase activity, triggers genetic reprogramming, and alleviates tumor malignancy. Furthermore, we find that a high level of GAC phosphorylation correlates with poor survival rate of lung cancer patients. These findings highlight a previously unappreciated mechanism for activation of GAC by phosphorylation and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

Topics: Animals; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Glutaminase; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Protein Kinase C-epsilon

Metabolic inhibition might sensitize tumors to irradiation. Here, we examined the effect of lonidamine (several metabolic effects, inhibiting hexokinase amongst others) and/or 968 (glutaminase inhibitor) on tumor cell metabolism, cell growth, cytotoxicity and radiosensitivity in NSCLC cell lines in vitro in relation to histology.. Adeno- (H23, HCC827, H1975) and squamous cell carcinoma (H520, H292, SW900) NSCLC cells were treated with lonidamine and/or 968 for 72 h under physiological levels of glucose (1.5 mM). Cells were irradiated with 0, 4 or 8 Gy. Cell growth of H2B-mCherry transduced cells and cytotoxicity (CellTox™ Green Cytotoxicity Assay) were measured using live cell imaging (IncuCyte). Inhibitory effects on metabolic profiles was determined using the Seahorse XF96 extracellular Flux analyzer.. NSCLC cell lines responded differently to glycolysis (lonidamine) and/or glutaminase (968) inhibition, largely corresponding with changes in glycolytic and mitochondrial metabolism upon treatment. Response patterns were not related to histology. 968 was cytotoxic in cell lines with high glutaminase C expression (H1975 and H520), whereas combination treatment was cytotoxic in KRAS mutated cell lines SW900 and H23. H292 and HCC827 were resistant to combination treatment. Treatment with 968 and especially lonidamine resulted in radiosensitization of H292 and HCC827 in terms of decreased relative cell growth and increased cytotoxicity.. NSCLC is a heterogeneous disease, which is reflected in the response of different cell lines to the treatment (combinations) reported here. Only a part of NSCLC patients may benefit from the combination of radiation therapy and metabolic inhibition, making stratification necessary.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Radiation; Enzyme Inhibitors; Glucose; Glutaminase; Glutamine; Glycolysis; Humans; Indazoles; Lung Neoplasms; Radiation Tolerance; X-Rays

Metabolic reprogramming is critical for cancer cell proliferation. Glutaminolysis which provides cancer cells with bioenergetics and intermediates for macromolecular synthesis have been intensively studied in recent years. Glutaminase C (GAC) is the first and rate-limiting enzyme in glutaminolysis and plays important roles in cancer initiation and progression. We previously screened a small molecule named 968, a specific inhibitor of GAC, to block the proliferation of human breast cancer cells. In this study, we found that 968 effectively inhibited NSCLC cell proliferation and migration and arrested G0/G1 phase of cell cycle. Furthermore, we demonstrated that 968 inhibited the EGFR/ERK pathway via decreasing the expression of EGFR and phospho-ERK. Apart from this, we discovered that 968 treatment induced autophagy to protect cells against apoptosis and the combination of 968 with autophagy inhibitor Chloroquine (CQ) had synergistic effects on the growth of NSCLC cells. Thus, our study pointed out a new therapeutic strategy for NSCLC treatment by combination of 968 with CQ.

Topics: Autophagy; Beclin-1; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glutaminase; Humans; Lung Neoplasms; Signal Transduction

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Benzeneacetamides; Carbon Radioisotopes; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; ErbB Receptors; Erlotinib Hydrochloride; Fluorodeoxyglucose F18; Glutaminase; Glutamine; Humans; Lung Neoplasms; Mice; Mice, SCID; Mutation; Radiopharmaceuticals; RNA Interference; Thiadiazoles; Transplantation, Heterologous

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib has been approved based on the clinical benefit in non-small cell lung cancer (NSCLC) patients over the past decade. Unfortunately, cancer cells become resistant to this agent via various mechanisms, and this limits the improvement in patient outcomes. Thus, it is urgent to develop novel agents to overcome erlotinib resistance. Here, we propose a novel strategy to overcome acquired erlotinib resistance in NSCLC by inhibiting glutaminase activity. Compound 968, an inhibitor of the glutaminase C (GAC), when combined with erlotinib potently inhibited the cell proliferation of erlotinib-resistant NSCLC cells HCC827ER and NCI-H1975. The combination of compound 968 and erlotinib not only decreased GAC and EGFR protein expression but also inhibited GAC activity in HCC827ER cells. The growth of erlotinib-resistant cells was glutamine-dependent as proved by GAC gene knocked down and rescue experiment. More importantly, compound 968 combined with erlotinib down-regulated the glutamine and glycolysis metabolism in erlotinib-resistant cells. Taken together, our study provides a valuable approach to overcome acquired erlotinib resistance by blocking glutamine metabolism and suggests that combination of EGFR-TKI and GAC inhibitor maybe a potential treatment strategy for acquired erlotinib-resistant NSCLC.

Topics: Apoptosis; Benzophenanthridines; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Erlotinib Hydrochloride; Flow Cytometry; Glutaminase; Humans; Lung Neoplasms; Mitochondria; Protein Kinase Inhibitors; RNA Interference; Time Factors

We found that non-small cell lung cancer (NSCLC) is remarkably sensitive to the regulation of glutamine supply by testing the metabolic dependency of 11 cancer cell lines against regulation of glycolysis, autophagy, fatty acid synthesis, and glutamine supply. Glutamine is known as a key supplement of cancer cell growth that is converted to α-ketoglutarate for anabolic biogenesis via glutamate by glutaminase 1 (GLS1). GLS1 inhibition using 10 μM of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) showed about 50% cell growth arrest by SRB assay. By testing the synergistic effects of conventional therapeutics, BPTES combined with 5-fluorouracil (5-FU), an irreversible inhibitor of thymidylate synthase, significant effects were observed on cell growth arrest in NSCLC. We found that GLS1 inhibition using BPTES reduced metabolic intermediates including thymidine and carbamoyl phosphate. Reduction of thymidine and carbamoyl-phosphate synthesis by BPTES treatment exacerbated pyrimidine supply by combination with 5-FU, which induced cell death synergistically in NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Glutaminase; Humans; Lung Neoplasms; Thymidine

Glutaminase 1 (GLS1) expression is increased in non-small cell lung cancer (NSCLC). GLS1 knockdown using siRNA or inhibition using bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) induced cell cycle arrest with significant reduction of ATP level while levels of reactive oxygen species or glutathione were not affected in NSCLC cell lines. Recently we found that NSCLC significantly depends on cytosol NADH for ATP production. GLS1 remarkably contributes to ATP production through transferring cytosolic NADH into mitochondria via malate-aspartate shuttle by supply of glutamate in NSCLC. Regulation of malate-aspartate shuttle by knockdown or inhibition of glutamic-oxaloacetic transaminase 2 or malate dehydrogenase 2 mimicked GLS1 knockdown, which induced cell death with ATP reduction in NSCLC. Therefore, GLS1 inhibition induced cell cycle arrest with ATP depletion by glutamate reduction. Dual inhibition with BPTES and thymidylate synthase inhibitor, 5-fluorouracil (5-FU), elicits cell death synergistically through cell cycle arrest in NSCLC. A preclinical xenograft model of NSCLC showed remarkable anti-tumour effect synergistically in the BPTES and 5-FU dual therapy group.

Topics: A549 Cells; Adenosine Triphosphate; Animals; Aspartic Acid; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Death; Cell Proliferation; Cell Survival; Cytosol; Drug Synergism; Fluorouracil; Gene Knockdown Techniques; Glutamic Acid; Glutaminase; Glutamine; Lung Neoplasms; Malates; Mice, Inbred BALB C; Mice, Nude; Molecular Targeted Therapy; NAD; Oxidation-Reduction; Sulfides; Thiadiazoles; Thymidylate Synthase; Xenograft Model Antitumor Assays

Topics: Aged; Biomarkers; Carcinoma, Non-Small-Cell Lung; Cognitive Dysfunction; Female; Glutamate Decarboxylase; Glutamate Dehydrogenase; Glutamic Acid; Glutaminase; Humans; Lung Neoplasms; Male; Middle Aged; Monocytes

Recent work has highlighted glutaminase (GLS) as a key player in cancer cell metabolism, providing glutamine-derived carbon and nitrogen to pathways that support proliferation. There is significant interest in targeting GLS for cancer therapy, although the gene is not known to be mutated or amplified in tumors. As a result, identification of tractable markers that predict GLS dependence is needed for translation of GLS inhibitors to the clinic. Herein we validate a small molecule inhibitor of GLS and show that non-small cell lung cancer cells marked by low E-cadherin and high vimentin expression, hallmarks of a mesenchymal phenotype, are particularly sensitive to inhibition of the enzyme. Furthermore, lung cancer cells induced to undergo epithelial to mesenchymal transition (EMT) acquire sensitivity to the GLS inhibitor. Metabolic studies suggest that the mesenchymal cells have a reduced capacity for oxidative phosphorylation and increased susceptibility to oxidative stress, rendering them unable to cope with the perturbations induced by GLS inhibition. These findings elucidate selective metabolic dependencies of mesenchymal lung cancer cells and suggest novel pathways as potential targets in this aggressive cancer type.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Epithelial-Mesenchymal Transition; Genetic Association Studies; Glutaminase; Humans; Lung Neoplasms; Molecular Targeted Therapy; Oxidative Stress; Sulfides; Thiadiazoles

One of the hallmarks of cancer is metabolic deregulation. Many tumors display increased glucose uptake and breakdown through the process of aerobic glycolysis, also known as the Warburg effect. Less studied in cancer development and progression is the importance of the glutamine (Gln) pathway, which provides cells with a variety of essential products to sustain cell proliferation, such as ATP and macromolecules for biosynthesis. To this end Gln dependency was assessed in a panel of non-small cell lung cancer lines (NSCLC). Gln was found to be essential for the growth of cells with high rates of glutaminolysis, and after exploring multiple genes in the Gln pathway, GLS1 was found to be the key enzyme associated with this dependence. This dependence was confirmed by observing the rescue of decreased growth by exogenous addition of downstream metabolites of glutaminolysis. Expression of the GLS1 splice variant KGA was found to be decreased in tumors compared with normal lung tissue. Transient knock down of GLS1 splice variants indicated that loss of GAC had the most detrimental effect on cancer cell growth. In conclusion, NSCLC cell lines depend on Gln for glutaminolysis to a varying degree, in which the GLS1 splice variant GAC plays an essential role and is a potential target for cancer metabolism-directed therapy.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Glutaminase; Glutamine; Glycolysis; Humans; Lung Neoplasms; Molecular Targeted Therapy; Protein Isoforms