Targeting mTORC2/HDAC3 Inhibits Stemness of Liver Cancer Cells Against Glutamine Starvation.

Cancer cells are addicted to glutamine. However, cancer cells often suffer from glutamine starvation, which largely results from the fast growth of cancer cells and the insufficient vascularization in the interior of cancer tissues. Herein, based on clinical samples, patient-derived cells (PDCs), and cell lines, it is found that liver cancer cells display stem-like characteristics upon glutamine shortage due to maintaining the stemness of tumor initiating cells (TICs) and even promoting transformation of non-TICs into stem-like cells by glutamine starvation. Increased expression of glutamine synthetase (GS) is essential for maintaining and promoting stem-like characteristics of liver cancer cells during glutamine starvation. Mechanistically, glutamine starvation activates Rictor/mTORC2 to induce HDAC3-mediated deacetylation and stabilization of GS. Rictor is significantly correlated with the expression of GS and stem marker OCT4 at tumor site, and closely correlates with poor prognosis of hepatocellular carcinomas. Inhibiting components of mTORC2-HDAC3-GS axis decrease TICs and promote xenografts regression upon glutamine-starvation therapy. Collectively, the data provides novel insights into the role of Rictor/mTORC2-HDAC3 in reprogramming glutamine metabolism to sustain stemness of cancer cells. Targeting Rictor/HDAC3 may enhance the efficacy of glutamine-starvation therapy and limit the rapid growth and malignant progression of tumors.

Leukemia Cells Resistant to Glutamine Deprivation Express Glutamine Synthetase Protein

Objective: Low glutamine levels have been shown in tumor environments for several cancer subtypes. Therefore, it has been suggested that cancer cells rewire their metabolism to adopt low nutrient levels for survival and proliferation. Although glutamine is a non-essential amino acid and can be synthesized de novo, many cancer cells including malignant hematopoietic cells have been indicated to be addicted to glutamine. This study aimed to investigate the proliferation of leukemia cell lines in glutamine-deprived conditions. Materials and Methods: Cell proliferation of K562, NB-4, and HL-60 cells was determined by calculating cell numbers in normal vs. low glutamine media. Changes in mRNA expressions were investigated using qRT-PCR. The glutamine synthetase (GS)-encoding GLUL gene was knocked out (KO) in HL-60 cells using the CRISPR/Cas9 method and protein expression was evaluated with immunoblotting. Results: The proliferation of all cell lines was decreased in glutamine-deprived medium. GS protein expression was increased in glutamine-limited medium although the mRNA level did not change. Increased protein expression was confirmed with inhibition of new protein synthesis by treating cells with cycloheximide. To further investigate the role of GS protein, the GS-encoding GLUL gene was KO in HL-60 cells using the CRISPR/Cas9 method. GS KO cells proliferated less compared to control cells in glutamine-limited medium. Conclusion: Our results indicate that upregulated GS protein expression is responsible for glutamine addiction of leukemia cell lines. Exploiting the genetic and metabolic mechanisms responsible for GS protein expression could lead to the identification of new anti-cancer drug targets.

Glutamine deprivation triggers NAGK-dependent hexosamine salvage.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

Glutamine deficiency induces lipolysis in adipocytes.

Glutamine is the most abundant amino acid in the body, and adipose tissue is one of the glutamine-producing organs. Glutamine has important and unique metabolic functions; however, its effects in adipocytes are still unclear. 3T3-L1 adipocytes produced and secreted glutamine dependent on glutamine synthetase, but preadipocytes did not. The inhibition of glutamine synthetase by l-methionine sulfoximine (MSO) impaired the differentiation of preadipocytes to mature adipocytes, and this inhibitory effect of MSO was rescued by exogenous glutamine supplementation. Glutamine concentrations were low, and Atgl gene expression was high in epididymal white adipose tissues of fasting mice in vivo. In 3T3-L1 adipocytes, glutamine deprivation induced Atgl expression and increased glycerol concentration in culture medium. Atgl expression is regulated by FoxO1, and glutamine deprivation reduced FoxO1 phosphorylation (Ser256), indicating the activation of FoxO1. These results demonstrate that glutamine is necessary for the differentiation of preadipocytes and regulates lipolysis through FoxO1 in mature adipocytes.

Comorbidity-associated glutamine deficiency is a predisposition to severe COVID-19.

SARS-CoV-2 vaccinations have greatly reduced COVID-19 cases, but we must continue to develop our understanding of the nature of the disease and its effects on human immunity. Previously, we suggested that a dysregulated STAT3 pathway following SARS-Co-2 infection ultimately leads to PAI-1 activation and cascades of pathologies. The major COVID-19-associated metabolic risks (old age, hypertension, cardiovascular diseases, diabetes, and obesity) share high PAI-1 levels and could predispose certain groups to severe COVID-19 complications. In this review article, we describe the common metabolic profile that is shared between all of these high-risk groups and COVID-19. This profile not only involves high levels of PAI-1 and STAT3 as previously described, but also includes low levels of glutamine and NAD+, coupled with overproduction of hyaluronan (HA). SARS-CoV-2 infection exacerbates this metabolic imbalance and predisposes these patients to the severe pathophysiologies of COVID-19, including the involvement of NETs (neutrophil extracellular traps) and HA overproduction in the lung. While hyperinflammation due to proinflammatory cytokine overproduction has been frequently documented, it is recently recognized that the immune response is markedly suppressed in some cases by the expansion and activity of MDSCs (myeloid-derived suppressor cells) and FoxP3+ Tregs (regulatory T cells). The metabolomics profiles of severe COVID-19 patients and patients with advanced cancer are similar, and in high-risk patients, SARS-CoV-2 infection leads to aberrant STAT3 activation, which promotes a cancer-like metabolism. We propose that glutamine deficiency and overproduced HA is the central metabolic characteristic of COVID-19 and its high-risk groups. We suggest the usage of glutamine supplementation and the repurposing of cancer drugs to prevent the development of severe COVID-19 pneumonia.

Targeting glutamine metabolism and autophagy: the combination for prostate cancer radiosensitization.

Radiotherapy is one of the curative mainstays of prostate cancer; however, its efficacy is often diminished by tumor radioresistance. Depending on the stage of disease, tumors can relapse in approximately 50% of patients with prostate cancer after radiotherapy. Nevertheless, the mechanisms that drive tumor cell survival are not fully characterized, and reliable molecular prognostic markers of prostate cancer radioresistance are missing. Similar to other tumor entities, prostate cancer cells are heterogeneous in their capability to maintain tumor growth. The populations of cancer stem cells (CSCs) with self-renewal and differentiation properties are responsible for tumor development and recurrence after treatment. Eradication of these CSC populations is of utmost importance for efficient tumor cure. In a recently published study, we showed that prostate cancer cells could be radiosensitized by glutamine deprivation, resulting in DNA damage, oxidative stress, epigenetic modifications, and depletion of CSCs. Conversely, prostate cancer cells with resistance to glutamine depletion show an activation of ATG-mediated macroautophagy/autophagy as a survival strategy to withstand radiation-induced damage. Thus, a combination of targeting glutamine metabolism and autophagy blockade lead to more efficient prostate cancer radiosensitization.Abbreviations: ATG5: autophagy related 5; CSCs: cancer stem cells; GLS: glutaminase; TCA cycle: tricarboxylic acid cycle.

Hepatoblastoma: glutamine depletion hinders cell viability in the embryonal subtype but high GLUL expression is associated with better overall survival.

PURPOSE: Glutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival. METHODS: We correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations. RESULTS: High GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival. CONCLUSION: Growth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

VHL regulates the sensitivity of clear cell renal cell carcinoma to SIRT4-mediated metabolic stress via HIF-1α/HO-1 pathway.

Clear cell renal cell carcinomas (ccRCC) reprogram carbon metabolism responses to hypoxia, thereby promoting utilization of glutamine. Recently, sirtuin 4 (SIRT4), a novel molecular has turned out to be related to alternating glutamine metabolism and modulating the tumor microenvironment. However, the role of SIRT4 in ccRCC remains poorly understood. Here, we illustrated that the expression of SIRT4 is markedly reduced in cancerous tissues, and closely associated with malignancy stage, grade, and prognosis. In ccRCC cells, SIRT4 exerted its proapoptotic activity through enhancing intracellular reactive oxygen species (ROS). Heme oxygenase-1 (HO-1) is part of an endogenous defense system against oxidative stress. Nevertheless, overexpression of SIRT4 hindered the upregulation of HO-1 in von Hippel-Lindau (VHL)-proficient cells and repressed its expression in VHL-deficient cells. This discrepancy indicated that competent VHL withstands the inhibitory role of SIRT4 on HIF-1α/HO-1. Functionally, overexpression of HO-1 counteracted the promotional effects of SIRT4 on ROS accumulation and apoptosis. Mechanistically, SIRT4 modulates ROS and HO-1 expression via accommodating p38-MAPK phosphorylation. By contrast, downregulation of p38-MAPK by SB203580 decreased intracellular ROS level and enhanced the expression of HO-1. Collectively, this work revealed a potential role for SIRT4 in the stimulation of ROS and the modulation of apoptosis. SIRT4/HO-1 may act as a potential therapeutic target, especially in VHL-deficient ccRCCs.

VCP relocalization limits mitochondrial activity, GSH depletion and ferroptosis during starvation in PC3 prostate cancer cells.

During periods of crisis, cells must compensate to survive. To this end, cells may need to alter the subcellular localization of crucial proteins. Here, we show that during starvation, VCP, the most abundant soluble ATPase, relocalizes and forms aggregate-like structures at perinuclear regions in PC3 prostate cancer cells. This movement is associated with a lowered metabolic state, in which mitochondrial activity and ROS production are reduced. VCP appears to explicitly sense glutamine levels, as removal of glutamine from complete medium triggered VCP relocalization and its addition to starvation media blunted VCP relocalization. Cells cultured in Gln(+) starvation media exhibited uniformly distributed VCP in the cytoplasm (free VCP) and underwent ferroptotic cell death, which was associated with a decrease in GSH levels. Moreover, the addition of a VCP inhibitor, CB-5083, in starvation media prevented VCP relocalization and triggered ferroptotic cell death. Likewise, expression of GFP-fused VCP proteins, irrespective of ATPase activities, displayed free VCP and triggered cell death during starvation. These results indicate that free VCP is essential for the maintenance of mitochondrial function and that PC3 cells employ a strategy of VCP self-aggregation to suppress mitochondrial activity in order to escape cell death during starvation, a novel VCP-mediated survival mechanism.

Asparagine sustains cellular proliferation and c­Myc expression in glutamine­starved cancer cells.

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c­Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c­Myc protein expression, leading to a reduction in the levels of the canonical c­Myc isoform and an increase in the expression of c­Myc 1, a c­Myc isoform translated from an in­frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c­Myc expression as in glutamine­fed cells, with high levels of canonical c­Myc and c­Myc 1 almost undetectable. In asparagine­fed cells, global protein translation was higher than in glutamine­starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine­starved asparagine­fed cells, the inhibition of c­Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c­Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.

Elongation factor eEF2 kinase and autophagy jointly promote survival of cancer cells.

Cells within solid tumours can become deprived of nutrients; in order to survive, they need to invoke mechanisms to conserve these resources. Using cancer cells in culture in the absence of key nutrients, we have explored the roles of two potential survival mechanisms, autophagy and elongation factor 2 kinase (eEF2K), which, when activated, inhibits the resource-intensive elongation stage of protein synthesis. Both processes are regulated through the nutrient-sensitive AMP-activated protein kinase and mechanistic target of rapamycin complex 1 signalling pathways. We find that disabling both autophagy and eEF2K strongly compromises the survival of nutrient-deprived lung and breast cancer cells, whereas, for example, knocking out eEF2K alone has little effect. Contrary to some earlier reports, we find no evidence that eEF2K regulates autophagy. Unexpectedly, eEF2K does not facilitate survival of prostate cancer PC3 cells. Thus, eEF2K and autophagy enable survival of certain cell-types in a mutually complementary manner. To explore this further, we generated, by selection, cells which were able to survive nutrient starvation even when autophagy and eEF2K were disabled. Proteome profiling using mass spectrometry revealed that these 'resistant' cells showed lower levels of diverse proteins which are required for energy-consuming processes such as protein and fatty acid synthesis, although different clones of 'resistant cells' appear to adapt in dissimilar ways. Our data provide further information of the ways that human cells cope with nutrient limitation and to understanding of the utility of eEF2K as a potential target in oncology.

Targeting SLC1A5 blocks cell proliferation through inhibition of mTORC1 in arsenite-treated human uroepithelial cells.

Arsenic is an environmental contaminant, which is widely distributed in soil, air, and water. There is sufficient evidence to indicate that arsenic increases the risk of bladder cancer in humans. However, its underlying mechanisms remain elusive. Glutamine (Gln) has multiple functions that promote carcinogenesis. Indeed, Gln transporters on cancer cells surface are often upregulated. Elevated expression levels of Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) have been reported in many types of human tumors. This study characterized the role of SLC1A5 in cell proliferation in arsenite-treated cells. In short-term experiments, SV-40 immortalized human uroepithelial (SV-HUC-1) cells were treated with Sodium arsenite (NaAsO2) (0, 0.5, 1, 2, 4, 8 µM) for 24 h. In long-term experiments, SV-HUC-1 cells were exposed to 0.5 µM NaAsO2 for 40 weeks. In both short-term and long-term experiments, arsenite increased expression of SLC1A5 by 1.89-fold and 2.25-fold, respectively. Arsenite increased Gln consumption of SV-HUC-1 cells, and Gln starvation inhibited cell proliferation in long-term arsenite-treated cells. Importantly, inhibiting SLC1A5 blocked cell proliferation by downregulating mTORC1 in long-term arsenite-treated cells. Moreover, SLC1A5 regulated mTORC1 in an αKG-dependent manner. Our results suggest that SLC1A5 plays an important role in cell proliferation of arsenite-treated SV-HUC-1 cells.

Glutamine deficiency links clindamycin-induced dysbiosis and intestinal barrier dysfunction in mice.

Antibiotics rank as the most powerful weapons against bacterial infection, but their use is often limited by antibiotic-associated diarrhoea (AAD). Here, we reported that glutamine deficiency might act as a new link between clindamycin-induced dysbiosis and intestinal barrier dysfunction during AAD progression. Using a mouse model, we demonstrated that glutamine became a conditionally essential amino acid upon persistent therapeutic-dose clindamycin exposure, evidenced by a dramatic decrease in intestinal glutamine level and glutaminase expression. Mechanistically, clindamycin substantially confounded the abundance of butyrate-producing strains, leading to the deficiency of faecal butyrate which is normally a fundamental fuel for enterocytes, and in turn increased the compensatory use of glutamine. In addition to its pivotal roles in colonic epithelial cell turnover, glutamine was required for nitric oxide production in classic macrophage-driven host defence facilitating pathogen removal. Importantly, oral administration of glutamine effectively attenuated clindamycin-induced dysbiosis and restored intestinal barrier dysfunction in mice. Collectively, the present study highlighted the importance of gut microbiota in host energy homoeostasis and provided a rationale for introducing glutamine supplementation to patients receiving long-term antibiotic treatment.

Dysregulated Glutamate Transporter SLC1A1 Propels Cystine Uptake via Xc- for Glutathione Synthesis in Lung Cancer.

Cancer cells need to generate large amounts of glutathione (GSH) to buffer oxidative stress during tumor development. A rate-limiting step for GSH biosynthesis is cystine uptake via a cystine/glutamate antiporter Xc-. Xc- is a sodium-independent antiporter passively driven by concentration gradients from extracellular cystine and intracellular glutamate across the cell membrane. Increased uptake of cystine via Xc- in cancer cells increases the level of extracellular glutamate, which would subsequently restrain cystine uptake via Xc-. Cancer cells must therefore evolve a mechanism to overcome this negative feedback regulation. In this study, we report that glutamate transporters, in particular SLC1A1, are tightly intertwined with cystine uptake and GSH biosynthesis in lung cancer cells. Dysregulated SLC1A1, a sodium-dependent glutamate carrier, actively recycled extracellular glutamate into cells, which enhanced the efficiency of cystine uptake via Xc- and GSH biosynthesis as measured by stable isotope-assisted metabolomics. Conversely, depletion of glutamate transporter SLC1A1 increased extracellular glutamate, which inhibited cystine uptake, blocked GSH synthesis, and induced oxidative stress-mediated cell death or growth inhibition. Moreover, glutamate transporters were frequently upregulated in tissue samples of patients with non-small cell lung cancer. Taken together, active uptake of glutamate via SLC1A1 propels cystine uptake via Xc- for GSH biosynthesis in lung tumorigenesis. SIGNIFICANCE: Cellular GSH in cancer cells is not only determined by upregulated Xc- but also by dysregulated glutamate transporters, which provide additional targets for therapeutic intervention.

Glutamine deprivation alters the origin and function of cancer cell exosomes.

Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.

Glutamine Deprivation Promotes the Generation and Mobilization of MDSCs by Enhancing Expression of G-CSF and GM-CSF.

Solid tumors are often challenged by hypoxic and nutrient-deprived tumor microenvironments (TME) as tumors progress, due to limited perfusion and rapid nutrient consumption. While cancer cells can demonstrate the ability to survive in nutrient-deprived conditions through multiple intrinsic alterations, it is poorly understood how nutrient-deprived cancer cells co-opt the TME to promote cancer cell survival and tumor progression. In the present study, we found that glutamine deprivation markedly potentiated the expression of G-CSF and GM-CSF in mouse mammary cancer cells. The IRE1α-JNK pathway, which is activated by glutamine starvation, was found to be important for the upregulation of these cytokines. G-CSF and GM-CSF are well-known facilitators of myelopoiesis and mobilization of hematopoietic progenitor cells (HPC). Consistently, as tumors progressed, we found that several myeloid HPC compartments were gradually decreased in the bone marrow but were significantly increased in the spleen. Mechanistically, the HPC-maintaining capacity of the bone marrow was significantly impaired in tumor-bearing mice, with lower expression of HPC maintaining genes (i.e., CXCL12, SCF, ANGPT1, and VCAM1), and reduced levels of mesenchymal stem cells and CXCL12-producing cells. Furthermore, the mobilized HPCs that displayed the capacity for myelopoiesis were also found to accumulate in tumor tissue. Tumor-infiltrating HPCs were highly proliferative and served as important sources of immunosuppressive myeloid-derived suppressor cells (MDSCs) in the TME. Our work has identified an important role for glutamine starvation in regulating the expression of G-CSF and GM-CSF, and in facilitating the generation of immunosuppressive MDSCs in breast cancer.

Inborn errors of enzymes in glutamate metabolism.

Glutamate is involved in a variety of metabolic pathways. We reviewed the literature on genetic defects of enzymes that directly metabolise glutamate, leading to inborn errors of glutamate metabolism. Seventeen genetic defects of glutamate metabolising enzymes have been reported, of which three were only recently identified. These 17 defects affect the inter-conversion of glutamine and glutamate, amino acid metabolism, ammonia detoxification, and glutathione metabolism. We provide an overview of the clinical and biochemical phenotypes of these rare defects in an effort to ease their recognition. By categorising these by biochemical pathway, we aim to create insight into the contributing role of deviant glutamate and glutamine levels to the pathophysiology. For those disorders involving the inter-conversion of glutamine and glutamate, these deviant levels are postulated to play a pivotal pathophysiologic role. For the other IEM however-with the exception of urea cycle defects-abnormal glutamate and glutamine concentrations were rarely reported. To create insight into the clinical consequences of disturbed glutamate metabolism-rather than individual glutamate and glutamine levels-the prevalence of phenotypic abnormalities within the 17 IEM was compared to their prevalence within all Mendelian disorders and subsequently all disorders with metabolic abnormalities notated in the Human Phenotype Ontology (HPO) database. For this, a hierarchical database of all phenotypic abnormalities of the 17 defects in glutamate metabolism based on HPO was created. A neurologic phenotypic spectrum of developmental delay, ataxia, seizures, and hypotonia are common in the inborn errors of enzymes in glutamate metabolism. Additionally, ophthalmologic and skin abnormalities are often present, suggesting that disturbed glutamate homeostasis affects tissues of ectodermal origin: brain, eye, and skin. Reporting glutamate and glutamine concentrations in patients with inborn errors of glutamate metabolism would provide additional insight into the pathophysiology.

Glutamine Deprivation Induces PD-L1 Expression via Activation of EGFR/ERK/c-Jun Signaling in Renal Cancer.

The programmed death-ligand 1/programmed death-1 (PD-L1/PD-1) pathway plays a pivotal role in the immune escape of tumors. Many tumor cells show "glutamine dependence." However, the relationship between glutamine metabolism and PD-L1 expression has not been reported. In this study, changes in PD-L1 expression in renal carcinoma cells were evaluated during glutamine deprivation and recovery. Although PD-L1 expression differed in two renal cancer cell lines, both cell lines upregulated PD-L1 during glutamine deprivation, and the upregulated PD-L1 was restored to normal after glutamine recovery. Mechanistically, glutamine deprivation resulted in activation of EGFR signaling via ERKs 1 and 2 (ERK1/2) and c-Jun. In addition, treatment of renal cancer cells with EGF also induced PD-L1 expression and ERK1/2 phosphorylation. Finally, inhibitors of EGFR, ERK, and c-Jun all inhibited phosphorylation of c-Jun and downregulated PD-L1 expression induced by glutamine deprivation. Taken together, the data suggest that glutamine regulates the expression of PD-L1 through the EGFR/ERK/c-Jun pathway in renal cancer. IMPLICATIONS: This study reveals glutamine deprivation induces PD-L1 expression via activation of EGFR/ERK/c-Jun signaling in renal cancer and provides novel markers for the treatment of renal cancer.

NF-YA overexpression protects from glutamine deprivation.

The heterotrimeric transcription factor NF-Y binds to CCAAT boxes of genes of glutamine metabolism. We set out to study the role of the regulatory NF-YA subunit in this pathway. We produced U2OS and A549 clones stably overexpressing -OE- the two splicing isoforms of NF-YA. NF-YA OE cells show normal growth and colony formation rates, but they become resistant to cell death upon glutamine deprivation. Increased mRNA and protein expression of the key biosynthetic enzyme GLUL in U2OS entails increased production of endogenous glutamine upon deprivation. The use of GLUL inhibitors dampens the NF-YA-mediated effect. NF-YA OE prevents activation of the pro-apoptotic transcription factor CHOP/DDIT3. Elevated basal levels of SERCA1/2, coding for the molecular target of Thapsigargin, correlate with resistance of NF-YA OE cells to the drug. The work represents a proof-of-principle that elevated levels of NF-YA, as found in some tumor types, helps altering cancer metabolic pathways.

Nutritional Stress in Head and Neck Cancer Originating Cell Lines: The Sensitivity of the NRF2-NQO1 Axis.

Nutritional stress disturbs the cellular redox-status, which is characterized by the increased generation of reactive oxygen species (ROS). The NRF2-NQO1 axis represents a protective mechanism against ROS. Its strength is cell type-specific. FaDu, Cal 27 and Detroit 562 cells differ with respect to basal NQO1 activity. These cells were grown for 48 hours in nutritional conditions (NC): (a) Low glucose-NC2, (b) no glucose, no glutamine-NC3, (c) no glucose with glutamine-NC4. After determining the viability, proliferation and ROS generation, NC2 and NC3 were chosen for further exploration. These conditions were also applied to IMR-90 fibroblasts. The transcripts/transcript variants of NRF2 and NQO1 were quantified and transcript variants were characterized. The proteins (NRF2, NQO1 and TP53) were analyzed by a western blot in both cellular fractions. Under NC2, the NRF2-NQO1 axis did not appear activated in the cancer cell lines. Under NC3, the NRF2-NQO1axis appeared slightly activated in Detroit 562. There are opposite trends with respect to TP53 nuclear signal when comparing Cal 27 and Detroit 562 to FaDu, under NC2 and NC3. The strong activation of the NRF2-NQO1 axis in IMR-90 resulted in an increased expression of catalytically deficient NQO1, due to NQO1*2/*2 polymorphism (rs1800566). The presented results call for a comprehensive exploration of the stress response in complex biological systems.