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In a mouse model of breast cancer, asparagine bioavailability strongly influences metastasis and this is correlated with the production of proteins that regulate the epithelial-to-mesenchymal transition, which provides at least one potential mechanism for how a single amino acid could regulate metastatic progression. Tumour spread assisted by asparagine Not all cells that derive from a primary tumour contribute to metastasis—the progression of cancer into other parts of the body. Gregory Hannon and colleagues find determinants of metastatic potential in a mouse model of breast cancer. They find that expression of the metabolic enzyme asparagine synthetase is associated with metastasis formation. Decreasing asparagine availability by treatment with ʟ-asparaginase or by dietary restriction reduced the development of metastasis. The authors show that asparagine availability promotes an epithelial-to-mesenchymal transition, a process that has been linked to metastasis. Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites 1 . A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient’s primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l -asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.

Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E . coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer.

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent L-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Asparagine synthetase (ASNS) catalyses the ATP-dependent conversion of aspartate to asparagine. However, both the regulation and biological functions of asparagine in tumour cells remain largely unknown. Here, we report that p53 suppresses asparagine synthesis through the transcriptional downregulation of ASNS expression and disrupts asparagine-aspartate homeostasis, leading to lymphoma and colon tumour growth inhibition in vivo and in vitro. Moreover, the removal of asparagine from culture medium or the inhibition of ASNS impairs cell proliferation and induces p53/p21-dependent senescence and cell cycle arrest. Mechanistically, asparagine and aspartate regulate AMPK-mediated p53 activation by physically binding to LKB1 and oppositely modulating LKB1 activity. Thus, we found that p53 regulates asparagine metabolism and dictates cell survival by generating an auto-amplification loop via asparagine-aspartate-mediated LKB1-AMPK signalling. Our findings highlight a role for LKB1 in sensing asparagine and aspartate and connect asparagine metabolism to the cellular signalling transduction network that modulates cell survival. Cancer cells can produce asparagine, yet both the regulation and biological functions of asparagine are unclear. Here the authors show that p53 suppresses asparagine synthesis and disrupts asparagine-aspartate homeostasis contributing to tumor growth inhibition, and that asparagine and aspartate modulates AMPK-mediated p53 activation by physically binding to and regulating LKB1.

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes. Working on human asparagine synthetase, Coricello et al show that combining 3D variability analysis (3DVA) of cryo-EM data with MD simulations is a valid approach to study conformation of amino acid side chains that might regulate enzyme function.

The availability of asparagine is the limitation of cell growth and metastasis. Asparagine synthetase (ASNS) was an essential enzyme for endogenous asparagine products. In our study, ASNS-induced asparagine products were essential to maintain tumor growth and colony formations in vitro. But mutated ASNS which defected endogenous asparagine products still upregulated cell invasiveness, which indicated that ASNS promoted invasiveness by alternative pathways. Mechanically, ASNS modulated Wnt signal transduction by promoting GSK3β phosphorylation on ser9 and stabilizing the β-catenin complex, as result, ASNS could promote more β-catenin translocation into nucleus independent of endogenous asparagine. At the same time, ASNS modulated mitochondrial response to Wnt stimuli with increased mitochondrial potential and membrane fusion. In summary, ASNS promoted metastasis depending on Wnt pathway and mitochondrial functions even without endogenous asparagine products. In the Wnt pathway, phosphorylation of GSK3β(S9) is very important to stabilize β-catenin complex, ASNS can upregulate phosphorylation of AKT on Ser473, then promote GSK3β phosphorylation on Ser9 to stabilize β-catenin complex. When Wnt pathway is activated, ASNS elevates p-DRP1(637) and decreases p-DRP1(616) to suppress mitochondrial fission.

Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4–ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns -ASO or Scr -ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns -ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns -ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD. Synopsis Metabolic Reprogramming, such as glycolysis and glutaminolysis, are key features of polycystic kidney disease (PKD). Asparagine synthetase drives glutamine utilization in this disease and it is upregulated in human and mouse tissues. Its inhibition retards disease progression and rescues metabolic derangement in PKD mice. Glutamine utilization and cyst expansion are driven by ASNS upregulation in PKD. PKD cystic phenotype and metabolic rewiring are hampered by Asns-ASO. Glutaminolysis, pyrimidine biosynthesis and proliferation are driven by a GCN2-ATF4-ASNS axis. PKD is further delayed by co-targeting glutaminolysis and glycolysis with Asns-ASO and 2DG. Metabolic Reprogramming, such as glycolysis and glutaminolysis, are key features of polycystic kidney disease (PKD). Asparagine synthetase drives glutamine utilization in this disease and it is upregulated in human and mouse tissues. Its inhibition retards disease progression and rescues metabolic derangement in PKD mice.

L-Asparagine, a crucial amino acid widely used in both food and medicine, presents pollution-related and side reaction challenges when prepared using chemical synthesis method. Although biotransformation methods offer significant advantages such as high efficiency and mild reaction conditions, they also entail increased costs due to the need for ATP supplementation. This study aimed to address the challenges associated with biopreparation of L-asparagine. Firstly, the functionality and characteristics of recombinant L-asparagine synthetase enzymes derived from Escherichia coli and Lactobacillus salivarius were evaluated to determine their practical applicability. Subsequently, recombinant expression of polyphosphate kinase from Erysipelotrichaceae bacterium was conducted. A reaction system for L-asparagine synthesis was established using a dual enzyme-coupled conversion approach. Under the optimal reaction conditions, a maximum yield of 11.67 g/L of L-asparagine was achieved, with an 88.43% conversion rate, representing a 5.03-fold increase compared to the initial conversion conditions. Notably, the initial addition of ATP was reduced to only 5.66% of the theoretical demand, indicating the effectiveness of our ATP regeneration system. These findings highlight the potential of our approach in enhancing the efficiency of L-asparagine preparation, offering promising prospects for the food and medical industries.

The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.

Background Sex-related differences in colorectal (CRC) incidence and mortality are well-documented. However, the impact of sex on metabolic pathways that drive cancer growth is not well understood. High expression of asparagine synthetase ( ASNS ) is associated with inferior survival for female CRC patients only. Here, we used a CRISPR/Cas9 technology to generate HCT116 ASNS −/− and HCT 116 ASNS +/+ cancer cell lines. We examine the effects of ASNS deletion on tumor growth and the subsequent rewiring of metabolic pathways in male and female Rag2/IL2RG mice. Results ASNS loss reduces cancer burden in male and female tumor-bearing mice (40% reduction, q < 0.05), triggers metabolic reprogramming including gluconeogenesis, but confers a survival improvement (30 days median survival, q < 0.05) in female tumor-bearing mice alone. Transcriptomic analyses revealed upregulation of G-protein coupled estrogen receptor ( GPER1 ) in tumors from male and female mice with HCT116 ASNS −/− xenograft. Estradiol activates GPER1 in vitro in the presence of ASNS and suppresses tumor growth. Conclusions Our study indicates that inferior survival for female CRC patients with high ASNS may be due to metabolic reprogramming that sustains tumor growth. These findings have translational relevance as ASNS / GPER1 signaling could be a future therapeutic target to improve the survival of female CRC patients.