ABSTRACT
Cancer vaccines have been developed as a promising way to boost cancer immunity. However, their clinical potency is often limited due to the imprecise delivery of tumor antigens. To overcome this problem, we conjugated an endogenous Toll-like receptor (TLR)2/6 ligand, UNE-C1, to human papilloma virus type 16 (HPV-16)-derived peptide antigen, E7, and found that the UNE-C1-conjugated cancer vaccine (UCV) showed significantly enhanced antitumor activity in vivo compared with the noncovalent combination of UNE-C1 and E7. The combination of UCV with PD-1 blockades further augmented its therapeutic efficacy. Specifically, the conjugation of UNE-C1 to E7 enhanced its retention in inguinal draining lymph nodes, the specific delivery to dendritic cells and E7 antigen-specific T cell responses, and antitumor efficacy in vivo compared with the noncovalent combination of the two peptides. These findings suggest the potential of UNE-C1 derived from human cysteinyl-tRNA synthetase 1 as a unique vehicle for the specific delivery of cancer antigens to antigen-presenting cells via TLR2/6 for the improvement of cancer vaccines.
ABSTRACT
Aminoacyl-tRNA synthetases (ARSs) are enzymes that catalyze the ligation of amino acids to tRNAs for translation. Beyond their traditional role in translation, ARSs have acquired regulatory functions in various biological processes (epi-translational functions). With their dual-edged activities, aberrant expression, secretion, and mutations of ARSs are associated with human diseases, including cancer, autoimmune diseases, and neurological diseases. The increasing numbers of newly unveiled activities and disease associations of ARSs have spurred interest in novel drug development, targeting disease-related catalytic and noncatalytic activities of ARSs as well as harnessing ARSs as sources for biological therapeutics. This review speculates how the translational and epi-translational activities of ARSs can be related and describes how their activities can be linked to diseases and drug discovery.
Subject(s)
Amino Acyl-tRNA Synthetases , Humans , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is known to associated with fibrosis through its catalytic activity to produce prolyl-tRNA. Although its catalytic inhibitor halofuginone (HF) has been known to inhibit the TGF-ß pathway as well as to reduce prolyl-tRNA production for the control of fibrosis, the underlying mechanism how EPRS1 regulates the TGF-ß pathway was not fully understood. Here, we show a noncatalytic function of EPRS1 in controlling the TGF-ß pathway and hepatic stellate cell activation via its interaction with TGF-ß receptor I (TßRI). Upon stimulation with TGF-ß, EPRS1 is phosphorylated by TGF-ß-activated kinase 1 (TAK1), leading to its dissociation from the multi-tRNA synthetase complex and subsequent binding with TßRI. This interaction increases the association of TßRI with SMAD2/3 while decreases that of TßRI with SMAD7. Accordingly, EPRS1 stabilizes TßRI by preventing the ubiquitin-mediated degradation of TßRI. HF disrupts the interaction between EPRS1 and TßRI, and reduces TßRI protein levels, leading to inhibition of the TGF-ß pathway. In conclusion, this work suggests the novel function of EPRS1 involved in the development of fibrosis by regulating the TGF-ß pathway and the antifibrotic effects of HF by controlling both of EPRS1 functions.
Subject(s)
Hepatic Stellate Cells , Receptors, Transforming Growth Factor beta , Humans , Hepatic Stellate Cells/metabolism , Receptor, Transforming Growth Factor-beta Type I/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/metabolism , Fibrosis , Signal Transduction/physiologyABSTRACT
Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.
Subject(s)
Amino Acyl-tRNA Synthetases , Humans , Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Fibrosis , Proline/genetics , Proline/metabolism , Protein BiosynthesisABSTRACT
All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.
Subject(s)
Acetylglucosamine , Glucose , Leucine-tRNA Ligase , Leucine , Mechanistic Target of Rapamycin Complex 1 , Acetylglucosamine/metabolism , Autophagy , Glucose/metabolism , Humans , Leucine/metabolism , Leucine-tRNA Ligase/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolismABSTRACT
Although key tumorigenic and tumor-suppressive factors have been unveiled over the last several decades, cancer remains the most life-threatening disease. Multiomic analyses of patient samples and an in-depth understanding of tumorigenic processes have rapidly revealed unexpected pathologic associations of new cellular factors previously overlooked in cancer biology. In this regard, the newly discovered activities of human aminoacyl-tRNA synthases (ARSs) deserve attention not only for their pathological significance in tumorigenesis but also regarding diagnostic and therapeutic implications. ARSs are not only essential enzymes covalently linking substrate amino acids to cognate tRNAs for protein synthesis but also function as regulators of cellular processes by sensing different cellular conditions. With their catalytic role in protein synthesis and their regulatory role in homeostasis, functional alterations or dysregulation of ARSs might be pathologically associated with tumorigenesis. This review focuses on the potential implications of ARS genes and proteins in different aspects of cancer based on various bioinformatic analyses and experimental data. We also review their diverse activities involving extracellular secretion, protein-protein interactions, and amino acid sensing, which are related to cancers. The newly discovered cancer-related activities of ARSs are expected to provide new opportunities for detecting, preventing and curing cancers.
Subject(s)
Amino Acyl-tRNA Synthetases , Neoplasms , Amino Acids/metabolism , Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Carcinogenesis , Humans , Neoplasms/pathology , Protein Biosynthesis , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNALeu for protein synthesis and plays an important role in mTORC1 activation by sensing intracellular leucine concentrations. Here, we describe a protocol for the purification, reductive methylation, binding affinity measurement by microscale thermophoresis, T i value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed us to build the RagD binding domain, which was shown to be a dynamic region of LARS1 refractory to crystallization. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).
Subject(s)
Cold Temperature , Cryoprotective Agents/chemistry , Crystallography, X-Ray/methods , Leucine-tRNA Ligase/chemistry , Crystallization , Leucine-tRNA Ligase/metabolism , Methylation , Protein BindingABSTRACT
Leucyl-tRNA synthetase 1 (LARS1) mediates activation of leucine-dependent mechanistic target of rapamycin complex 1 (mTORC1) as well as ligation of leucine to its cognate tRNAs, yet its mechanism of leucine sensing is poorly understood. Here we describe leucine binding-induced conformational changes of LARS1. We determine different crystal structures of LARS1 complexed with leucine, ATP, and a reaction intermediate analog, leucyl-sulfamoyl-adenylate (Leu-AMS), and find two distinct functional states of LARS1 for mTORC1 activation. Upon leucine binding to the synthetic site, H251 and R517 in the connective polypeptide and 50FPYPY54 in the catalytic domain change the hydrogen bond network, leading to conformational change in the C-terminal domain, correlating with RagD association. Leucine binding to LARS1 is increased in the presence of ATP, further augmenting leucine-dependent interaction of LARS1 and RagD. Thus, this work unveils the structural basis for leucine-dependent long-range communication between the catalytic and RagD-binding domains of LARS1 for mTORC1 activation.
Subject(s)
Leucine-tRNA Ligase/metabolism , Leucine/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Humans , Models, Molecular , Signal TransductionABSTRACT
BACKGROUND: The generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive immune responses by modulating the function of antigen-presenting cells (APCs). Despite their therapeutic potential, the development of TLR ligands for immunotherapy is often hampered due to rapid systemic toxicity. Regarding the safety concerns of currently available TLR ligands, finding a new TLR agonist with potent efficacy and safety is needed. METHODS: A unique structural domain (UNE-C1) was identified as a novel TLR2/6 in the catalytic region of human cysteinyl-tRNA synthetase 1 (CARS1) using comprehensive approaches, including RNA sequencing, the human embryonic kidney (HEK)-TLR Blue system, pull-down, and ELISA. The potency of its immunoadjuvant properties was analyzed by assessing antigen-specific antibody and CTL responses. In addition, the efficacy of tumor growth inhibition and the presence of the tumor-infiltrating leukocytes were evaluated using E.G7-OVA and TC-1 mouse models. The combined effect of UNE-C1 with an immune checkpoint inhibitor, anti-CTLA-4 antibody, was also evaluated in vivo. The safety of UNE-C1 immunization was determined by monitoring splenomegaly and cytokine production in the blood. RESULTS: Here, we report that CARS1 can be secreted from cancer cells to activate immune responses via specific interactions with TLR2/6 of APCs. A unique domain (UNE-C1) inserted into the catalytic region of CARS1 was determined to activate dendritic cells, leading to the stimulation of robust humoral and cellular immune responses in vivo. UNE-C1 also showed synergistic efficacy with cancer antigens and checkpoint inhibitors against different cancer models in vivo. Further, the safety assessment of UNE-C1 showed lower systemic cytokine levels than other known TLR agonists. CONCLUSIONS: We identified the endogenous TLR2/6 activating domain from human cysteinyl-tRNA synthetase CARS1. This novel TLR2/6 ligand showed potent immune-stimulating activity with little toxicity. Thus, the UNE-C1 domain can be developed as an effective immunoadjuvant with checkpoint inhibitors or cancer antigens to boost antitumor immunity.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Cancer Vaccines/administration & dosage , Immunity, Cellular/immunology , Immunotherapy/methods , Neoplasms, Experimental/therapy , Toll-Like Receptor 2/immunology , Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/immunology , Animals , Cancer Vaccines/immunology , Catalytic Domain , Dendritic Cells/immunology , Female , Humans , Immunization , Ligands , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , T-Lymphocytes, Cytotoxic/immunology , Toll-Like Receptor 2/chemistry , Toll-Like Receptor 2/metabolismABSTRACT
Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.
Subject(s)
Energy Metabolism , Glucose/metabolism , Leucine-tRNA Ligase/metabolism , Leucine/metabolism , Animals , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Fibroblasts , HEK293 Cells , HeLa Cells , Humans , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Monomeric GTP-Binding Proteins/metabolism , Phosphorylation , Signal TransductionABSTRACT
A fundamental question in biology is how vertebrates evolved and differ from invertebrates, and little is known about differences in the regulation of translation in the two systems. Herein, we identify a threonyl-tRNA synthetase (TRS)-mediated translation initiation machinery that specifically interacts with eIF4E homologous protein, and forms machinery that is structurally analogous to the eIF4F-mediated translation initiation machinery via the recruitment of other translation initiation components. Biochemical and RNA immunoprecipitation analyses coupled to sequencing suggest that this machinery emerged as a gain-of-function event in the vertebrate lineage, and it positively regulates the translation of mRNAs required for vertebrate development. Collectively, our findings demonstrate that TRS evolved to regulate vertebrate translation initiation via its dual role as a scaffold for the assembly of initiation components and as a selector of target mRNAs. This work highlights the functional significance of aminoacyl-tRNA synthetases in the emergence and control of higher order organisms.
Subject(s)
Peptide Chain Initiation, Translational , Threonine-tRNA Ligase/metabolism , Amino Acid Sequence , Animals , Blood Vessels/growth & development , Blood Vessels/metabolism , Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factor-4F/metabolism , Eukaryotic Initiation Factor-4G/metabolism , HEK293 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice, Inbred C57BL , Protein Binding , RNA Cap-Binding Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Species Specificity , Threonine-tRNA Ligase/chemistry , Vertebrates/growth & development , Vertebrates/metabolism , ZebrafishABSTRACT
Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches.
Subject(s)
Leucine-tRNA Ligase/metabolism , Leucine/analogs & derivatives , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Leucine/chemistry , Leucine/metabolism , Leucine/pharmacology , Mechanistic Target of Rapamycin Complex 1/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
According to recent studies, leucyl-tRNA synthetase (LRS) acts as a leucine sensor and modulates the activation of the mammalian target of rapamycin complex 1 (mTORC1) activation. Because overactive mTORC1 is associated with several diseases, including colon cancer, LRS-targeted mTORC1 inhibitors represent a potential option for anti-cancer therapy. In this work, we developed a series of simplified leucyladenylate sulfamate analogues that contain the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine moiety to replace the adenine group. We identified several compounds with comparable activity to previously reported inhibitors and exhibited selective mTORC1 inhibition and anti-cancer activity. This study further supports the hypothesis that LRS is a promising target to modulate the mTORC1 pathway.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Leucine-tRNA Ligase/antagonists & inhibitors , Leucine/analogs & derivatives , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Leucine/chemical synthesis , Leucine/chemistry , Leucine/pharmacology , Leucine-tRNA Ligase/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Molecular Structure , Structure-Activity RelationshipABSTRACT
A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating "ON" switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an "OFF" switch by controlling GTP hydrolysis of RagB in the Rag GTPase-mTORC1 axis. The LRS-RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP-GDP cycle of the RagD-RagB pair, rather than the RagC-RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD-RagB pair can overcome the absence of the RagC-RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD-RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.
Subject(s)
Leucine-tRNA Ligase/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Cell Line/metabolism , GTP Phosphohydrolases/metabolism , Humans , Leucine/metabolism , Lysosomes/metabolism , Multiprotein Complexes/metabolism , Nuclear Proteins/metabolism , Protein Binding , Protein Biosynthesis , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
Mucin1 (MUC1), a heterodimeric oncoprotein, containing tandem repeat structures with a high proportion of threonine, is aberrantly overexpressed in many human cancers including pancreatic cancer. Since the overall survival rate of pancreatic cancer patients has remained low for several decades, novel therapeutic approaches are highly needed. Intestinal mucin has been known to be affected by dietary threonine supply since de novo synthesis of mucin proteins is sensitive to luminal threonine concentration. However, it is unknown whether biosynthesis of MUC1 is regulated by threonine in human cancers. In this study, data provided suggests that threonine starvation reduces the level of MUC1 and inhibits the migration of MUC1-expressing pancreatic cancer cells. Interestingly, knockdown of threonyl-tRNA synthetase (TRS), an enzyme that catalyzes the ligation of threonine to its cognate tRNA, also suppresses MUC1 levels but not mRNA levels. The inhibitors of TRS decrease the level of MUC1 protein and prohibit the migration of MUC1-expressing pancreatic cancer cells. In addition, a positive correlation between TRS and MUC1 levels is observed in human pancreatic cancer cells. Concurrent with these results, the bioinformatics data indicate that co-expression of both TRS and MUC1 is correlated with the poor survival of pancreatic cancer patients. Taken together, these findings suggest a role for TRS in controlling MUC1-mediated cancer cell migration and provide insight into targeting TRS as a novel therapeutic approach to pancreatic cancer treatment.
Subject(s)
Mucin-1/biosynthesis , Pancreatic Neoplasms/pathology , Threonine-tRNA Ligase/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Enzyme Inhibitors/pharmacology , Fatty Alcohols/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Mucin-1/metabolism , Pancreatic Neoplasms/mortality , Survival Analysis , Threonine/metabolism , Threonine/pharmacology , Threonine-tRNA Ligase/antagonists & inhibitors , Threonine-tRNA Ligase/genetics , Tissue Array AnalysisABSTRACT
Leucyl-tRNA synthetase (LRS) is known to function as leucine sensor in the mammalian target of rapamycin complex 1 (mTORC1) pathway. However, the pathophysiological significance of its activity is not well understood. Here, we demonstrate that the leucine sensor function for mTORC1 activation of LRS can be decoupled from its catalytic activity. We identified compounds that inhibit the leucine-dependent mTORC1 pathway by specifically inhibiting the GTPase activating function of LRS, while not affecting the catalytic activity. For further analysis, we selected one compound, BC-LI-0186, which binds to the RagD interacting site of LRS, thereby inhibiting lysosomal localization of LRS and mTORC1 activity. It also effectively suppressed the activity of cancer-associated MTOR mutants and the growth of rapamycin-resistant cancer cells. These findings suggest new strategies for controlling tumor growth that avoid the resistance to existing mTOR inhibitors resulting from cancer-associated MTOR mutations.Leucyl-tRNA synthetase (LRS) is a leucine sensor of the mTORC1 pathway. Here, the authors identify inhibitors of the GTPase activating function of LRS, not affecting its catalytic activity, and demonstrate that the leucine sensor function of LRS can be a new target for mTORC1 inhibition.
Subject(s)
Leucine-tRNA Ligase/metabolism , Leucine/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Neoplasms/enzymology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Humans , Leucine-tRNA Ligase/genetics , Mechanistic Target of Rapamycin Complex 1/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Monomeric GTP-Binding Proteins/genetics , Neoplasms/genetics , Neoplasms/metabolism , Protein Binding/drug effects , Signal Transduction/drug effects , Sirolimus/pharmacologyABSTRACT
Leucyl-tRNA synthetase (LRS) has been reported to be a possible mediator of intracellular amino acids signaling to mTORC1. Given that mTORC1 is associated with cell proliferation and tumorigenesis, the LRS-mediated mTORC1 pathway may offer an alternative strategy in anticancer therapy. In this study, we developed a series of simplified analogues of leucyladenylate sulfamate (1) as LRS-targeted mTORC1 inhibitors. We replaced the adenylate group with a N-(3,4-dimethoxybenzyl)benzenesulfonamide (2a) or a N-(2-phenoxyethyl)benzenesulfonamide groups (2b) that can maintain specific binding, but has more favorable physicochemical properties such as reduced polarity and asymmetric centers. Among these simplified analogues, compound 16 and its constrained analogue 22 effectively inhibited S6K phosphorylation in a dose-dependent manner and exhibited cancer cell specific cytotoxicity against six different types of cancer cells. This result supports that LRS is a viable target for novel anticancer therapy.
Subject(s)
Drug Discovery , Leucine-tRNA Ligase/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Sulfonic Acids/chemistry , Sulfonic Acids/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , HEK293 Cells , Humans , Mass Spectrometry/methods , Phosphorylation , Proton Magnetic Resonance Spectroscopy , Ribosomal Protein S6 Kinases/metabolismABSTRACT
Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-α and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.
Subject(s)
Caspase 8/metabolism , Colorectal Neoplasms/enzymology , Exosomes/enzymology , Inflammation Mediators/metabolism , Lysine-tRNA Ligase/metabolism , Animals , Caspase 8/genetics , Chemotaxis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Exosomes/genetics , Exosomes/metabolism , Exosomes/pathology , HCT116 Cells , Humans , Lysine-tRNA Ligase/genetics , Macrophages/metabolism , Mice , Multienzyme Complexes , PDZ Domains , Protein Binding , RAW 264.7 Cells , RNA Interference , Signal Transduction , Syntenins/metabolism , Time Factors , Transfection , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Despite the explosion in the numbers of cancer genomic studies, metastasis is still the major cause of cancer mortality. In breast cancer, approximately one-fifth of metastatic patients survive 5 years. Therefore, detecting the patients at a high risk of developing distant metastasis at first diagnosis is critical for effective treatment strategy. We hereby present a novel systems biology approach to identify driver mutations escalating the risk of metastasis based on both exome and RNA sequencing of our collected 78 normal-paired breast cancers. Unlike driver mutations occurring commonly in cancers as reported in the literature, the mutations detected here are relatively rare mutations occurring in less than half metastatic samples. By supposing that the driver mutations should affect the metastasis gene signatures, we develop a novel computational pipeline to identify the driver mutations that affect transcription factors regulating metastasis gene signatures. We identify driver mutations in ADPGK, NUP93, PCGF6, PKP2 and SLC22A5, which are verified to enhance cancer cell migration and prompt metastasis with in vitro experiments. The discovered somatic mutations may be helpful for identifying patients who are likely to develop distant metastasis.