Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation.

Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1's enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs. Pharmacological inhibition or RNAi-mediated depletion of the protein deacetylase SIRT2 increases ENO1's acetylation and enhances its RNA binding. Similarly, induction of mESC differentiation leads to increased ENO1 acetylation, enhanced RNA binding, and inhibition of glycolysis. Stem cells expressing mutant forms of ENO1 that escape or hyper-activate this regulation display impaired germ layer differentiation. Our findings uncover acetylation-driven riboregulation of ENO1 as a physiological mechanism of glycolytic control and of the regulation of stem cell differentiation. Riboregulation may represent a more widespread principle of biological control.

Single-cell RNA sequencing unveils unique transcriptomic signatures of endothelial cells and role of ENO1 in response to disturbed flow.

Flow patterns exert significant effects on vascular endothelial cells (ECs) to lead to the focal nature of atherosclerosis. Using a step flow chamber to investigate the effects of disturbed shear (DS) and pulsatile shear (PS) on ECs in the same flow channel, we conducted single-cell RNA sequencing analyses to explore the distinct transcriptomic profiles regulated by DS vs. PS. Integrated analysis identified eight cell clusters and demonstrated that DS induces EC transition from atheroprotective to proatherogenic phenotypes. Using an automated cell type annotation algorithm (SingleR), we showed that DS promoted endothelial-to-mesenchymal transition (EndMT) by inducing the transcriptional phenotypes for inflammation, hypoxia responses, transforming growth factor-beta (TGF-ß) signaling, glycolysis, and fatty acid synthesis. Enolase 1 (ENO1), a key gene in glycolysis, was one of the top-ranked genes in the DS-induced EndMT cluster. Pseudotime trajectory analysis revealed that the kinetic expression of ENO1 was significantly associated with EndMT and that ENO1 silencing repressed the DS- and TGF-ß-induced EC inflammation and EndMT. Consistent with these findings, ENO1 was highly expressed in ECs at the inner curvature of the mouse aortic arch (which is exposed to DS) and atherosclerotic lesions, suggesting its proatherogenic role in vivo. In summary, we present a comprehensive single-cell atlas of ECs in response to different flow patterns within the same flow channel. Among the DS-regulated genes, ENO1 plays an important role in DS-induced EC inflammation and EndMT. These results provide insights into how hemodynamic forces regulate vascular endothelium in health and disease.

Enolase 1 of Candida albicans binds human CD4+ T cells and modulates naïve and memory responses.

To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies, we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 monoclonal antibodies tested in parallel. CaEno1 binding to naïve human CD4+ T cells skewed cytokine secretion toward a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4+ T-cell recall responses. Therapeutically, CD4+ T cells transduced with a p41/Crf1-specific T-cell receptor developed for adoptive T-cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4+ T cells with CaEno1 modulated host CD4+ T-cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through the direct modulation of CD4+ T-cell responses.

Glycolytic enzyme Enolase-1 regulates insulin gene expression in pancreatic ß-cell.

Enolase-1 (Eno1) plays a critical role in regulating glucose metabolism; however, its specific impact on pancreatic islet ß-cells remains elusive. This study aimed to provide a preliminary exploration of Eno1 function in pancreatic islet ß-cells. The findings revealed that the expression of ENO1 mRNA in type 2 diabetes donors was significantly increased and positively correlated with HbA1C and negatively correlated with insulin gene expression. A high level of Eno1 in human insulin-secreting rat INS-1832/13 cells with co-localization with intracellular insulin proteins was accordingly observed. Silencing of Eno1 using siRNA or inhibiting Eno1 protein activity with an Eno1 antagonist significantly reduced insulin secretion and insulin content in ß-cells, while the proinsulin/insulin content ratio remained unchanged. This reduction in ß-cells function was accompanied by a notable decrease in intracellular ATP and mitochondrial cytochrome C levels. Overall, our findings confirm that Eno1 regulates the insulin secretion process, particularly glucose metabolism and ATP production in the ß-cells. The mechanism primarily involves its influence on insulin production, suggesting that Eno1 represents a potential target for ß-cell protection and diabetes treatment.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Tracking Prostate Carcinogenesis over Time through Urine Proteome Profiling in an Animal Model: An Exploratory Approach.

Prostate cancer (PCa) is one of the most lethal diseases in men, which justifies the search for new diagnostic tools. The aim of the present study was to gain new insights into the progression of prostate carcinogenesis by analyzing the urine proteome. To this end, urine from healthy animals and animals with prostate adenocarcinoma was analyzed at two time points: 27 and 54 weeks. After 54 weeks, the incidence of pre-neoplastic and neoplastic lesions in the PCa animals was 100%. GeLC-MS/MS and subsequent bioinformatics analyses revealed several proteins involved in prostate carcinogenesis. Increased levels of retinol-binding protein 4 and decreased levels of cadherin-2 appear to be characteristic of early stages of the disease, whereas increased levels of enolase-1 and T-kininogen 2 and decreased levels of isocitrate dehydrogenase 2 describe more advanced stages. With increasing age, urinary levels of clusterin and corticosteroid-binding globulin increased and neprilysin levels decreased, all of which appear to play a role in prostate hyperplasia or carcinogenesis. The present exploratory analysis can be considered as a starting point for studies targeting specific human urine proteins for early detection of age-related maladaptive changes in the prostate that may lead to cancer.

Salidroside Induces Apoptosis in Human Gastric Cancer Cells via the Downregulation of ENO1/PKM2/GLUT1 Expression.

Salidroside is reported to have a wide range of pharmacological properties and has been proven to play a key anti-cancer effect. This study investigated the effects of purified salidroside, an ingredient of Rhodiola rosea, on the proliferation of two human gastric cancer cell lines and further investigating its possible molecular mechanisms. We verified that salidroside exerts a dose-dependent inhibitory effect on the proliferation of SGC-7901 and MKN-45 human gastric cancer cells. Moreover, salidroside can induce cell apoptosis, which was accompanied by an increase in nuclear fragmentation. In addition, salidroside inhibited glycolysis, as evidenced by the reduced expression levels of the glycolysis-related enzymes pyruvate kinase isoenzyme M2 (PKM2), enolase 1 (ENO1) and glucose transporter 1 (GLUT1), which could play important roles in the metabolism of gastric cancer cells. Further investigation showed that salidroside exerted potent anti-proliferative effects by inhibiting glycolysis in human gastric cancer cells in vitro. In vivo, xenograft tumors treated with salidroside were significantly smaller than those in the control animals. Therefore, salidroside could be a promising therapeutic prospect in the treatment of gastric cancer.

Impact of Tissue Enolase 1 Protein Overexpression in Esophageal Cancer Progression.

Enolase (ENO) 1 is a key glycolytic enzyme and important player in tumorigenesis. ENO1 overexpression has been correlated with tumor progression and/or worse prognosis in several solid malignancies. However, data concerning the impact of ENO1 in cancer conflict. The study correlated local and circulating ENO1 protein levels in esophageal cancer (EC) with clinicopathological data, to assess its potential clinical value. ENO1 expression was analyzed by immunohistochemistry in paired tumor and non-tumor tissue samples from 40 EC cases and mucosal biopsies from 45 Barrett's esophagus (BE) cases, plus in plasma from these patients and 25 matched healthy controls. ENO1 was abnormally elevated in cancer-cell cytoplasm in both EC types, in esophageal squamous cell carcinoma and in adenocarcinoma (EAC), increasing significantly with tumor stage progression and the transition from BE to EAC. EAC patients exhibited significantly lower ENO1 plasma concentrations than normal subjects. Neither local nor systemic ENO1 expression levels were significantly associated with overall survival. These results indicate ENO1 as potential biomarker, delineating a population of patients with Barrett's esophagus at high risk of cancer, and as new therapeutic opportunity in EC patient management. However, further confirmation might be necessary.

The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection.

Candida albicans is an opportunistic fungus that is part of the normal microflora commonly found in the human digestive tract and the normal mucosa or skin of healthy individuals. However, in immunocompromised individuals, it becomes a serious health concern and a threat to their lives and is ranked as the leading fungal infection in humans worldwide. As existing treatments for this infection are non-specific or under threat of developing resistance, there is a dire necessity to find new targets for designing specific drugs to defeat this fungus. Some authors reported the presence of the transglutaminase activity in Candida and Saccharomyces, but its identity remains unknown. We report here the phenotypic effects produced by the inhibition of transglutaminase enzymatic activity with cystamine, including growth inhibition of yeast cells, induction of autophagy in response to damage caused by cystamine, alteration of the normal yeast division pattern, changes in cell wall, and inhibition of the yeast-to-mycelium transition. The latter phenomenon was also observed in the C. albicans ATCC 26555 strain. Growth inhibition by cystamine was also determined in other Candida strains, demonstrating the importance of transglutaminase in these species. Finally, we identified enolase 1 as the cell wall protein responsible for TGase activity. After studying the inhibition of enzymatic activities with anti-CaEno1 antibodies and through bioinformatics studies, we suggest that the enolase and transglutaminase catalytic sites are localized in different domains of the protein. The aforementioned data indicate that TGase/Eno1 is a putative target for designing new drugs to control C. albicans infection.

Enolase1 overexpression regulates the growth of gastric cancer cells and predicts poor survival.

Gastric cancer has become the third most common cancer around the world. In patients with gastric cancer, the 5-year survival rate is still low. However, the mechanism underlying gastric cancer remains largely unknown. As a glycolytic enzyme, enolase 1 (ENO1) is widely expressed in most tissues. The functions of ENO1 have been reported in various types of cancer. Here in this study, we identified that ENO1 promoted the growth of gastric cancer cells through diverse mechanisms. Our immunohistochemical, bioinformatic and Western blot data showed that ENO1 was significantly overexpressed in human gastric cancer cell lines and tissues. The survival analysis revealed that ENO1 overexpression predicted poor survival in the patients suffering gastric cancer. Knockdown of ENO1 expression repressed the rate of proliferation and capacity of colony formation in two human gastric cancer cell lines (MGC-803 and MKN-45). In addition, knockdown of the expression of ENO1 led to the arrest of the cell cycle at the G1 phase and promoted the apoptosis of MKN-45 and MGC-803 cells. The further microarray and bioinformatic analysis revealed that ENO1 regulated the expression of diverse genes, many of which are involved in the progress of cancer. Taken together, our data demonstrated that ENO1 was an oncogene-like factor and might serve as a promising target for the treatment of human gastric cancer.

Nano-LC based proteomic approach identifies that E6AP interacts with ENO1 and targets it for degradation in breast cancer cells.

E6AP (E6 associated protein) is a HECT domain containing protein having dual E3 ligase and ERα coactivation activity in breast cancer cells. Although E6AP is known to possess antitumorigenic activity, the underlying molecular mechanism is poorly understood. In the present study, we applied nano-LC based proteomics approach to identify E6AP-interacting proteins where we performed GST-pull down using GST-E6AP from whole cell extracts of MCF7 cells, resolved the differentially interacting proteins on 1D-SDS-PAGE, excised the gel bands that were trypsin digested followed by fractionation and spotting on MALDI-TOF/TOF plate through Nano-LC MALDI spotter. Subsequently, fractionated and spotted peptides were identified using MALDI-TOF/TOF. We identified several E6AP interacting proteins including previously reported such as HSP70 and new ones such as Enolase-1. We further confirmed that E6AP and Enolase1 interacted and colocalized more in the cytoplasmic periphery in breast cancer cells and further demonstrated that E6AP also targeted ENO1 for ubiquitin-mediated degradation in these cells.

Enolase-1 expression in the stratum corneum is elevated with parakeratosis of atopic dermatitis and disrupts the cellular tight junction barrier in keratinocytes.

OBJECTIVE: Previous studies have shown that enolase-1 (ENO1) in the stratum corneum (SC) is more highly expressed in patients with atopic dermatitis (AD) than in healthy individuals, suggesting that it is a novel biomarker for evaluating skin condition in patients with AD. However, the mechanism underlying high ENO1 expression in the SC and its pathological relevance in AD are unclear. In this study, the relationship between ENO1 expression and keratinization of epidermis was investigated, and the role of high ENO1 expression in keratinocytes was characterized. METHODS: ENO1 expression and morphological characteristics were examined in SC from the cheeks of 24 patients with AD. Additionally, the localization of ENO1 in the excised human epidermis was observed. Moreover, to analyse the role of ENO1 in cellular barrier function, tight junction proteins (TJs) and transepithelial electrical resistance (TEER) in keratinocytes with ENO1 overexpression were evaluated. Furthermore, the localization of ENO1 and plasminogen in keratinocytes was evaluated by immunostaining, and the cellular barrier function in keratinocytes was examined after treatment with tranexamic acid (TXA). RESULTS: ENO1 expression was substantially correlated with the rate of nucleated corneocytes in AD. In addition, ENO1 localized in the basal to spinous layers, but was its expression dramatically decreased in healthy human SC. ENO1 overexpression in human epidermal keratinocytes reduced the expression of TJs (claudin-4, E-cadherin, tricellulin, and occludin) and TEER, and treatment with anti-ENO1 IgG reversed these effects. ENO1 colocalized with plasminogen in keratinocytes. Treatment with TXA rescued the ENO1-induced reductions in TJ and TEER expression. CONCLUSION: We found a substantial correlation between ENO1 expression and the rate of nucleated corneocytes in AD and decreased ENO1 expression with nuclear disappearance. These results suggest that high ENO1 expression in the SC of AD is caused by deficient keratinization, which is an AD characteristic. Moreover, ENO1 overexpression in keratinocytes promoted dysfunction of TJ dynamics, leading to reduced integrity of the cellular barrier, and these effects might be mediated by plasmin activity. We propose that ENO1 is a useful indicator of parakeratosis and might have a potential role in cellular TJ barrier function in the epidermis.

Analysis of A549 cell proteome alteration in response to recombinant influenza A virus nucleoprotein and its interaction with cellular proteins, a preliminary study.

Influenza A virus undergoes frequent changes of antigenicity and contributes to seasonal epidemics or unpredictable pandemics. Nucleoprotein, encoded by gene segment 5, is an internal protein of the virus and is conserved among strains of different host origins. In the current study, we analyzed the differentially expressed proteins in A549 cells transiently transfected with the recombinant nucleoprotein of influenza A virus by 2D gel electrophoresis. The resolved protein spots on gel were identified by MALDI-TOF/Mass spectrometry analysis. The majority of the host proteins detected to be differentially abundant in recombinant nucleoprotein-expressing cells as compared to vector-transfected cells are the proteins of metabolic pathways, glycolytic enzymes, molecular chaperones and cytoskeletal proteins. We further demonstrated the interaction of virus nucleoprotein with some of the identified host cellular proteins. In vitro binding assay carried out using the purified recombinant nucleoprotein (pET29a+NP-His) and A549 cell lysate confirmed the interaction between nucleoprotein and host proteins, such as alpha enolase 1, pyruvate kinase and ß-actin. The preliminary data of our study provides the information on virus nucleoprotein interaction with proteins involved in glycolysis. However, studies are ongoing to understand the significance of these interactions in modulating the host factors during virus replication.

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization.

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca(2+) chelator BAPTA or an inhibitor of endoplasmic reticulum Ca(2+)-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca(2+) entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca(2+) channel-mediated Ca(2+) influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.

PGK1, a glucose metabolism enzyme, may play an important role in rheumatoid arthritis.

BACKGROUND: Some studies have indicated that glucose metabolism plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to find the novel genes affecting glucose metabolism in RA. MATERIALS/METHODS: Synovial tissues of collagen-induced arthritis (CIA) were analyzed with Rat Glucose Metabolism RT(2) Profiler™ PCR Array to screen those genes with special expressions in glucose metabolism. Real-time PCR, western blotting, and ELISA were used to confirm the result in synovial tissues and blood of human RA. Culture synovial fibroblast cells (RASF) was treated with siRNA to suppress expressions of the target genes. CCK-8 cell proliferation assay and two-compartment transwell system were performed to examine cell proliferation and cell migration of the treated RASF. RESULTS: Both PCR array and real-time PCR detected the up-regulation of ENO1, HK2, and PGK1 and the down-regulation of PCK1 and PDK4 in synovial tissues of CIA rats. Real-time PCR and western blotting detected the increased expression of ENO1 and PGK1 in RA synovial tissues. ELISA detected a high level of PGK1 in the blood of RA patients. Decreased cell proliferation and cell migration capabilities were significantly detected in RASF following treatment of anti-PGK1 siRNA. IL-1ß and IFN-γ rather than TNF-α and IL-1α levels were significantly declined in supernatants of the treated RASF. CONCLUSIONS: PGK1, a glycolytic enzyme catalyzing the conversion of 3-phosphoglycerate into 2-phosphoglycerate, has increased expression in synovial tissues and blood of RA, which may be involved in pro-inflammation and synovial hyperplasia of the disease.

Enolase1 (ENO1) and glucose-6-phosphate isomerase (GPI) are good markers to predict human sperm freezability.

Sperm cryopreservation is a method to preserve sperm samples for a long period. However, the fertility of sperm decreases markedly after freezing and thawing in a certain amount of samples. The aim of the present study was to find useful and reliable predictive biomarkers of the capacity to withstand the freeze-thawing process in human ejaculates. Previous researches have shown that enolase1 (ENO1) and glucose-6-phosphate isomerase (GPI) are closely related to spermatozoa quality. We chose the two proteins as probable markers of sperm freezing capacity. Ejaculate samples were separated into good freezability ejaculates (GFE) and poor freezability ejaculates (PFE) according to progressive motility of the sperm after thawing. Before starting cryopreservation protocols, the two proteins from each group were compared using western blot analysis and immunofluorescence. Results showed that normalized content of ENO1 (P<0.05) and GPI (P<0.01) were both significantly higher in GFE than in PFE. The association of ENO1 and GPI with postthaw sperm viability and motility was confirmed using Pearson's linear correlation. In conclusion, ENO1 and GPI can be used as markers of human sperm freezability before starting the cryopreservation procedure.

Investigation of the anti-cancer potential of epoxyazadiradione in neuroblastoma: experimental assays and molecular analysis.

Neuroblastoma, the most common childhood solid tumor, originates from primitive sympathetic nervous system cells. Epoxyazadiradione (EAD) is a limonoid derived from Azadirachta indica, belonging to the family Meliaceae. In this study, we isolated the EAD from Azadirachta indica seed and studied the anti-cancer potential against neuroblastoma. Herein, EAD demonstrated significant efficacy against neuroblastoma by suppressing cell proliferation, enhancing the rate of apoptosis and cycle arrest at the SubG0 and G2/M phases. EAD enhanced the pro-apoptotic Caspase 3 and Caspase 9 and inhibited the NF-kß translocation in a dose-dependent manner. In order to identify the specific EAD target, a gel-free quantitative proteomics study on SH-SY5Y cells using Liquid Chromatography with tandem mass spectrometry was done in a dose-dependent manner, followed by detailed bioinformatics analysis to identify effects on protein. Proteomics data identified that Enolase1 and HSP90 were up-regulated in neuroblastoma. EAD inhibited the expression of Enolase1 and HSP90, validated by mRNA expression, immunoblotting, Enolase1 and HSP90 kit and flow-cytometry based bioassay. Molecular docking study, Molecular dynamic simulation, and along with molecular mechanics/Poisson-Boltzmann surface area analysis also suggested that EAD binds at the active site of the proteins and were stable throughout the 100 ns Molecular dynamic simulation study. Overall, this study suggested EAD exhibited anti-cancer activity against neuroblastoma by targeting Enolase1 and HSP90 pathways.Communicated by Ramaswamy H. Sarma.

Unrevealed roles of extracellular enolase­1 (ENO1) in promoting glycolysis and pro­cancer activities in multiple myeloma via hypoxia­inducible factor 1α.

The involvement of enolase­1 (ENO1), intracellularly or extracellularly, has been implicated in cancer development. Moreover, anticancer activities of an ENO1­targeting antibody has demonstrated the pathological roles of extracellular ENO1 (surface or secreted forms). However, although ENO1 was first identified as a glycolytic enzyme in the cytosol, to the best of our knowledge, extracellular ENO1 has not been implicated in glycolysis thus far. In the present study, the effects of extracellular ENO1 on glycolysis and other related pro­cancer activities were investigated in multiple myeloma (MM) cells in vitro and in vivo. Knockdown of ENO1 expression reduced lactate production, cell viability, cell migration and surface ENO1 expression in MM cells. Notably, addition of extracellular ENO1 protein in cancer cell culture enhanced glycolytic activity, hypoxia­inducible factor 1­α (HIF­1α) expression, glycolysis­related gene (GRG) expression and pro­cancer activities, such as cell migration, cell viability and tumor­promoting cytokine secretion. Consistently, these extracellular ENO1­induced cellular effects were inhibited by an ENO1­specific monoclonal antibody (mAb). In addition, extracellular ENO1­mediated glycolysis, GRG expression and pro­cancer activities were also reduced by HIF­1α silencing. Lastly, administration of an ENO1 mAb reduced tumor growth and serum lactate levels in an MM xenograft model. These results suggested that extracellular ENO1 (surface or secreted forms) enhanced a HIF­1α­mediated glycolytic pathway, in addition to its already identified roles. Therefore, the results of the present study highlighted the therapeutic potential of ENO1­specific antibodies in treating MM, possibly via glycolysis inhibition, and warrant further studies in other types of cancer.

Seven oxidative stress-related genes predict the prognosis of hepatocellular carcinoma.

Predicting the prognosis of hepatocellular carcinoma (HCC) is a major medical challenge and of guiding significance for treatment. This study explored the actual relevance of RNA expression in predicting HCC prognosis. Cox's multiple regression was used to establish a risk score staging classification and to predict the HCC patients' prognosis on the basis of data in the Cancer Genome Atlas (TCGA). We screened seven gene biomarkers related to the prognosis of HCC from the perspective of oxidative stress, including Alpha-Enolase 1(ENO1), N-myc downstream-regulated gene 1 (NDRG1), nucleophosmin (NPM1), metallothionein-3, H2A histone family member X, Thioredoxin reductase 1 (TXNRD1) and interleukin 33 (IL-33). Among them we measured the expression of ENO1, NGDP1, NPM1, TXNRD1 and IL-33 to investigate the reliability of the multi-index prediction. The first four markers' expressions increased successively in the paracellular tissues, the hepatocellular carcinoma samples (from patients with better prognosis) and the hepatocellular carcinoma samples (from patients with poor prognosis), while IL-33 showed the opposite trend. The seven genes increased the sensitivity and specificity of the predictive model, resulting in a significant increase in overall confidence. Compared with the patients with higher-risk scores, the survival rates with lower-risk scores are significantly increased. Risk score is more accurate in predicting the prognosis HCC patients than other clinical factors. In conclusion, we use the Cox regression model to identify seven oxidative stress-related genes, investigate the reliability of the multi-index prediction, and develop a risk staging model for predicting the prognosis of HCC patients and guiding precise treatment strategy.

RNA-binding protein ENO1 promotes the tumor progression of gastric cancer by binding to and regulating gastric cancer-related genes.

Background: This study sought to identify the downstream target genes of enolase 1 (ENO1), clarify the role of ENO1 in gastric cancer (GC), and provide novel insights into the regulatory mechanisms of ENO1 in the occurrence and development of GC. Methods: We performed RNA-immunoprecipitation sequencing in MKN-45 cells to study the types and abundance of pre-messenger RNA (mRNA)/mRNA bound by ENO1, the binding sites and motifs, the relationship between ENO1 binding and its regulation of transcription level, and alternative splicing level by combining with RNA-sequencing (RNA-seq) data to further clarify the role of ENO1 in GC. Results: We found that ENO1 stabilized the expression of SRY-box transcription factor 9 (SOX9), vascular endothelial growth factor A (VEGFA), G protein-coupled receptor class C group 5 member A (GPRC5A), and myeloid cell leukemia-1 (MCL1) by binding to their mRNA, which increased the growth of GC. In addition, ENO1 interacted with some other long non-coding RNAs (lncRNAs) or small-molecule kinases, such as NEAT1, LINC00511, CD44, and pyruvate kinase M2 (PKM2), to regulate their expression to affect cell proliferation, migration, and apoptosis. Conclusions: ENO1 may play a role in GC by binding to and regulating GC-related genes. Our findings extend understandings of its mechanism as a clinical therapeutic target.