Alpha-enolase Research Articles

Integrative transcriptomic and TMT-based proteomic analysis reveals the mechanism by which AtENO2 affects seed germination under salt stress.

Seed germination is critical for plant survival and agricultural production and is affected by many cues, including internal factors and external environmental conditions. As a key enzyme in glycolysis, enolase 2 (ENO2) also plays a vital role in plant growth and abiotic stress responses. In our research, we found that the seed germination rate was lower in the AtENO2 mutation (eno2- ) than in the wild type (WT) under salt stress in Arabidopsis thaliana, while there was no significant difference under normal conditions. However, the mechanisms by which AtENO2 regulates seed germination under salt stress remain limited. In the current study, transcriptome and proteome analyses were used to compare eno2- and the WT under normal and salt stress conditions at the germination stage. There were 417 and 4442 differentially expressed genes (DEGs) identified by transcriptome, and 302 and 1929 differentially expressed proteins (DEPs) qualified by proteome under normal and salt stress conditions, respectively. The combined analysis found abundant DEGs and DEPs related to stresses and hydrogen peroxide removal were highly down-regulated in eno2- . In addition, several DEGs and DEPs encoding phytohormone transduction pathways were identified, and the DEGs and DEPs related to ABA signaling were relatively greatly up-regulated in eno2- . Moreover, we constructed an interactive network and further identified GAPA1 and GAPB that could interact with AtENO2, which may explain the function of AtENO2 under salt stress during seed germination. Together, our results reveal that under salt stress, AtENO2 mainly affects the expression of genes and proteins related to the phytohormone signal transduction pathways, stress response factors, and reactive oxygen species (ROS), and then affects seed germination. Our study lays the foundation for further exploration of the molecular function of AtENO2 under salt stress at the seed germination stage in Arabidopsis thaliana.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

MafA-positive insulin-producing cells derived from rat adipose stem cells are efficient in glucose hemostasis in vitro

Objective: MafA has been recognized as a specific pancreatic β-cells activator for Insulin expression. This study investigated the changes in glucose transport and consumption by MafA-positive Insulin-producing cells resulting from the differentiation of rat adipose tissue-derived mesenchymal stem cells. Methods: Adipose tissue-derived mesenchymal stem cells were differentiated into Insulin-producing cells in a 14-day protocol using nicotinamide and ITS. The ability to produce Insulin was confirmed by adipogenesis differentiation capacity and expression of Oct4, Nanog, and Sox2. Insulin and Glucagon concentrations in Insulin-producing cells were determined by ELISA. The ability of Insulin-producing cells for the presence of MafA, Glut2, ENO1, and IGF-I receptor genes was determined by Western blotting analysis. The amounts of Glucokinase and Pyruvate Carboxylase activities were measured by Fluorometric and Colorimetric methods, respectively. Results: The un-differentiated cells showed a promising capacity to differentiate toward adipocytes. The adipose tissue mesenchymal stem cells revealed significantly elevated levels of Oct4, Nanog, and Sox2 compared to differentiated adipocytes. The cells that differentiated into Insulin-producing cells secreted considerably more Insulin and Glucagon than undifferentiated cells.

Sulfenylation of ENOLASE2 facilitates H2O2-conferred freezing tolerance in Arabidopsis.

H2O2 affects the expression of genes that are involved in plant responses to diverse environmental stresses; however, the underlying mechanisms remain elusive. Here, we demonstrate that H2O2 enhances plant freezing tolerance through its effect on a protein product of lowexpression of osmotically responsive genes2 (LOS2). LOS2 is translated into a major product, cytosolic enolase2 (ENO2), and sometimes an alternative product, the transcription repressor c-Myc-binding protein (MBP-1). ENO2, but not MBP-1, promotes cold tolerance by binding the promoter of C-repeat/DRE binding factor1 (CBF1), a central transcription factor in plant cold signaling, thus activating its expression. Overexpression of CBF1 restores freezing sensitivity of a LOS2 loss-of-function mutant. Furthermore, cold-induced H2O2 increases nuclear import and transcriptional binding activity of ENO2 by sulfenylating cysteine 408 and thereby promotes its oligomerization. Collectively, our results illustrate how H2O2 activates plant cold responses by sulfenylating ENO2 and promoting its oligomerization, leading to enhanced nuclear translocation and transcriptional activation of CBF1.

Tissue-Based Proteomic Profiling in Patients with Hyperplasia and Endometrial Cancer.

Uterine cancers are among the most prevalent gynecological malignancies, and endometrial cancer (EC) is the most common in this group. This study used tissue-based proteomic profiling analysis in patients with endometrial cancer and hyperplasia, and control patients. Conventional 2D gel electrophoresis, followed by a mass spectrometry approach with bioinformatics, including a network pathway analysis pipeline, was used to identify differentially expressed proteins and associated metabolic pathways between the study groups. Thirty-six patients (twelve with endometrial cancer, twelve with hyperplasia, and twelve controls) were enrolled in this study. The mean age of the participants was 46–75 years. Eighty-seven proteins were significantly differentially expressed between the study groups, of which fifty-three were significantly differentially regulated (twenty-eight upregulated and twenty-five downregulated) in the tissue samples of EC patients compared to the control (Ctrl). Furthermore, 26 proteins were significantly dysregulated (8 upregulated and 18 downregulated) in tissue samples of hyperplasia (HY) patients compared to Ctrl. Thirty-two proteins (nineteen upregulated and thirteen downregulated) including desmin, peptidyl prolyl cis-trans isomerase A, and zinc finger protein 844 were downregulated in the EC group compared to the HY group. Additionally, fructose bisphosphate aldolase A, alpha enolase, and keratin type 1 cytoskeletal 10 were upregulated in the EC group compared to those in the HY group. The proteins identified in this study were known to regulate cellular processes (36%), followed by biological regulation (16%). Ingenuity pathway analysis found that proteins that are differentially expressed between EC and HY are linked to AKT, ACTA2, and other signaling pathways. The panels of protein markers identified in this study could be used as potential biomarkers for distinguishing between EC and HY and early diagnosis and progression of EC from hyperplasia and normal patients.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.

Abstract 1799: Inhibition of triple negative breast cancer metastasis via Enolase-1 modulation

Abstract Triple-negative breast cancer (TNBC) is one of the aggressive forms of breast cancer and frequently relapses and metastases. Currently, very few options are available for TNBC treatment. Cancer cells exploit glycolytic machinery for the Warburg effect and aerobic glycolysis. Enolase 1 (ENO1) is the glycolytic enzyme expressed in the majority of tissues and many cancer cells have its higher expression. Apart from the glycolytic role in the cytosol, ENO1 also plays different roles in cancer cells including function as a surface receptor. We have developed a chemical small molecule (CET12) that strongly binds to ENO1 and restrain its activity and subcellular localization. CET12 treatment arrests the TNBC cells in the mitotic phase and leads the apoptotic cell death via AMPK activation. Global proteome profiling suggested that CET12 pushes the cells toward oxidative phosphorylation. It also inhibits cell migration and invasion in vitro. In vivo studies using 4T1 and EMT6 syngeneic mouse models are also in-line with in vitro results and revealed that SU0212 treatment inhibits tumor progression and metastasis. These results were further supported by tail vain lung metastasis assay and intracardiac injection mouse model. This study provides compelling preclinical data for further development of CET12 for the treatment of TNBC. Citation Format: Dhanir Tailor, Arpit Dheeraj, Fernando Jose Garcia-Marques, Mallesh Pandrala, Abel Bermudez, Sharon Pitteri, Sanjay V. Malhotra. Inhibition of triple negative breast cancer metastasis via Enolase-1 modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1799.

Abstract 1481: A microRNA classifier for predicting neuroendocrine differentiation in castration-resistant prostate cancer patients

Abstract Therapy-induced neuroendocrine prostate cancer (NEPC) is an extremely aggressive variant of castration-resistant prostate cancer (CRPC) that is increasing in incidence with the widespread use of second generation of androgen receptor (AR)-pathway inhibitors (APIs) such as Enzalutamide (ENZ) and Abiraterone. This aggressive variant arises from CRPC-Adenocarcinomas (CRPC-Adeno) via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED) wherein cells undergo a lineage switch and exhibit neuroendocrine (NE) features, characterized by expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA) and synaptophysin (SYP). Currently, histopathological assessment combined with immunohistochemical detection in PCa tissues/serum levels of neuronal markers including SYP, NSE, CHGA and CD56 is used to monitor NED in CRPC patients. However, these markers are not sufficiently specific, highlighting the urgent need of novel molecular markers to assess emergence of NED in CRPC patients. We demonstrated that progression of CRPC-Adeno to CRPC-NE states is associated with a characteristic set of miRNA alterations. Here we validate a ‘novel miRNA classifier’ to robustly stratify CRPC-NE tumors from CRPC-Adenocarcinomas in independent clinical cohorts and deduce the optimal miRNA genes required for NEPC diagnosis. Methods: Human FFPE sections from CRPC-Adeno and CRPC-NE patients were microdissected, RNA were extracted and small RNA sequencing was performed using an Illumina NextSeq 500 platform. Sequencing data were analyzed and machine learning algorithms were applied (random forest machine learning technique with leave-pair-out cross validation (LPOCV)). The performance of classifier was measured using receiver operating characteristic (ROC) analysis with area under the curve (AUC) as the primary evaluation metric. Results: Unsupervised analysis of sequencing data by principal component analyses (PCA) revealed distinct clustering of the CRPC-NE tumors from CRPC-Adenocarcinomas based on miRNA profiles suggesting that miRNA profiles can be used to stratify these tumor types. We applied the ‘43-miRNA classifier data’ we deduced earlier to these validation cohorts. Our analyses showed that a set of 5 miRNAs of the classifier are important in distinguishing between CRPC-Adeno vs CRPC-NE with an AUC=0.8318. Conclusions: A ‘5- miRNA’ classifier was validated to be of significance in two independent validation cohorts employing clinical samples from two independent sites. We propose this miRNA classifier as an important tool for diagnosing NED in CRPC patients. Citation Format: Sharanjot Saini, Amritha Sreekumar, Jin Tae Lee, Nikhil Patel, Ashok Sharma. A microRNA classifier for predicting neuroendocrine differentiation in castration-resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1481.

A Novel Enolase-1 Antibody Targets Multiple Interacting Players in the Tumor Microenvironment of Advanced Prostate Cancer.

Prostate cancer is one of the most common causes of cancer death in men worldwide, and the treatment options are limited for patients with advanced stages of prostate cancer. Upon oncogenic or inflammatory stimulation, tumor cells or immune cells express cell surface enolase-1 (ENO1) as plasminogen receptor to facilitate their migration via plasmin activation. Little is known about the roles of ENO1 in prostate cancer, especially in the tumor microenvironment (TME). We hypothesized that targeting surface ENO1 with specific mAbs would exert multifactorial therapeutic potentials against prostate cancer. In vivo, we showed ENO1 mAb (HuL227) reduced the growth of subcutaneous PC-3 xenograft, monocytes recruitment, and intratumoral angiogenesis. In a PC-3 intratibial implantation model, HuL227 reduced tumor growth and osteoclast activation in the bone. To investigate the antitumor mechanism of ENO1 mAb, we found that blocking surface ENO1 significantly reduced VEGF-A-induced tube formation of endothelial cells in vitro. Furthermore, HuL227 inhibited inflammation-enhanced osteoclasts activity and the secretion of invasion-related cytokines CCL2 and TGFβ from osteoclasts. In addition, inflammation-induced migration and chemotaxis of androgen-independent prostate cancer cells were dose-dependently inhibited by HuL227. In summary, we showed that, ENO1 mAb targets multiple TME niches involved in prostate cancer progression and bone metastasis via a plasmin-related mechanism, which may provide a novel immunotherapy approach for men with advanced prostate cancer.

MOLECULAR BASIS OF THE DEVELOPMENT OF INSULIN RESISTANCE IN OBESE ADOLESCENT AND ADULT MEN

The aim of this work was to study the association between the expression of glucose metabolism related genes and insulin resistance, which expression is changed in obese adolescents and adult men with and without insulin resistance, for better understanding the molecular basis of the development of obesity complications and evaluation of possible contribution of these genes in development of insulin resistance. Methods. The expression level of genes related to glucose metabolism and their regulations was studied by real-time qPCR in adipose tissue and blood cells using SYBRGreen Mix and specific for each mRNA forward and reverse primers. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). Results. It was shown that in obese patients with insulin resistance the expression level of IRS1 (insulin receptor substrate 1), HK2 (hexokinase 2), PFKFB2 (6-phosphofructokinase/fructose-2,6-bisphosphatase 2) and PFKFB3 as well as circadian factors CLOCK and ARNTL genes in subcutaneous adipose tissue is significantly decreased as compared to obese men with normal sensitivity to insulin. At the same time, the development of insulin resistance in obese patients leads to up-regulation of PFKFB4, PER1, HSPA6, ALDH1A3, COL5A1, TIMP1, TIMP2, SPARC, and VCAN gene expressions in subcutaneous adipose tissue. The expression level of IGF1 (insulin-like growth factor 1) and IGFBP5 (IGF binding protein 5) as well as ENO1 (enolase 1) and ENO2 is down-regulated in the blood of obese adolescent with insulin resistance, but IGFBP2 and IGFBP7 gene expressions are significantly increased in these patients. Conclusions. The results of this investigation provide evidence that the development of insulin resistance in obese patients is associated with gene specific changes in the expression of many very important regulatory genes, which are endoplasmic reticulum stress responsible.

Role of MicroRNAs in Neuroendocrine Prostate Cancer.

Therapy-induced neuroendocrine prostate cancer (t-NEPC/NEPC) is an aggressive variant of prostate cancer (PCa) that frequently emerges in castration-resistant prostate cancer (CRPC) under the selective pressure of androgen receptor (AR)-targeted therapies. This variant is extremely aggressive, metastasizes to visceral organs, tissues, and bones despite low serum PSA, and is associated with poor survival rates. It arises via a reversible trans-differentiation process, referred to as ‘neuroendocrine differentiation’ (NED), wherein PCa cells undergo a lineage switch and exhibit neuroendocrine features, characterized by the expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA), and synaptophysin (SYP). The molecular and cellular mechanisms underlying NED in PCa are complex and not clearly understood, which contributes to a lack of effective molecular biomarkers for diagnosis and therapy of this variant. NEPC is thought to derive from prostate adenocarcinomas by clonal evolution. A characteristic set of genetic alterations, such as dual loss of retinoblastoma (RB1) and tumor protein (TP53) tumor suppressor genes and amplifications of Aurora kinase A (AURKA), NMYC, and EZH2, has been reported to drive NEPC. Recent evidence suggests that microRNAs (miRNAs) are important epigenetic players in driving NED in advanced PCa. In this review, we highlight the role of miRNAs in NEPC. These studies emphasize the diverse role that miRNAs play as oncogenes and tumor suppressors in driving NEPC. These studies have unveiled the important role of cellular processes such as the EMT and cancer stemness in determining NED in PCa. Furthermore, miRNAs are involved in intercellular communication between tumor cells and stromal cells via extracellular vesicles/exosomes that contribute to lineage switching. Recent studies support the promising potential of miRNAs as novel diagnostic biomarkers and therapeutic targets for NEPC.

CircRPN2 Inhibits Aerobic Glycolysis and Metastasis in Hepatocellular Carcinoma.

The circRNA circRPN2 is a potential prognostic biomarker and therapeutic target in hepatocellular carcinoma that suppresses aerobic glycolysis and metastasis.

A microRNA-based classifier for identification of neuroendocrine differentiation in metastatic castration-resistant prostate cancer.

149 Background: Therapy-induced neuroendocrine prostate cancer (NEPC) is an extremely aggressive variant of castration-resistant prostate cancer (CRPC) that is increasing in incidence with the widespread use of second generation of androgen receptor (AR)-pathway inhibitors (APIs) such as enzalutamide (ENZ) and abiraterone. This aggressive variant arises from CRPC-Adenocarcinomas (CRPC-Adeno) via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED) wherein cells undergo a lineage switch and exhibit neuroendocrine (NE) features, characterized by expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA) and synaptophysin (SYP). Currently, histopathological assessment combined with immunohistochemical detection in PCa tissues/serum levels of neuronal markers including SYP, NSE, CHGA and CD56 is currently used to monitor NED in CRPC patients. However, these markers are not sufficiently specific, highlighting the urgent need of novel molecular markers to assess emergence of NED in CRPC patients. Recently, we demonstrated that progression of advanced CRPC with adenocarcinoma characteristics to CRPC-NE is associated with a characteristic set of miRNA alterations that promote plasticity of advanced PCa to NEPC (Bhagirath et al., Oncogene, 2020). Importantly, we could develop a ‘novel miRNA classifier’ to robustly stratify CRPC-NE tumors from CRPC-Adenocarcinomas. Here we further validate the classifier in independent clinical cohorts and deduce the optimal miRNA genes required for NEPC diagnosis. Methods: Human clinical samples with corresponding clinical information were procured from two independent sites of Prostate Cancer Biorepository Network (PCBN). FFPE sections from clinical samples were microdissected, RNA were extracted and small RNA sequencing was performed using an Illumina NextSeq 500 platform. Sequencing data were analyzed and machine learning algorithms were applied. The performance of classifier was measured using receiver operating characteristic (ROC) analysis with area under the curve (AUC) as the primary evaluation metric. Results: Unsupervised analysis of sequencing data by principal component analyses (PCA) revealed distinct clustering of the CRPC-NE tumors from CRPC-Adenocarcinomas based on miRNA profiles suggesting that miRNA profiles can be used to stratify these tumor types. We applied the ‘43-miRNA classifier data’ we deduced earlier to these validation cohorts. Our analyses showed that a set of 5 miRNAs of the classifier are important in distinguishing between CRPC-Adeno vs CRPC-NE with an AUC=0.8318. Conclusions: A ‘5- miRNA’ classifier was validated to be of significance in two independent validation cohorts. We propose this miRNA classifier as an important tool for diagnosing NED in CRPC patients.

The essential roles of m6A RNA modification to stimulate ENO1-dependent glycolysis and tumorigenesis in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m6A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m6A modification in LUAD tumorigenesis is unknown.MethodsGlobal m6A levels and expressions of m6A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m6A on LUAD. The RNA-protein interactions, translation, putative m6A sites and glycolysis were explored in the investigation of the mechanism underlying how m6A stimulates tumorigenesis.ResultsThe elevation of global m6A level in most human LUAD specimens resulted from the combined upregulation of m6A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m6A level was associated with a poor overall survival in LUAD patients. Reducing m6A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m6A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m6A methylated at 359 A, which facilitated it’s binding with the m6A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m6A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m6A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m6A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD.ConclusionsThe m6A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m6A-dependent LUAD.

Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway.

BACKGROUNDHepatic stellate cell (HSC) hyperactivation is a central link in liver fibrosis development. HSCs perform aerobic glycolysis to provide energy for their activation. Focal adhesion kinase (FAK) promotes aerobic glycolysis in cancer cells or fibroblasts, while FAK-related non-kinase (FRNK) inhibits FAK phosphorylation and biological functions.AIMTo elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODSMouse liver fibrosis models were established by administering CCl4, and the effect of FRNK on the degree of liver fibrosis in the model was evaluated. Transforming growth factor-β1 was used to activate LX-2 cells. Tyrosine phosphorylation at position 397 (pY397-FAK) was detected to identify activated FAK, and the expression of the glycolysis-related proteins monocarboxylate transporter 1 (MCT-1) and enolase1 (ENO1) was assessed. Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region, which were validated with chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.RESULTSThe pY397-FAK level was increased in human fibrotic liver tissue. FRNK knockout promoted liver fibrosis in mouse models. It also increased the activation, migration, proliferation and aerobic glycolysis of primary hepatic stellate cells (pHSCs) but inhibited pHSC apoptosis. Nevertheless, opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells. Mechanistically, the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis, which was inhibited by exogenous FRNK.CONCLUSIONFRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway, thereby improving liver fibrosis. FRNK might be a potential target for liver fibrosis treatment.

Low Sperm Motility Is Determined by Abnormal Protein Modification during Epididymal Maturation.

PurposeDuring epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility.Materials and MethodsTo determine the differential protein expression in caput and cauda epididymal spermatozoa from low and high motility bulls, western blot analysis was performed. Moreover, signaling pathways were identified to understand the mechanisms of regulation of sperm motility through the differential protein expression associated with fertility-related proteins.ResultsWe found that ENO1 was substantially higher in the caput spermatozoa from low motility bulls than the caput and cauda spermatozoa from high motility bulls. However, ENO1 expression in low motility bull spermatozoa was downregulated to a level comparable to that in the high motility bull spermatozoa during epididymal maturation. Moreover, there was a lack of modification of mitochondrial proteins, including glutathione peroxidase 4 and NADH:Ubiquinone Oxidoreductase Core Subunit S8, in low motility bull spermatozoa during epididymal maturation, whereas active changes were detected in high motility bull spermatozoa.ConclusionsIrregular modifications of mitochondrial proteins during epididymal sperm maturation may increase excessive ROS production and premature activation of spermatozoa during epididymal maturation. Consequently, spermatozoa may lose their motility by the earlier consumption of their energy source and may be damaged by ROS during epididymal maturation, resulting in a decline in sperm motility and bull fertility.

Уровень протеолитических фрагментов IgG как дополнительный прогностический биомаркер рака предстательной железы

Introduction. Prostate cancer (PC) leads the world structure of male cancer mortality. Understanding the prognosis for PC patients is vitally important and requires diagnostic enhancements such as developing biomarker panels. The studyaimed to estimate sensitivity, specificity, and prognostic significance of the level of immunoglobulin G (IgG) proteolysis products in the blood serum depending on PC Gleason score and detect how the levels of IgG proteolytic fragments were associated with the diagnostic marker expression. Materials and methods. The study included 90 PC patients. We used the Gleason score to evaluate tumor grade. The level of the IgG proteolysis products and total prostate-specific antigen (PSA) in the serum was detected with ELISA. We performed an immunohistochemical assay with Ki-67, plasminogen-binding protein enolase-1 (ENO-1), and prostate-specific membrane antigen (PSMA). Results. ROC analysis demonstrated 100% specificity of the IgG proteolysis products test regardless of Gleason score, while the sensitivity reached 72.2%, 57.5%, 52.6%, and 46.2% for ISUP stages 1, 2, 3, and 4, respectively. An intergroup comparison showed significant differences between patient groups according to Gleason score, preoperative PSA and IgG proteolysis product serum levels and Ki-67 index. We detected a positive correlation between the level of IgG proteolysis products in the PC patients’ serum and ENO-1 expression and a negative correlation with the Gleason score and PSMA expression. Conclusion. Preoperative detection of high levels of proteolytic IgG fragments in the serum of PC patients even with low Gleason score and PSMA expression may help predict a more aggressive disease course and choose the management strategy. Keywords: prostate cancer, plasminogen activation system, proteolytic fragments of immunoglobulin G, oncomarkers

E4 engages uPAR and enolase-1 and activates urokinase to exert antifibrotic effects.

Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin, known as E4, prevented and reversed both dermal and pulmonary fibrosis. Our goal was to identify the mechanism by which E4 abrogates fibrosis and its cell surface binding partner(s). Our findings show that E4 activated the urokinase pathway and increased the urokinase plasminogen activator (uPA) to type 1 plasminogen activator inhibitor (PAI-1) ratio. In addition, E4 substantially increased MMP-1 and MMP-3 expression and activity. In vivo, E4 reversed bleomycin induction of PAI-1 and increased uPA activity. In patients with SSc, the uPA/PAI-1 ratio was decreased in both lung tissues and pulmonary fibroblasts compared with normal donors. Proteins bound to biotinylated-E4 were identified as enolase-1 (ENO) and uPA receptor (uPAR). The antifibrotic effects of E4 required uPAR. Further, ENO mediated the fibrotic effects of TGF-β1 and exerted TGF-β1–independent fibrotic effects. Our findings suggest that the antifibrotic effect of E4 is mediated, in part, by regulation of the urokinase pathway and induction of MMP-1 and MMP-3 levels and activity in a uPAR-dependent manner, thus promoting extracellular matrix degradation. Further, our findings identify a moonlighting function for the glycolytic enzyme ENO in fibrosis.

Mitochondrial Ndufa4l2 Enhances Deposition of Lipids and Expression of Ca9 in the TRACK Model of Early Clear Cell Renal Cell Carcinoma.

Mitochondrial dysfunction and aberrant glycolysis are hallmarks of human clear cell renal cell carcinoma (ccRCC). Whereas glycolysis is thoroughly studied, little is known about the mitochondrial contribution to the pathology of ccRCC. Mitochondrial Ndufa4l2 is predictive of poor survival of ccRCC patients, and in kidney cancer cell lines the protein supports proliferation and colony formation. Its role in ccRCC, however, remains enigmatic. We utilized our established ccRCC model, termed Transgenic Cancer of the Kidney (TRACK), to generate a novel genetically engineered mouse model in which dox-regulated expression of an shRNA decreases Ndufa4l2 levels specifically in the renal proximal tubules (PT). This targeted knockdown of Ndufa4l2 reduced the accumulation of neutral renal lipid and was associated with decreased levels of the ccRCC markers carbonic anhydrase 9 (CA9) and Enolase 1 (ENO1). These findings suggest a link between mitochondrial dysregulation (i.e. high levels of Ndufa4l2), lipid accumulation, and the expression of ccRCC markers ENO1 and CA9, and demonstrate that lipid accumulation and ccRCC development can potentially be attenuated by inhibiting Ndufa4l2.

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.