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The involvement of enolase-1 (ENO1), intracellularly or extracellularly, has been implicated in cancer development. Moreover, anticancer activities of an ENO1-targeting 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 pro-cancer 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, hypoxia-inducible factor 1-α (HIF-1α) expression, glycolysis-related gene (GRG) expression and pro-cancer activities, such as cell migration, cell viability and tumor-promoting cytokine secretion. Consistently, these extracellular ENO1-induced cellular effects were inhibited by an ENO1-specific monoclonal antibody (mAb). In addition, extracellular ENO1-mediated glycolysis, GRG expression and pro-cancer activities were also reduced by HIF-1α 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 HIF-1α-mediated glycolytic pathway, in addition to its already identified roles. Therefore, the results of the present study highlighted the therapeutic potential of ENO1-specific antibodies in treating MM, possibly via glycolysis inhibition, and warrant further studies in other types of cancer.

Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer's disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review.

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-β 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.

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.

Enolase-α (ENO-1) is a key glycolytic enzyme that has been used as a diagnostic marker to identify human lung cancers. To investigate the role of ENO-1 in breast cancer diagnosis and therapy, the mRNA levels of ENO-1 in 244 tumor and normal paired tissue samples and 20 laser capture-microdissected cell clusters were examined by quantitative real-time PCR analysis. Increased ENO-1 mRNA expression was preferentially detected in estrogen receptor-positive (ER+) tumors (tumor/normal ratio >90-fold) when compared to ER-negative (tumor/normal ratio >20-fold) tumor tissues. The data presented here demonstrate that those patients whose tumors highly expressed ENO-1 had a poor prognosis with greater tumor size (>2 cm, *P = .017), poor nodal status (N > 3, *P = .018), and a shorter disease-free interval (≦1 year, *P < .009). We also found that higher-expressing ENO-1 tumors confer longer distance relapse (tumor/normal ratio = 82.8-92.4-fold) when compared to locoregional relapse (tumor/normal ratio = 43.4-fold) in postsurgical 4-hydroxy-tamoxifen (4-OHT)-treated ER+ patients (*P = .014). These data imply that changes in tumor ENO-1 levels are related to clinical 4-OHT therapeutic outcome. In vitro studies demonstrated that decreasing ENO-1 expression using small interfering RNA (siRNA) significantly augmented 4-OHT (100 nM)-induced cytotoxicity in tamoxifen-resistant (Tam-R) breast cancer cells. These results suggest that downregulation of ENO-1 could be utilized as a novel pharmacological approach for overcoming 4-OHT resistance in breast cancer therapy.

In the present study, we investigated the effect of reduced enolase-1 expression in human umbilical vein endothelial cells (HUVECs)/MDA-MB-231 cells on the response to hypoxia and the possible mechanisms involved. Breast cancer cells transfected with enolase-1 siRNA were injected into mice to establish a tumor-bearing mouse model, and the correlation between enolase-1 expression and breast cancer angiogenesis, as well as its effect on the efficacy of radiation therapy were assessed. HUVECs were cultured in vitro, and transfected with enolase-1 siRNA. Following stable passage, 1.0% O2 was used to induce hypoxia. The growth, proliferation, division and angiogenesis of HUVECs were observed using MTT assay, flow cytometry (FCM) and time-lapse video microscopy. The key regulatory molecules were detected using western blot analysis, two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The breast cancer cell line, MDA-MB-231, was cultured in vitro, and transfected with enolase-1 siRNA. The cells were injected into nude mice, and radiation therapy was administered. Tumor growth, angiogenesis in tumor tissues and apoptosis were observed, and the expression of the endogenous hypoxia marker, hypoxia inducible factor-1α (HIF-1α), was detected using immunohistochemistry after the mice were sacrificed. A significant reduction in the hypoxia-induced apoptosis of HUVECs was observed in the control group compared with the endothelial cells transfected with enolase-1 siRNA. After the enolase-1 transfected breast cancer cells were injected into nude mice, tumor growth significantly declined, and the tumor volume and weight were reduced. Following treatment with radiation therapy, tumor size significantly decreased in both groups, and the highest reduction was observed in the transfected group. The reduction in enolase-1 expression significantly decreases the response to hypoxia and enhances the sensitivity of the cells to radiation therapy; therefore, enolase-1 may be a drug target for the treatment of breast cancer.

Hepatic fibrosis is an inevitable process in the progression of chronic HBV infection to hepatic cirrhosis, but its detailed mechanism is still unknown. Clinic serum biomarkers of HBV hepatic cirrhosis were scanned by proteomic methods. We used two-dimensional electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) to separate and identify the proteins which were differentially expressed in the serum of patients with hepatic fibrosis compared to HBV carriers. We identified 27 differentially expressed proteins, of which 19 proteins were up-regulated and 8 proteins were down-regulated in the serum of patients with hepatic fibrosis compared to HBV carriers. The expression level of enolase-1 (α-enolase) was decreased while the level of thrombospondin-1 (TSP-1) increased in the serum of patients with hepatic fibrosis by western blot. Enolase-1 and TSP-1 may be useful as biomarkers for the clinic diagnosis of hepatic fibrosis, but further study is necessary.

Cell surface-associated proteolysis mediated by plasmin (PLA) is an essential feature of wound healing, angiogenesis and cell invasion, processes that are dysregulated in cancer development, progression and systemic spread. The generation of PLA, initiated by the binding of its precursor plasminogen (PLG) to the cell surface, is regulated by an array of activators, inhibitors and receptors. In this review, we will highlight the importance of the best-characterized components of the PLG/PLA cascade in the pathogenesis of cancer focusing on the role of the cell surface-PLG receptors (PLG-R). PLG-R overexpression has been associated with poor prognosis of cancer patients and resistance to chemotherapy. We will also discuss recent findings on the molecular mechanisms regulating cell surface expression and distribution of PLG-R.