Overexpression Of ENO1 Research Articles

Targeting Endothelial ENO1 (Enolase) -PI3K-Akt-mTOR Axis Alleviates Hypoxic Pulmonary Hypertension.

It has been shown that glycolytic protein alpha-ENO1 (enolase) contributes to the pathogenesis of pulmonary hypertension through acting smooth muscle cells; however, the roles of ENO1-caused endothelial and mitochondrial dysfunctions in Group 3 pulmonary hypertension remain unexplored. PCR array and RNA sequencing were used to screen and decipher the differential gene expression by hypoxia-treated ECs. Techniques of small-interfering RNA, specific inhibitor and plasmids carrying gene of ENO1, interventions with specific inhibitor and AAV-ENO1 delivery were employed to explore the role of ENO1 in hypoxic pulmonary hypertension in vitro and in vivo, respectively. Assays for cell proliferation, angiogenesis, and adhesion were employed to analyze cell behaviors, while seahorse analysis was used to measure mitochondrial function of human pulmonary artery endothelial cells. PCR array data showed that ENO1 expression increased in human pulmonary artery endothelial cells exposed to hypoxia, as well as in lung tissues from patients with chronic obstructive lung disease-pulmonary hypertension and murine model of hypoxic pulmonary hypertension. Inhibition of ENO1 restored the hypoxia-induced endothelial dysfunction, including excessive proliferation, angiogenesis, and adhesion, while overexpression of ENO1 promotes these disorders of human pulmonary artery endothelial cells. RNA-seq showed that ENO1 targets mitochondrion-related genes and PI3K-Akt signaling pathway. Mice treated with ENO1 inhibitor exhibited ameliorated pulmonary hypertension and improved right ventricular failure induced by hypoxia. A reversal effect was observed in mice exposed to hypoxia and inhaled adenoassociated virus overexpressing ENO1. These results indicate that hypoxic pulmonary hypertension is associated with an increased level of ENO1 and that targeting ENO1 might reduce experimental hypoxic pulmonary hypertension by improving endothelial and mitochondrial dysfunction via PI3K-Akt-mTOR signaling pathway.

Knockout of ENO1 leads to metabolism reprogramming and tumor retardation in pancreatic cancer.

The shift in glucose utilization from oxidative phosphorylation to glycolysis is the hallmark of tumor cells. The overexpression of ENO1, one of the key enzymes in the glycolysis process, has been identified in several cancers, however, its role in pancreatic cancer (PC) is yet unclear. This study identifies ENO1 as an indispensable factor in the progression of PC. Interestingly, ENO1-knockout could inhibit cell invasion and migration and prevent cell proliferation in pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1 and MIA PaCa-2); meanwhile, tumor cell glucose uptake and lactate excretion also decreased significantly. Furthermore, ENO1-knockout reduced colony formation and tumorigenesis in both in vitro and in vivo tests. In total, after ENO1 knockout, 727 differentially expressed genes (DEGs) were identified in PDAC cells by RNA-seq. Gene Ontology enrichment analysis revealed that these DEGs are mainly associated with components such as the 'extracellular matrix' and 'endoplasmic reticulum lumen', and participate in the regulation of signal receptor activity. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the identified DEGs are associated with pathways, such as 'fructose and mannose metabolism', 'pentose phosphate pathway, and 'sugar metabolism for amino and nucleotide. Gene Set Enrichment Analysis showed that ENO1 knockout promoted the upregulation of oxidative phosphorylation and lipid metabolism pathways-related genes. Altogether, these results indicated that ENO1-knockout inhibited tumorigenesis by reducing cell glycolysis and activating other metabolic pathways by altering the expression of G6PD, ALDOC, UAP1, as well as other related metabolic genes. Concisely, ENO1, which plays a vital role in the abnormal glucose metabolism in PC, can be exploited as a target to control carcinogenesis by reducing aerobic glycolysis.

NEDD4L inhibits glycolysis and proliferation of cancer cells in oral squamous cell carcinoma by inducing ENO1 ubiquitination and degradation

ABSTRACT Glycolysis contributes to cell metabolism and facilitates cell proliferation of oral squamous cell carcinoma (OSCC), the most common type of oral cancer. Understanding the regulatory mechanisms involved in the glycolysis of OSCC cells may provide important therapeutic inspirations. Immunohistochemistry was used to examine protein localization patterns in human OSCC tissues and Western blot was conducted to gauge protein level. Lentivirus transduction was used to overexpress or silence genes of interest. Cell proliferation was assessed by Cell Counting Kit (CCK)-8 assay while glycolysis was examined via measurement of extracellular acidification rate, oxygen consumption rate, and lactate and ATP production. In vivo cancer development was evaluated with a mouse tumor growth model. OSCC tissues displayed reduced expression of NEDD4L compared with normal tissues. NEDD4L expression positively correlated with 5-year patient survival rate, indicating that NEDD4L may be a prognosis marker for OSCC. NEDD4L overexpression suppressed proliferation, cell cycle transition, and glycolysis in OSCC cells, and inhibited in vivo tumor growth. UbiBrowser identified ENO1, an enzyme that catalyzes glycolysis, as a substrate of NEDD4L. Overexpression of NEDD4L resulted in the ubiquitination and subsequent degradation of ENO1 whereas overexpression of ENO1 reversed the functional effects of NEDD4L overexpression, restoring proliferation, cell cycle transition, and glycolysis in OSCC cells. NEDD4L elicits tumor-suppressive functions via inhibition of OSCC cell proliferation, cell cycle transition, and glycolysis by stimulating ENO1 ubiquitination and degradation. Our results unraveled a signaling axis important for OSCC cell survival and metabolism, which can serve as a potential therapeutic target.

Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma-An In Vitro and a Clinical Study.

Simple SummaryAlpha-enolase (ENO1) undergoes accentuated overexpression in several solid cancers, but little is known about its status in cutaneous melanoma. The aim of this study was to investigate the prognostic significance of ENO1 in surgical resections from melanoma patients and to assess its expression and enzymatic activity in several melanoma cell lines. In clinical analysis, the overexpression of ENO1 in melanoma cells was significantly correlated with advanced clinical stage, presence of metastases in regional lymph nodes, and shorter cancer-specific overall survival and disease-free survival. We also demonstrated high expression of ENO1 in melanoma cell lines compared with normal melanocytes. Our study, which extends previous in vitro research, makes the alpha-enolase a candidate for a promising diagnostic and therapeutic target for various types of cancers. Consequently, additional testing of ENO1 as a target for melanoma therapy is necessary.Alpha-enolase (ENO1) is a glycolytic metalloenzyme, and its overexpression occurs in numerous cancers, contributing to cancer cell survival, proliferation, and maintenance of the Warburg effect. Patients with an overexpression of ENO1 have a poor prognosis. The aim of the present study was to investigate the prognostic significance of ENO1 in surgical resections from 112 melanoma patients and to assess its expression and enzymatic activity in normoxia and hypoxia in several melanoma cell lines. Overexpression of ENO1 in tumor cells from patients was correlated with unfavorable prognosticators such as Breslow thickness, Clark level, mitotic activity, and the presence of ulceration. The expression of ENO1 also positively correlated with a greater thickness of the neoplastic infiltrate and a worse long-term prognosis for patients with cutaneous melanoma. We report significantly higher expression of ENO1 in melanoma cell lines in comparison to normal melanocytes. To conclude, our in vitro and clinical models showed that overexpression of ENO1 promotes invasiveness of melanoma cells and correlates with aggressive clinical behavior. These observations open the way to further search of a potential prognostic and therapeutic target in cutaneous melanoma.

ENO1 Promotes Lung Cancer Metastasis via HGFR and WNT Signaling-Driven Epithelial-to-Mesenchymal Transition.

ENO1 (α-enolase) expression is significantly correlated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in carcinogenesis remains elusive. In this study, we found that high expression of ENO1 is present in metastatic lung cancer cell lines and malignant tumors and is associated with poor overall survival of patients with lung cancer. Knockdown of ENO1 decreased cancer cell proliferation and invasiveness, whereas overexpression of ENO1 enhanced these processes. Moreover, ENO1 expression promoted tumor growth in orthotopic models and enhanced lung tumor metastasis in tail-vein injection models. These effects were mediated by upregulation of mesenchymal markers N-cadherin and vimentin and the epithelial-to-mesenchymal transition regulator SLUG, along with concurrent downregulation of E-cadherin. Mechanistically, ENO1 interacted with hepatocyte growth factor receptor (HGFR) and activated HGFR and Wnt signaling via increased phosphorylation of HGFR and the Wnt coreceptor LRP5/6. Activation of these signaling axes decreased GSK3β activity via Src-PI3K-AKT signaling and inactivation of the β-catenin destruction complex to ultimately upregulate SLUG and β-catenin. In addition, we generated a chimeric anti-ENO1 mAb (chENO1-22) that can decrease cancer cell proliferation and invasion. chENO1-22 attenuated cancer cell invasion by inhibiting ENO1-mediated GSK3β inactivation to promote SLUG protein ubiquitination and degradation. Moreover, chENO1-22 prevented lung tumor metastasis and prolonged survival in animal models. Taken together, these findings illuminate the molecular mechanisms underlying the function of ENO1 in lung cancer metastasis and support the therapeutic potential of a novel antibody targeting ENO1 for treating lung cancer. SIGNIFICANCE: This study shows that ENO1 promotes lung cancer metastasis via HGFR and WNT signaling and introduces a novel anti-ENO1 antibody for potential therapeutic use in lung cancer.

Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration

BackgroundA protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication.ResultsHere we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication.ConclusionsThese results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.

Enolase 1 differentially contributes to cell transformation in lung cancer but not in esophageal cancer.

Enolase transforms 2-phospho-D-glycerate into phosphoenolpyruvate during glycolysis. The human enolase (ENO) family comprises three members named ENO3, which is restricted to muscle tissues, ENO2, which is neuron- and neuroendocrine tissue-specific, and ENO1, which is expressed in almost all tissues. ENO1 is involved in various types of human cancer, including retinoblastoma, hepatocellular carcinoma, pancreatic cancer, renal cell carcinoma, cholangiocarcinoma and gastric cancer. Furthermore, ENO1 enhances cell transformation in numerous cancer cell lines. It has been reported that ENO1 is involved in various activities that are detrimental to cell transformation, including apoptosis and differentiation. However, a few studies demonstrated that ENO1 can be down- or upregulated in various types of lung cancer, which suggests that ENO1 has an ambiguous role in the development of lung cancer. The present study aimed to investigate the differential influences of ENO1 on various types of cancer, and to clarify the role of ENO1 in lung cancer in particular. Western blotting was performed to assess ENO1 protein expression levels in lung cancer and esophageal cancer tissues. Furthermore, exogenous ENO1 was overexpressed in cell lines derived from various tissues and single cell proliferation, flowcytometric analysis, and western blotting were performed to determine the cell proliferation rate, cell transformation status, cell cycle progression and the expression of cell cycle regulators, such as cyclins and cyclin-dependent kinases, and survival factors, such as MAPK and AKT. The results demonstrated that ENO1 was upregulated in collected panels of lung cancer tissues, but not in esophageal cancer tissues. In addition, overexpression of ectopic ENO1 promoted cell proliferation and survival in lung cancer cell lines, which was not the case in other cells, including an esophageal cell line. Furthermore, mechanistic analyses revealed that ENO1 enhanced cell proliferation by accelerating G1 progression and upregulating G1 phase cyclin-dependent kinase 6 (CDK6), and improved cell survival by upregulating p38 in the MAPK cascade and increasing p-AKT in the AKT cascade, in particular in lung cancer cell lines. Overall, the results from the present study demonstrated that ENO1 may contribute to the development of lung cancers, but not esophageal cancers.

Single-cell landscape in mammary epithelium reveals bipotent-like cells associated with breast cancer risk and outcome

Adult stem-cells may serve as the cell-of-origin for cancer, yet their unbiased identification in single cell RNA sequencing data is challenging due to the high dropout rate. In the case of breast, the existence of a bipotent stem-like state is also controversial. Here we apply a marker-free algorithm to scRNA-Seq data from the human mammary epithelium, revealing a high-potency cell-state enriched for an independent mammary stem-cell expression module. We validate this stem-like state in independent scRNA-Seq data. Our algorithm further predicts that the stem-like state is bipotent, a prediction we are able to validate using FACS sorted bulk expression data. The bipotent stem-like state correlates with clinical outcome in basal breast cancer and is characterized by overexpression of YBX1 and ENO1, two modulators of basal breast cancer risk. This study illustrates the power of a marker-free computational framework to identify a novel bipotent stem-like state in the mammary epithelium.

Alpha-enolase regulates the malignant phenotype of pulmonary artery smooth muscle cells via the AMPK-Akt pathway

The molecular mechanisms underlying the metabolic shift toward increased glycolysis observed in pulmonary artery smooth muscle cells (PASMC) during the pathogenesis of pulmonary arterial hypertension (PAH) are not fully understood. Here we show that the glycolytic enzyme α-enolase (ENO1) regulates the metabolic reprogramming and malignant phenotype of PASMC. We show that ENO1 levels are elevated in patients with associated PAH and in animal models of hypoxic pulmonary hypertension (HPH). The silencing or inhibition of ENO1 decreases PASMC proliferation and de-differentiation, and induces PASMC apoptosis, whereas the overexpression of ENO1 promotes a synthetic, de- differentiated, and apoptotic-resistant phenotype via the AMPK-Akt pathway. The suppression of ENO1 prevents the hypoxia-induced metabolic shift from mitochondrial respiration to glycolysis in PASMC. Finally, we find that pharmacological inhibition of ENO1 reverses HPH in mice and rats, suggesting ENO1 as a regulator of pathogenic metabolic reprogramming in HPH.

α‐enolase promotes tumorigenesis and metastasis via regulating AMPK/mTOR pathway in colorectal cancer

The α-enolase (ENO1) plays pivotal roles in several types of cancer, but its clinical significance, functional role, and possible mechanism in colorectal cancer (CRC) have remained unclear. Expression level of ENO1 in CRC tissues was examined by qRT-PCR, Western blot, and immunohistochemistry. The effects of ENO1 on cell growth were investigated by MTT, colony formation, flow cytometry assays, and in vivo tumorigenic capacity analysis. The impacts of ENO1 on cell migration and invasion were also explored by scratch-healing, Transwell or Matrigel chamber assays, and in vivo metastatic capacity analysis. Our results showed that the expression level of ENO1 was significantly elevated in CRC tissues. High expression level of ENO1 was associated with disease progression in CRC patients. Overexpression of ENO1 in HCT116 cell line promoted cell proliferation, migration, and invasion in vitro as well as tumorigenesis and metastasis in vivo. In other hand, ablation of ENO1 in HCT116 cells led to totally reverse effects. Mechanistically, we revealed ENO1 could regulate AMPK/mTOR signaling pathway. AMPK pathway activation or mTOR pathway suppression blocked these ENO1 induced alterations. Together, our results demonstrated that ENO1 is a potent promoter of CRC genesis and metastasis at least in part though regulating AMPK/mTOR pathway. These findings also suggested that ENO1 may be a promising therapeutic target in CRC patients.

Proteomic analysis of docetaxel resistance in human nasopharyngeal carcinoma cells using the two-dimensional gel electrophoresis method.

Docetaxel-based chemotherapy has been recommended for advanced nasopharyngeal carcinoma (NPC). However, treatment failure often occurs because of acquired drug resistance. In this study, a docetaxel-resistant NPC cell line CNE-2R was established with increasing doses of docetaxel for more than 6 months. Two-dimensional gel electrophoresis and ESI-Q-TOF-MS were used to compare the differential expression of docetaxel-resistance-associated proteins between human NPC CNE-2 cells and docetaxel-resistant CNE-2R cells. As a result, 24 differentially expressed proteins were identified, including 11 proteins with increased expression and 13 proteins with decreased expression. These proteins function in diverse biological processes such as metabolism, signal transduction, calcium ion binding, immune response, proteolysis, and so on. Among these, α-enolase (ENO1), significantly upregulated in CNE-2R, was selected for detailed analysis. Inhibition of ENO1 by shRNA restored CNE-2R cells' sensitivity to docetaxel. Moreover, overexpression of ENO1 could facilitate the development of acquired resistance of docetaxel in CNE-2 cells. Western blot and reverse-transcription PCR data of clinical samples confirmed that α-enolase was upregulated in docetaxel-resistant human NPC tissues. Finding such proteins might improve interpretation of the molecular mechanisms leading to the acquisition of docetaxel chemoresistance.

RETRACTED: Proteomics-based identification of α-enolase as a potential prognostic marker in cholangiocarcinoma

To investigate the association of expression status of α-enolase (ENO1) and clinicopathological outcomes of CCA patients. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to compare differential expressed protein profiles of four human CCA cell lines and H69, a non-malignant biliary cell line, as a control. Immunohistochemical analysis was carried out in tissue-microarray of human CCA tissues (n=301). We identified ENO1 in all CCA cell lines but not H69 by proteomics based. About 75% of patients with CCA showed over-expression of ENO1 in hyperplastic bile duct and the tumors compared with that in tumor-adjacent normal tissue counterparts. Moreover, over-expression of ENO1 is significantly associated with poor prognosis and tumor invasion of CCA patients. ENO1 may serve as a prognostic marker to monitor the disease progression of these patients.

Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma

Backgroundα-Enolase (ENO1) is a key glycolytic enzyme implicated in the development of many human cancers including breast cancer. Increased expression of ENO1 has recently been reported in estrogen (ER)-positive human breast cancer patients. The present study examined the expression of ENO1 and assessed its significance in canine mammary carcinoma.ResultsImmunohistochemical staining was employed to investigate the expression of ENO1 in 82 cases of canine mammary tumor (32 benign tumors and 50 carcinomas). Quantification of immunohistochemistry was carried out using Quick score and the results showed cytoplasmic ENO1 overexpression in 9 of the 50 carcinomas (18%). Overexpression of ENO1 correlated significantly with shorter cause-specific survival (P = 0.019), but was not associated with ER positivity in canine mammary carcinoma.ConclusionsOur findings suggest that overexpression of ENO1 may be used as a prognostic marker for poor outcome in canine mammary carcinoma.

Differential proteomic profiling of chordomas and analysis of prognostic factors

The recurrence rate of chordoma is high, and the prognosis is poor. Differential proteomic analysis was performed on chordomas and adjacent normal tissues, with verification by Western blot. Protein expression was evaluated by immunohistochemistry of 37 chordomas. Association of candidate protein expression with clinical parameters, disease-free survival, and overall survival were analyzed. We identified 14 up-regulated and 5 down-regulated proteins in chordomas. Expression of alpha enolase (ENO1), pyruvate kinase M2 (PKM2), and gp96 was higher in recurrences than in primary tumors. Univariate analysis showed that significantly adverse factors for disease-free survival were overexpression of ENO1 and PKM2, involvement of contiguous vertebral levels, and inadequate surgical margin at initial surgery. Inadequate surgical margin without radiotherapy, involvement of contiguous vertebral levels, and cervical spine location were adverse factors for overall survival. By multivariate analysis, independent adverse prognostic factors were inadequate surgical margin and involvement of multiple contiguous vertebral levels for recurrence; upper cervical spine location and involvement of contiguous vertebral levels for tumor-related death. Multivariate analysis failed to show the significance of the proteins. Involvements of multiple contiguous vertebral levels and upper cervical spine, rather than overexpression of ENO1, PKM2, or gp96, are independent prognostic indicators for chordomas.