Role of ENO1 and its targeted therapy in tumors

Abstract

ENO1, also called 2-phospho-D-glycerate hydrolase in cellular glycolysis, is an enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate and plays an important role in the Warburg effect. In various tumors, ENO1 overexpression correlates with poor prognosis. ENO1 is a multifunctional oncoprotein that, when located on the cell surface, acts as a “moonlighting protein” to promote tumor invasion and metastasis. When located intracellularly, ENO1 facilitates glycolysis to dysregulate cellular energy and sustain tumor proliferation. Additionally, it promotes tumor progression by activating oncogenic signaling pathways. ENO1 is a tumor biomarker and represents a promising target for tumor therapy. This review summarizes recent advances from 2020 to 2024 in understanding the relationship between ENO1 and tumors and explores the latest targeted therapeutic strategies involving ENO1.

Introduction

In normoxic conditions, glucose is fully metabolized into carbon dioxide and water by the tricarboxylic acid cycle and oxidative phosphorylation, creating large amounts of ATP. However, even under well-oxygenated conditions, tumor cells generate energy by aerobic glycolysis rather than oxidative phosphorylation, a phenomenon known as the Warburg effect [1]. Several studies have investigated the role of glycolysis-related genes in cancer development and progression, which has led to the development of promising targeted drugs. These genes include enolase (ENO), M2-type pyruvate kinase (PKM2), glucose transporters (GLUTs), and lactate dehydrogenase A (LDHA).

As one of the important enzymes catalyzing glycolysis, the primary function of enolase is to convert 2-phosphoglycerate (2-PG) into phosphoenolpyruvate (PEP) [2]. The three highly conserved isoforms of enolase in mammals are α-enolase (ENO1), β-enolase (ENO3), and γ-enolase (ENO2). Among them, ENO1 accounts for 90% of the glycolytic pathway’s cytosolic enolase activity [3]. Furthermore, ENO1 overexpression is associated with poor prognosis in many types of cancer [4]. ENO2, predominantly localized in the cytoplasm of neurons and neuroendocrine cells, has been identified as a serum marker for tumors of neuronal lineage, such as glioblastoma (GBM) and medulloblastoma [5, 6]. Recent studies, however, have revealed that ENO2 is not only highly expressed in neurological tumors but also plays a critical role in the progression of other malignancies, including lung adenocarcinoma (LUAD) and breast cancer. A multiomic analysis demonstrated that 3D tumor spheroid growth implies the overexpression of ENO2 and another glycolytic enzyme, ALDOC, concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate, in lung and breast cancer cell lines in different nutrient environmental conditions [7]. ENO3 is predominantly expressed in muscle tissue, with studies indicating that 86% of rhabdomyosarcomas test positive for ENO3 [8]. Furthermore, recent research has shown that ENO3 expression is upregulated in colorectal cancer (CRC), where it promotes cell proliferation and migration by facilitating glycolysis [9]. Conversely, in hepatocellular carcinoma (HCC), ENO3 inhibits the proliferation, migration, invasion, tumor growth, and lung metastasis of HCC cells by suppressing the epithelial-mesenchymal transition (EMT) process and the Wnt/β-catenin pathway [10].

Apart from its function as a glycolytic enzyme, ENO1 has multiple biological functions as a moonlighting protein, depending on its localization. In the cytoplasm, ENO1 catalyzes glycolysis and promotes a variety of pro-tumorigenic effects, including tumorigenesis, cell proliferation, invasion, migration, and chemoresistance [11,12,13,14]. ENO1 also regulates multiple intracellular pro-tumor signaling pathways. By acting as a plasminogen receptor on the cell surface, it enhances migration and invasion of tumor cells by promoting extracellular matrix (ECM) degradation [15]. ENO1 can also be secreted extracellularly and interact with other immune cells, such as tumor-associated macrophages, to promote tumor progression [16].

Given the aerobic glycolytic properties of tumor cells and the functional alterations of ENO1, treatments targeting ENO1 may be effective against cancer. Therefore, exploring the relationship between ENO1 function and tumor progression in cancer therapy is crucial. This article summarizes the functions and roles of ENO1 in cancer progression from 2020 to 2024, as well as reported ENO1-targeted drugs. This paper aims to provide insights and aid in targeting ENO1 in cancer therapy.

Clinical relevance of ENO1

ENO1 is overexpressed in various cancers and is associated with malignant progression of tumors, poor prognosis, pathological stage of tumors, immune cell infiltration, and tumor purity [4]. In GBM, elevated ENO1 expression is associated with shorter patient survival and higher glioma tumor grade, suggesting a poor prognosis [17, 18]. ENO1 is also associated with low T-cell, B-cell, and natural killer cell infiltration, high immunosuppressive cell infiltration, and promotes microglia M2 polarization and malignant progression of GBM [19]. In CRC, elevated ENO1 expression predicts poor prognosis and correlates with CRC stage; high ENO1 levels predict poor survival and serve as a biomarker for radiation response [13, 20]. In HCC, high ENO1 expression predicts poor prognosis, shorter survival [21], and correlates with tumor differentiation grade and tumor lymph node metastasis (TNM) stage [22]. As one of the hypoxia prognostic model genes, a high hypoxia score indicates a poor prognosis in HCC [23]. In bladder cancer, aberrant ENO1 overexpression correlates with clinicopathological features, poorer survival, poor prognosis, and has independent prognostic value in tumor progression [24,25,26,27]. A high expression of ENO1 in pancreatic cancer is associated with a shorter survival time [28]. Pancreatic ductal adenocarcinoma with high ENO1 expression has a poor prognosis; a high expression of ENO1 is strongly correlated with histological differentiation and tumor invasion; high ENO1 autoantibodies (aAb) in patients predict poor prognosis and are associated with reduced survival [29,30,31,32]. Moreover, to construct a prognostic model, ENO1 was selected, and the high-risk group had a worse prognosis [33]. In melanoma, ENO1 overexpression in melanoma cells correlates with unfavorable prognostic factors, such as Clark levels, mitotic activity, Breslow thickness, and ulcers. It also correlates with increased tumor infiltration thickness and poorer long-term prognosis in patients with skin melanoma [34]. In breast cancer, elevated ENO1 expression predicts poorer recurrence-free survival and overall survival, particularly in the aggressive triple-negative breast cancer (TNBC). ENO1 can also serve as a prognostic marker for breast cancer radiotherapy efficacy [35,36,37]. However, ENO1 is positively correlated with various anti-tumor infiltrating immune cells, such as NK cells, M1 macrophages, helper T cells, CD8 ⁺ T cells and B cells in patients with stage I/II breast cancer. In early-stage breast cancer, higher ENO1 expression correlates with a better prognosis [38]. ENO1 was highly expressed in malignant lobular tumors of the breast and correlated with hypoxia [39]. In lung cancer, ENO1 in sputum can be used as a biomarker for early detection of lung cancer [40]. Moreover, ENO1 is strongly associated with poor prognosis in LUAD and sarcoma [41,42,43]. In osteosarcoma, a biomarker for immunodiagnosis and osteosarcoma progression can be determined by ENO1, which triggers an autoimmune response [44]. ENO1 autoantibody levels in patient sera are higher than in healthy individuals; predictive modeling of autoantibodies can help in the early detection of osteosarcoma, serving as a promising and powerful tool in clinical practice [45]. Analysis of gene expression profiling databases of multiple myeloma patients revealed a negative correlation between overall survival andENO1 expression [46]. In esophageal squamous cell carcinoma (ESCC), ENO1 is abnormally elevated in all cancer cytoplasm, significantly increasing with tumor stage progression and transition from Barrett’s esophagus to esophageal adenocarcinoma [47], and the expression level of ENO1 is positively correlated with the clinicopathological TNM stage [48]. High expression of ENO1 in prostate cancer is significantly associated with shorter patient survival [49]. ENO1 is upregulated in the hair of female cervical cancer patients, according to metabolomic analysis [50]. In acute myeloid leukemia, high ENO1 expression predicts poor overall survival [51]  (Table 1).

Role of ENO1 in tumorigenesis and progression

Fig. 1. ENO1 exerts significant pro-tumor effects in a variety of tumor types, including (1)promoting tumorigenesis [52], (2)enhancing tumor cell proliferation [53], (3)inducing EMT, promoting invasion, migration and metastasis [14, 54], (4)promoting chemoresistance [55], (5)stimulating angiogenesis [56], (6)shaping the immune microenvironment [38], (7)augmenting cancer stem cells stemness [57, 58], (8)inducing ferroptosis [59], (9)promoting resistance to radiotherapy [35], and (10)modulating cellular autophagy [30].

Role of ENO1 in tumorigenesis

ENO1 can promote tumorigenesis by activating cellular signaling pathways and increasing cellular glycolysis. In breast cancer, ENO1 promotes glucose uptake through the PI3K/AKT pathway, thereby enhancing tumorigenesis [52]. In CRC, upregulation of ENO1 enhances cellular aerobic glycolysis, which subsequently promotes tumorigenesis [11]. In LUAD, ENO1 mRNA is methylated by m⁶A at A359, enhancing its translation. The m⁶A -dependent increase in ENO1 promotes glycolysis and tumorigenesis in LUAD [60]. In pancreatic cancer, ENO1 knockdown reduces cellular glycolysis and activates oxidative phosphorylation and lipid metabolism pathways by altering the expression of UAP1, ALDOC, G6PD, and other related metabolic genes, thereby suppressing tumorigenesis [61]. In HCC, ENO1 binds to yes-associated protein 1 (YAP1) mRNA, promoting its translation, which activates arachidonic acid metabolism and prostaglandin E2 accumulation. A significant increase in ENO1/YAP1 activation in clinical samples of hepatocellular carcinoma suggests that ENO1 may play a role in cancer development via its regulation of arachidonic acid metabolism [62] (Fig. 1).

Role of ENO1 in tumor growth, anti-apoptosis, and tumor cell proliferation

Numerous studies have demonstrated that ENO1 enhances glycolysis and proliferation, while suppressing apoptosis in various tumors. ENOblock, a specific inhibitor of ENO1, significantly decreased F. nucleatum-induced cell proliferation in CRC. In a CRC xenograft mouse model, ENOblock treatment significantly diminished the tumor growth of F. nucleatum-cultured tumor cells [53]. Furthermore, knockdown of ENO1 hindered the proliferative capacity of CRC cells [14]. Another study revealed that decreased levels of ubiquitinated degradation of ENO1 promoted ERK phosphorylation and glycolytic activity in CRC cells. Inhibition of ENO1 reduces cellular proliferation [12]. Post-translational modifications of ENO1 are crucial in fundamental biological processes and are closely associated with tumor progression. Lysine crotonylation at K420 on ENO1 promotes CRC cell growth by enhancing ENO1 activity and regulating the expression of tumor-related genes [63]. In pancreatic cancer, the knockdown of ENO1 inhibited pancreatic cancer cell proliferation and colon-forming ability, and ENO1-KO significantly reduced tumor growth in a mouse model [28, 64]. Hyperlipidemia enhances tumor growth and increases the expression level of ENO1 in a subcutaneous xenograft mouse model [65]. ENO1 knockdown hinders the proliferative capacity of pancreatic ductal adenocarcinoma cells [61], and hypoxia induces elevated ENO1 expression, which enhances ERK phosphorylation and suppresses apoptosis, leading to cell survival [31]. In gastric cancer, ubiquitination and degradation of ENO1 diminished AKT1 binding and subsequently inactivated AKT1, resulting in cell proliferation [66]. In gastric cancer, ENO1 binds to the mRNAs of vascular endothelial growth factor A (VEGFA), myeloid cell leukemia-1 (MCL1), SRY-box transcription factor 9 (SOX9) and G-protein-coupled receptor class C group 5 member A (GPRC5A), thereby stabilizing their expression and promoting gastric cancer cell growth. Additionally, ENO1 interacts with several small-molecule kinases (e.g., CD44, PKM2, NEAT1, and LINC00511) or other long-chain non-coding RNAs (lncRNAs) to regulate their expression and affect cell apoptosis and proliferation [67]. Elevated ENO1 activity regulates the PI3K/AKT pathway and promotes lung cancer cell proliferation [68, 69]. Elevated ENO1 activity regulates the flux of the oxidized pentose phosphate pathway and the glycolytic pathway in human lung cancer, thereby promoting tumor growth [70]. In LUAD, regulation of the expression of the glycolysis-related gene ENO1 suppresses tumor activity [71], and inhibition of ENO1 expression suppresses the proliferation of LUAD cells [72], and down-regulation of ENO1 expression reduces the activation of AKT1 phosphorylation, which subsequently inactivates cell cycle signaling and inhibits cell proliferation [73]. In non-small cell lung cancer (NSCLC), decreased levels of ENO1 phosphorylation lead to reduced phosphorylation of PI3K and AKT, thereby further inhibiting cell proliferation and tumor progression [74]. In breast cancer, promoting m⁶A methylation of ENO1 RNA can influence the glycolytic activity of breast cancer cells [75], and ENO1 contributes to breast cancer growth by promoting efficient glycolysis [76]. In TNBC, targeted depletion of ENO1 prevents TNBC cell colony formation, proliferation, and organoid tumor growth, and increases cell death [36]. ENO1 involvement in TNBC cell apoptosis and proliferation has been observed [37, 77]. In prostate cancer, targeting cell surface ENO1 reduces the growth of subcutaneous xenografts [56]. Increased transcription of ENO1 promotes glycolysis in castration-resistant prostate cancer, subsequently promoting tumor growth [78]. In skin melanoma, activation of AKT-mTOR signaling promotes the expression of ENO1, thereby enhancing the growth and proliferation of melanoma cells [79]. Overexpression of ENO1 promotes proliferation of skin melanoma cells [80]. In clear cell renal cell carcinoma, upregulation of ENO1 expression promotes tumor cell proliferation and xenograft growth [81]. ENO1 upregulates levels of tumor cell glycolysis in leukemia, promoting cell proliferation and enhancing colony formation of hematopoietic stem and progenitor cells [82]. In ovarian cancer, stabilization of ENO1 mRNA in an m⁶A -dependent manner by ENO1 promotes tumor growth and glycolysis [83], and the activation of ENO1 dimers increases glycolytic flux, accelerating tumor growth [84]. Activation of ENO1 promotes proliferation of hepatocellular carcinoma [85]. In HCC, exosomal ENO1 promotes HCC cell growth by upregulating integrin α6β4 expression and activating the FAK/Src-p38MAPK pathway [22]. In hepatopancreatobiliary cancer patient-derived organoids, ENO1 promotes their growth through the AKT/PI3K pathway [86]. In cholangiocarcinoma, ENO1 promotes aerobic glycolysis for tumor proliferation [87]. In bladder cancer, analysis of epithelial cells suggests ENO1’s involvement in promoting bladder cancer progression through metabolism, cell cycle, and some pro-tumor pathways [27]. In cervical cancer, increased transcription of ENO1 induces tumor cell glycolysis and promotes cancer progression [88]. In endometrial cancer, upregulation of ENO1 expression promotes cell proliferation [89]. The RNA deconjugating enzyme DHX33 promotes cancer cell proliferation by regulating several key genes involved in the Warburg effect such as ENO1 [90]. In head and neck squamous carcinoma (HNSCC), ENO1 regulates glycolysis, which is involved in the proliferation of HNSCC cells [91], and inhibition of ENO1 degradation promotes glycolysis and reduces ROS production in tumor cells [92]. In oral squamous cell carcinoma (OSCC), ubiquitination and degradation of ENO1 inhibit OSCC cell cycle transition, proliferation, and glycolysis, thereby triggering tumor suppressor functions [93]. Moreover, in OSCC, ENO1 levels are higher than those in other oral malignant diseases such as oral lichen planus [94].

Role of ENO1 in tumor cell invasion, migration and metastasis

EMT is an important biological mechanism in primary tumor metastasis. During EMT, epithelial cells undergo a transformation characterized by the downregulation of epithelial markers (such as E-cadherin) and the upregulation of mesenchymal markers (such as N-cadherin and Vimentin). This transformation can enhance tumor metastasis by promoting migration and invasion [95, 96]. In CRC, upregulation of ENO1 expression promotes CRC cells invasion, while knockdown of ENO1 inhibits the cellular EMT process and the migration ability of CRC cells [14, 54]. Additionally, blocking ENO1 partially inhibits invasion and metastasis of CRC [13]. Moreover, lysine crotonylation at the ENO1 K420 site enhances ENO1 activity, thereby promoting migration and invasion of CRC cells [63]. Reduced ubiquitination-mediated degradation of ENO1 promotes ERK phosphorylation and glycolytic activity in CRC cells. ENO1 inhibition reduces cell migration [12]. Lung cancer cell migration is promoted by ENO1 through the regulation of the PI3K/AKT pathway [69]. ENO1 promotes lung cancer cells EMT process and contributes to lung tumor metastasis in a tail vein injection model [68]. AL355338 regulates the interaction between ENO1 and EGFR, thereby activating EGFR-AKT signaling and modulating invasiveness in NSCLC [97]. Breast cancer metastasis is fueled by ENO1 through the enhancement of cellular glycolysis [76]. In TNBC, elevated ENO1 expression activates glycolysis in TNBC cells, thereby promoting TNBC cell migration, invasion, and EMT [98]. ENO1 is instrumental in activating the EMT pathway in various tumor cells [37]. Additionally, ENO1 interacts with several small molecule kinases (e.g., LINC00511, PKM2, NEAT1, and CD44) or other lncRNAs, thereby regulating their expression and influencing cell migration [67]. In melanoma, overexpression of ENO1 promotes melanoma cell invasiveness and correlates with aggressive clinical behavior. Furthermore, it induces tumor cell invasion, migration, and elevated levels of MMP-9 and MMP-13 [34, 80]. ENO1 promotes HCC metastasis [85], while degradation of ENO1 inhibits metastasis by promoting glycolytic reprogramming via the AKT/mTOR pathway [99]. Exosomal ENO1 promotes HCC metastasis through activation of the FAK/Src-p38MAPK pathway and the upregulation of integrin α6β4 expression [22]. Knockdown of ENO1 in pancreatic cancer cells inhibits migration, invasion, and EMT [64]. Knockdown of ENO1 inhibits cell invasion and migration in pancreatic ductal adenocarcinoma cells [61]. Hypoxia increases ENO1 expression in pancreatic ductal adenocarcinoma, thereby promoting tumor cell migration and invasion [31]. Upregulation of ENO1 expression promotes clear cell renal cell carcinoma cells migration [81]. In multiple myeloma, extracellular ENO1 enhances tumor cell glycolytic activity, the expression of glycolysis-related genes, pro-carcinogenic activity, cell migration, cell viability, and secretion of VEGF and TGF-β via HIF-1α [100]. In ovarian cancer, ENO1 stabilizes its mRNA in an m⁶A manner to promote tumor metastasis [83]. ENO1 promotes migration and invasion of endometrial cancer cells [89]. In uveal melanoma with BAP1 inactivating mutation, inhibition of hypoxia- and ECM-related pathways, including the ENO1 gene, in vivo, suppresses cancer liver metastasis in BAP1 mutants [101]. ENO1 promotes bladder cancer cell invasion [24]. Furthermore, a study of epithelial cells revealed that ENO1 may promote bladder cancer progression through cell cycle, metabolism, and other oncogenic pathways [27]. In ESCC, ENO1 promotes tumor EMT, migration, invasion, and metastasis [102]. ENO1 secreted by OSCC cells promotes macrophage IL-6 secretion via toll-like receptor 4 (TLR4). In turn, secreted IL-6 inversely promotes OSCC invasion, migration, and EMT [16]. ENO1 regulates glycolysis, which is involved in HNSCC cell migration and invasion [91].

Role of ENO1 in chemoresistance

Chemotherapy is widely employed in cancer treatment; however, the resistance of tumor cells to chemotherapeutic agents markedly diminishes the clinical efficacy of this approach. Several studies have indicated that overexpression of ENO1 can contribute to antitumor resistance by accelerating glycolytic energy metabolism or by activating drug-resistant cell pathways (Fig. 2).

ENO1 expression was significantly elevated in five types of 5-FU-resistant CRC cells. Transfection of HCT-116/5-FU and SW620/5-FU cells with ENO1-shRNA resulted in decreased viability of 5-FU-resistant tumor cells, reduced proliferative and colony-forming abilities, and diminished migration ability and EMT levels. The impact of ENO1 on EMT progression was inhibited by the glycolysis inhibitor 2-DG, suggesting that ENO1’s regulation of tumor proliferation, migration, and drug resistance may be linked to energy metabolism [14]. In TNBC, ENO1 knockdown by siRNA or 2-DG treatment in ANRIL-overexpressing TNBC cells demonstrated that ENO1 siRNA or 2-DG reversed adriamycin/doxorubicin (ADR) resistance in lncRNA ANRIL upregulated TNBC cells, indicating that ENO1 enhances ADR resistance by promoting glucose metabolism [104]. In liver cancer, scRNA-seq analysis identified ENO1 among five key energy metabolism-related genes in hepatocellular carcinoma. Drug sensitivity analyses showed that patients with lower scores in these genes were more responsive to metabolism-related chemotherapeutic agents and immune checkpoint inhibitors, such as methotrexate, metformin, and AICAR [105]. The methylation of the ENO1 R9/372 locus increased activity, and heightened ENO1 activity modulates the flux of the pentose phosphate pathway (PPP) and glycolysis in human lung cancer, contributing to cisplatin resistance [70]. This underscores the significant potential of ENO1 in regulating glucose energy metabolism in tumor cells. However, the mechanism by which ENO1-mediated enhancement of glycolysis promotes chemoresistance in tumor cells requires further investigation.

ENO1 not only promotes tumor drug resistance by regulating tumor cell metabolism but also activates associated cell signaling pathways to confer chemotherapy resistance. In GBM, temozolomide (TMZ)-resistant tumor cells secrete exosomes containing lnc-TALC. Lnc-TALC in these exosomes binds to ENO1 in microglia, which subsequently activates the p38 MAPK signaling pathway. p38 MAPK then interacts with the transcription factor MEF2C, enhancing its transcriptional activity and promoting C5a transcription. Elevated C5a in microglia is released into the tumor microenvironment and binds to C5aR on TMZ-sensitive tumor cells, facilitating TMZ-induced DNA damage repair and resulting in chemoresistance [55]. ENO1 is overexpressed in pancreatic cancer cells, and ENO1 knockdown enhances the cytotoxicity of gemcitabine while reducing YAP1 expression, a key downstream effector of the Hippo pathway. YAP1 promotes autophagy, thereby protecting pancreatic cancer cells from gemcitabine-induced apoptosis. This suggests that ENO1 overexpression increases gemcitabine resistance by upregulating YAP1, which promotes tumor growth and progression [30]. In OSCC, ENO1 activates the AKT/GSK3β axis within the Wnt signaling pathway, leading to increased β-catenin expression. This β-catenin binds to the transcription factors TCF/LEF in the nucleus and promotes the expression of CD44, C-Myc, and CCND1, which in turn enhances cisplatin resistance in OSCC cells [107].

Given the role of ENO1 in tumor chemotherapy resistance, targeting ENO1 represents a promising strategy to counteract chemotherapy resistance that develops during tumor progression. For instance, ENOblock, a well-known ENO1 inhibitor, has been shown to inhibit glycolysis and stemness in gastric cancer cells, thereby increasing their sensitivity to cisplatin [58]. In CRC, ENOblock significantly enhanced the tumor suppression effects of oxaliplatin and 5-FU in CRC cells inoculated with Fusobacterium nucleatum. Animal studies demonstrated that ENOblock treatment markedly improved tumor suppression efficacy in hormonally induced mice treated with oxaliplatin [53]. In small cell lung cancer (SCLC), ENO1 is highly expressed in chemoresistant cells and regulates chemoresistance through activation of the PI3K/AKT pathway. Treatment with ENOblock effectively reversed this ENO1-mediated chem22oresistance, highlighting its potential as a valuable therapeutic agent for ENO1-mediated chemoresistance and tumor treatment [106].

Fig. 2. ENO1 promotes chemoresistance in tumors by promoting glycolysis [14] and activating the PI3K/AKT [106], YAP1 [30], and Wnt/β-catenin signaling pathways [107]. ENOblock, an inhibitor of ENO1, blocks this chemoresistant effect by inhibiting ENO1 [58, 106].

Role of ENO1 in angiogenesis

Tumor angiogenesis provides essential oxygen and nutrients to rapidly proliferating tumor cells and is a hallmark of tumor progression.

In prostate cancer, targeting ENO1 with ENO1 mAb HuL227 reduced angiogenesis in a PC-3 subcutaneous xenograft model. In vitro, ENO1 mAb HuL227 inhibited VEGF-A-mediated angiogenesis in HUVEC cells, and this inhibition was ENO1-dependent and possibly related to fibrinolysis. However, the mechanism through which ENO1 upregulates VEGF-A and promotes angiogenesis requires further investigation [56].

ENO1 also plays an important role in angiogenesis in other diseases beyond cancer. ENO1 expression was elevated in hypoxic human pulmonary artery endothelial cells and in lung tissues of chronic obstructive pulmonary disease-associated pulmonary hypertension and hypoxic pulmonary hypertension mouse models. Elevated ENO1 promotes the angiogenesis by activating the PI3K/AKT/mTOR signaling pathway and inducing mitochondrial dysfunction [108]. ENO1-driven glycolysis promotes pathologic angiogenesis in small bowel vascular malformation [109]. In non-tumor diseases, ENO1 can promote angiogenesis by activating the PI3K/AKT/mTOR signaling pathway or by promoting glycolysis, raising the thought of whether ENO1 utilizes the similar mechanism to promote angiogenesis in the tumor environment.

Role of ENO1 in the tumor immune microenvironment

ENO1 correlates with the infiltration of several immune cells in tumor microenvironment and can act on immune cells to promote several tumor progression [24]. ENO1 was associated with depletion of CD8⁺T cells through large amounts of RNA-seq and scRNA-seq [27]. In prostate cancer, targeting cell surface ENO1 reduces monocyte recruitment in subcutaneous xenografts [56]. In glioblastoma, ENO1 is associated with low levels of NK cells, T cells and B cells, and in addition, ENO1 promotes M2 polarization of microglia [19]. ENO1 positively correlates with CD4⁺ T cell infiltration in colon adenocarcinoma [110]. OSCC cells produce IL-8 in response to ENO1, which activates STAT3 signaling and inhibits Jurkat T cell proliferation [111]. OSCC-secreted ENO1 promotes macrophage IL-6 secretion via TLR4 [16]. In pancreatic cancer, knockdown of ENO1 in the mouse pancreatic cancer cell line PAN02 by CRISPR/CAS9 reduced the number of Treg cells in the tumor microenvironment and up-regulated the expression of STAT1, TNF-α, and granzyme B, and down-regulated the expression of typical anti-inflammatory and pro-apoptotic cytokines, including IL-10, ARG-1, TGF-β, IL-13, and BCL-2 [28]. In breast cancer, ENO1 expression is high and positively correlates with a variety of anti-tumor infiltrating immune cells in stage I/II patients, such as M1 macrophages, NK-cells, B-cells, helper T-cells, and CD8⁺T-cells [38].

Role of ENO1 in promoting stemness in cancer stem cells

ENO1 has been shown to be positively associated with cancer stem cell (CSC) stemness and plays a key role in tumor invasion, migration, and chemoresistance, as indicated by recent studies. Expression of ENO1 was upregulated in a subset of stem cell-like glioblastomas. mRNA expression of ENO1 positively correlated with stemness markers, such as CD133/PROM1 and SOX2, in tumors of GBM patients [112]. In gastric cancer, ENO1 enhances the stemness of stem cells by increasing cellular glycolytic activity, which includes self-renewal ability, cell migration, invasion, chemotherapy resistance, and even tumorigenicity of gastric cancer cells. ENO1 is an important biomarker associated with the stemness of gastric cancer cells [57, 58]. In lung cancer stem cells, ENO1 activates AMPK/mTOR signaling, which increases self-renewal, growth, and invasion [113]. ENO1 is specifically expressed on the invasive surface of highly invasive and pro-metastatic cancer stem cell subpopulations in gastric and prostate cancers. Inhibition of ENO1 surface localization impairs invasive pseudopod biogenesis, protein hydrolysis, and CSC invasiveness [114].

Role of ENO1 in cellular ferroptosis

Cell death induced by excessive lipid peroxidation, known as ferroptosis, is associated with the development of various tumor types and their response to therapy. Impaired ferroptosis triggers inflammation-related immunosuppression in the tumor microenvironment, thus favoring tumor growth [115]. In HCC, CNOT6 is recruited by ENO1 to accelerate the mRNA decay of iron-regulated protein 1 by binding to RNA. This process suppresses mitochondrial ferritin 1 expression and subsequently inhibits mitochondrial iron-induced ferroptosis [59]. The silencing of ENO1 inhibits glycolysis and proliferation by inactivating AKT/STAT3 pathway, while promoting ferroptosis in CRC cells [116].

Role of ENO1 in radiosensitivity

Cancer treatment primarily involves surgery, radiotherapy, chemotherapy, and the rapidly evolving immunotherapy [117]. Although surgery is the preferred approach, radiotherapy continues to hold a relevant and significant role in cancer treatment. For example, many patients with advanced rectal or sigmoid colon cancer require both radiotherapy and initial surgical treatment [118].

Recently, a growing number of studies have demonstrated that ENO1 contributes to radiation resistance. A proteomic analysis suggested that metabolic proteins regulating glycolysis, including ENO1, could serve as biomarkers of CRC radioresponsiveness [20]. TCGA gene analysis showed that ENO1 expression was significantly upregulated in CRC tissues [20]. In breast cancer, RNA-seq analysis of the BC cell line MDA-MB-231 (ionizing radiation-sensitive, IR-S) and its IR-R counterpart, performed using the GEO database, revealed that ENO1 was the most highly expressed gene in IR-R BC cells [35]. The high expression of ENO1 in radiotherapy-resistant IR-R cells was verified at the gene and protein levels, respectively. This suggests that ENO1 can be used as a prognostic marker for the efficacy of radiotherapy in breast cancer. ENO1 reduces reactive oxygen species (ROS) production and apoptosis in vitro and in vivo by regulating mitochondrial homeostasis, which enhances therapeutic resistance to radiotherapy in breast cancer [35]. However, further research is required to elucidate the specific mechanism by which ENO1 drives tumor radiotherapy resistance.

Role of ENO1 in cellular autophagy

During autophagy, damaged organelles, pathogens, and aggregated proteins are translocated to the lysosome for degradation, maintaining cellular homeostasis [119]. Autophagy can keep tumor cells alive under stress during the tumor development stage and seems to play a significant role in tumor cell metastasis [120]. Silencing of ENO1 inhibits pancreatic cancer cells autophagy [30]. Inhibition of glycolysis, which includes ENO1, leads to autophagy induction in a subset of stem cell-like GBM tumors [112].

Role of ENO1 in anti-tumor immunity

However, ENO1 is not always a promoter of tumor progression, and recent reports suggest that ENO1 has an important role in exerting anti-tumor immunity. Condition medium (CM) of such tumor cells (breast, prostate, and pancreatic) was collected, and genome-wide proteomic analysis was performed. An analysis of the proteomic profile of CM revealed that it enriched with ENO1 and had significant tumor suppressor effects [121, 122]. ENO1 inhibits tumor progression through cell adhesion receptors and interacts with the CSC marker CD44 [121,122,123].

According to Di Ge et al., ENO1 also plays a crucial role in antitumor immunity. By promoting ubiquitination and subsequent proteasomal degradation of programmed cell death ligand 1 (PD-L1) in lung cancer cells, ENO1 destabilizes and reduces PD-L1 expression. Moreover, ENO1 overexpression enhances antitumor immunity mediated by T-cells by sensitizing tumor cells to specific T-cell killing [124]. However, given the antitumor effects of ENO1, more studies are needed to clarify the specific antitumor mechanisms.

Table 1 Summary of tumor clinical correlation and biological function of ENO1

Specific mechanisms

Fig. 3. ENO1 is stimulated by extracellular signals or binds to intracellular proteins and activates relevant oncogenic pathways in tumor cells, such as the PI3K/AKT signaling pathway [64], Wnt/β-catenin signaling pathway [107, 125], MAPK signaling pathway [12, 22, 31], AMPK signaling pathway [113], and NF-κB signaling pathway [13]. These pathways promote tumor cell proliferation, anti-apoptosis, invasion and metastasis, chemoresistance, and self-renewal, growth and invasion of cancer stem cells.

ENO1 and PI3K/AKT/mTOR signaling pathway

The PI3K/AKT/mTOR signaling pathway is a highly conserved network in eukaryotic cells promoting cell survival, cycle progression and growth. The PI3K/AKT signaling pathway primarily drives glycolysis in cancer cells. ENO1 acts as a key activator of the PI3K/AKT signaling pathway. ENO1 promotes aerobic glycolysis, tumorigenesis, proliferation, invasion, migration, metastasis, and chemoresistance of tumor cells through the activation of the PI3K/AKT/mTOR pathway. In Xie et al.‘s study, ENO1 promotes breast tumorigenesis through the activation of PI3K/AKT [52]. In hepatocellular carcinoma, circRPN2 binds to ENO1, accelerating its degradation, and inhibits HCC metastasis by reducing aerobic glycolysis via the AKT/mTOR pathway [99]. In pancreatic cancer, the knockdown of ENO1 inhibits the invasion, migration, and proliferation of pancreatic cancer cells via the PI3K/AKT pathway [64]. In gastric cancer, CCDC65 binds to ENO1, contributing to tumor pathogenesis through its structural domain (a.a. 130–484), and further promoting the ubiquitination of ENO1 through the recruitment of the E3 ubiquitin ligase FBXW7. And then ENO1 reduces its binding to and further inactivates AKT1, leading to a reduction in cell proliferation and the loss of EMT signaling [66]. Lactate promotes growth via the HIF1α/ENO1/PI3K/AKT pathway in patient-derived organoids of hepatopancreatobiliary carcinoma [86]. In lung cancer, B7-H3 (CD276) interacts directly with ENO1 in lung cancer cells and regulates the PI3K/AKT pathway through ENO1, thereby promoting the proliferation and migration of lung cancer cells [69]. In SCLC, fibroblast growth factor receptor-like 1 regulates chemotherapy resistance by interacting with ENO1 and modulating its downstream PI3K/AKT signaling pathway [106]. In NSCLC, reduced ENO1 phosphorylation results in reduced PI3K and AKT phosphorylation, which is associated with a decrease in cell proliferation and tumor progression [74] (Fig. 3).

ENO1 not only positively regulates the PI3K/AKT/mTOR signaling pathway, in turn the PI3K pathway, a key activator of the Warburg effect in tumor cells, also upregulates the expression of various key glycolytic genes, including ENO1, thereby mediating tumor progression. The PI3K signaling pathway affects the expression of glycolytic genes, including ENO1, in glioblastoma [17]. In clear cell renal cell carcinoma, the knockdown of PB1 activates AKT-mTOR signaling and increases the expression of key glycolytic enzymes, including ENO1, at both the gene and protein levels [81].

ENO1 and Wnt/β-catenin signaling pathway

Wnt signaling regulates essential developmental gene expression programs by increasing nuclear and cytoplasmic β-catenin. Abnormal activation of the Wnt/β-catenin signaling pathway promotes self-renewal of cancer stem cells, cell proliferation, and other oncogenic processes, thereby promoting tumor progression [126]. In OSCC, ENO1 drives cisplatin resistance through the AKT/GSK3β axis modulating Wnt signaling [107]. Moreover, metformin and anti-ENO1 antibody may alleviate CSC resistance by blocking the Wnt/β-catenin pathway [125].

ENO1 and MAPK signaling pathway

The mitogen-activated protein kinase (MAPK) pathway transports extracellular signals from membranes to intracellular targets. It is implicated in various tumor biological functions, including tumor invasion, metastasis, cell proliferation, apoptosis, cell cycle progression, cellular metabolism, and ultimately cell death or survival [127]. In GBM, temozolomide-associated lnc-RNA binds to ENO1, leading to the phosphorylation of p38 MAPK in microglia. This promotes the release of C5a into the tumor microenvironment, enhancing the chemoresistance of GBM to TMZ [55]. In hepatocellular carcinoma, exosomal ENO1 increases the proliferation and metastasis of HCC cells by stimulating the production of integrin α6β4 and turning on the FAK/Src-p38MAPK pathway [22].

The RAF-MEK-ERK signaling cascade is a well-characterized MAPK pathway that participates in cell proliferation and survival [128]. In pancreatic ductal adenocarcinoma, hypoxia induces an increase in ENO1 expression, which in turn promotes ERK phosphorylation and inhibits tumor cell apoptosis, resulting in cell survival and invasion [31]. CD47 interacts with ENO1, and then promotes ERK phosphorylation and glycolytic activity in CRC cells, thus enhancing tumor cell growth and migration [12].

ENO1 and AMPK signaling pathway

AMP-activated protein kinase (AMPK) maintains energetic homeostasis by coordinating various cellular processes such as adipogenesis, glycolysis, the TCA cycle, cell cycle progression, and mitochondrial dynamics [129]. ENO1 enhances lung cancer stem cells self-renewal, growth, and invasion via the AMPK/mTOR pathway [113].

ENO1 and NF-κB signaling pathway

Activation of NF-κB may promote tumor cell proliferation, survival, angiogenesis, and invasion during tumorigenesis and progression [130]. In CRC, ENO1 binds to fibroblast activation protein α (FAP) and activates the ENO1-dependent NF-κB signaling pathway. FAP-mediated pro-tumor invasive and metastatic effects are partly mitigated by blocking ENO1 [13].

ENO1-targeted tumor therapy

Considering the significant role of ENO1 in tumors, therapeutic strategies targeting ENO1 have gained widespread attention. In recent years, ENO1 inhibitors and other targeted therapies, including specific antibody and DNA vaccine, have demonstrated remarkable efficacy in pharmacological studies and preclinical trials.

ENOblock

ENOblock, also known as AP-III-a4, functions as a non-enzymatic active site inhibitor by binding to enolase directly without the need for a substrate analog [131]. AP-III-a4 has been extensively utilized to elucidate the role of ENO1 in cancer development [35, 46, 53, 58]. ENOblock inhibits F. nucleatum-induced CRC cell proliferation, and it significantly reduces tumor growth of F. nucleatum-cultured tumor cells in a CRC xenograft mouse model [53]. ENOblock inhibits the proliferation of tumor cells and the growth of tumor spheres in vitro while enhancing the tumor cell sensitivity to chemotherapy drugs. For example, in gastric cancer, ENOblock increases the sensitivity of GC cells to cisplatin by inhibiting their stemness and levels of glycolysis [58]. In addition, ENOblock exhibits a synergistic anti-tumor effect when used in combination with radiotherapy. In breast cancer, ENO1 enhances radiation therapy resistance by modulating mitochondrial homeostasis in vitro and in vivo to reduce ROS production and inhibit apoptosis. In contrast, radiation therapy sensitivity is restored in breast cancer cells by ENOblock. This suggests that ENOblock can produce strong synergistic anti-tumor effects when used in combination with radiation therapy [35] (Fig. 4 and Table 2).

Specific antibody

The use of anti-ENO1 monoclonal antibodies is a promising targeted therapy. In GC and NSCLC, metformin combined with anti-ENO1 antibody can overcome CSC resistance by suppressing the Wnt/β-catenin pathway [125]. In addition to directly impacting tumor cells, ENO1 antibodies can inhibit tumor progression by modulating the tumor microenvironment. One effective and potentially beneficial approach to targeted therapy is the use of anti-ENO1 antibodies. In prostate cancer, the use of ENO1 mAb (HuL227) reduces subcutaneous xenograft growth, monocyte recruitment, and intratumoral angiogenesis by targeting cell surface ENO1 via a fibrinolytic-associated mechanism within the tumor microenvironmental ecosystems involved in prostate cancer progression and bone metastasis, which could provide a new immunotherapeutic approach for prostate cancer patients [56].

DNA vaccine

Therapeutic DNA cancer vaccines are now recognized as a very promising strategy to activate the immune system against cancer. Developing a therapeutic cancer vaccine, Ag85B-ENO146-82, according to the ENO1 tumor-associated antigen, which prevents the growth of lung cancer in vivo. Anti-tumor effects were shown in tumor-loaded immune-competent C57BL/6 mice treated with Ag85B-ENO146-82 for Lewis lung cancer (LLC). Ag85B-ENO146-82 therapy boosted tumor-specific IFN-γ and TNF-α released by CD8⁺ T cells, generated dense infiltration of CD4⁺ and CD8⁺ T cells in tumors, and promoted M1 phenotypic polarization of macrophages. Analysis using flow cytometry revealed that CD8⁺ T effector memory cells and central memory cells were upregulated. qPCR and ELISA tests revealed that the expression of TNF-α and IFN-γ was upregulated, while the expression of IL1β, IL6, and IL10 was downregulated [132].It highlights the potential of the ENO1 DNA vaccine as an innovative immunotherapy for Lewis lung carcinoma patients. Additionally, ENO1 DNA vaccine combined with chemotherapy has been shown to significantly enhance the therapeutic efficacy against tumors.

Recent studies have demonstrated favorable therapeutic effects of ENO1 DNA vaccine in combination with chemotherapy in the treatment of pancreatic cancer. As compared to mice vaccinated or treated with gemcitabine alone, mice treated with gemcitabine prior to ENO1 DNA vaccination developed CD4⁺ T cell antitumor activity and significantly impaired tumor progression [133]. This study supports the potential of combination therapy in cancer treatment.

POMHEX

Recently, HEX and its prodrug POMHEX, possessing high cellular and blood-brain barrier permeability, have been rapidly developed. The small molecule enolase inhibitor POMHEX selectively kills ENO1-deficient glioma cells at low nanomolar concentrations and eradicates intracranial in situ ENO1-deficient tumors in mice at doses that are well tolerated by nonhuman primates, exhibiting a notable safety profile, indicating their potential for clinical applications [134].

Traditional Chinese medicine

Traditional Chinese medicine extract components also play a significant role in targeting ENO1 in tumors. In glioblastoma, the expression of ENO1 was significantly reduced in tumor cells treated with curcumin, and ENO1 emerged as a potential target gene affected by curcumin for glioblastoma prognosis. Curcumin also inhibited migration and invasion while promoting apoptosis in GBM cells [18]. The development of electrical pulse (EP)-mediated turmeric-silver nanoparticle (TurNP) therapy in triple-negative breast cancer; high-throughput, label-free quantitative proteomics analysis reveals the downregulation of ENO1 expression by TurNP + EP treatment [135]. CAD-14, a cinnamaldehyde (CA) compound, inhibits the p38 pathway in melanoma and suppresses tumor growth by inhibiting ENO1 expression, which induces apoptosis [136]. Moreover, the covalent binding of CA to ENO1 alters ENO1 protein stability and impacts glycolytic activity. Additionally, a combination of dacarbazine (DTIC) and CA significantly inhibited melanoma cell growth both in vitro and vivo by enhancing S-phase cell cycle arrest. Since CA inhibits ENO1 covalently, it may offer benefits to patients who are resistant to antimelanoma treatment when combined with DTIC [137]. In hepatocellular carcinoma, CA covalently binds with ENO1, impacting both the stability and activity of ENO1 and altering the dynamic balance of glucose metabolism. Blockading ENO1-induced gluconeogenic reflux enhances the TCA cycle, ultimately resulting in lower blood glucose levels and increased mitochondrial efficiency [138]. In CRC, isoglycyrrhizin-loaded nanoliposomes (ISL-NLs) inhibit tumor progression by modulating ENO1 in glycolysis and suppressing lactate production via the AMPK/mTOR pathway [139].Salidroside suppresses tumor cell proliferation by inhibiting glycolysis through the suppression of ENO1 expression in gastric cancer cells [140]. Mechanistically, these traditional Chinese medicines inhibit the glycolysis of tumor cells by downregulating ENO1 expression, thereby inhibiting tumor cell proliferation, invasion, and migration. This highlights the potential of traditional Chinese medicine extracts in targeting ENO1 for cancer treatment; however, the mechanisms by which they downregulate ENO1 expression require further investigation.

Others

Aspirin decreases the activity of ENO1 by modulating its lysine 2-hydroxyisobutyrylation (Khib) level, thereby attenuating both glycolysis and proliferation of hepatocellular carcinoma cells [141]. Additionally, aspirin delays tumorigenesis of hepatocellular carcinoma via ENO1/YAP [62]. Macrosphelide A inhibits the proliferation of cells and induces apoptosis in hepatocellular carcinoma, leukemia, and breast cancer by targeting key glycolytic enzymes, including ENO1 [142]. In gastric cancer, metformin significantly inhibits cell proliferation mediated by the ENO1-AKT1 complex and EMT signaling, ultimately suppressing the malignant phenotype of gastric cancer cells [66]. In bladder cancer, melatonin inhibits tumorigenesis as well as enhances the resistance of bladder cancer cells to gemcitabine through PPARγ/ENO1-mediated glycolysis [143]. Triterpenoids promote the proliferation and inhibit the apoptosis of CRC cells by targeting ENO1 along with other key targets involved in glycolytic and glutaminolytic pathways [144].

Given that ENO1 plays a significant role in the development of various tumors, it would be beneficial to address potential clinical drugs targeting ENO1. ENO1 inhibitors, including ENOblock and POMHEX, have been developed. However, all ENO1 inhibitors remain in the preclinical research stage and have not yet entered clinical treatment. Immunotherapies targeting ENO1, including monoclonal antibodies and DNA vaccines, have also been developed. Beyond specific ENO1 inhibitors, antibodies and vaccines, several potential drugs may indirectly target ENO1. Some traditional Chinese medicines, such as curcumin, cinnamaldehyde, and salidroside, have been found to reduce ENO1 expression in tumors, exerting anti-tumor effects, though the mechanisms require further investigation. Additionally, common drugs such as aspirin, metformin, and melatonin have also been shown to reduce ENO1 expression in tumors, but available data are limited and further research is needed.

ENO1 inhibitors have demonstrated promising results in preclinical studies. More complex combination therapies, such as the integration of DNA vaccines with chemotherapy, have shown potential. Nevertheless, certain challenges remain, such as suboptimal drug targeting. Given that ENO1 is widely expressed in various tissues and cells, future studies should prioritize optimizing drug targeting, reducing side effects, and exploring the combined application with other therapeutic approaches. In conclusion, ENO1-targeted therapeutic agents hold promise, but further research and optimization are required to fully realize their clinical potential.

Table 2 Targeted therapeutic strategies of ENO1 in various tumors

Figure 4. Role of ENO1 in tumor progression and its treatment, including inhibitor therapy, immunotherapy, traditional Chinese medicine therapy, and common medicine therapy.

Conclusion and future perspectives

In summary, ENO1 is associated with a poor prognosis and overexpressed in a variety of human malignant tumors, and can be used as a tumor biomarker. ENO1 exhibits a variety of biological functions in tumor progression, which include promotion of tumorigenesis, increase in glycolysis, stimulation of cell EMT, migration, invasion, metastasis and proliferation, activation of oncogenesis-related signaling pathways, angiogenesis, and chemoresistance. Thus, ENO1 can be regarded as a crucial oncoprotein that facilitates and sustains tumor progression. In recent years, comprehensive studies of clinical and physiological functions have identified ENO1 as a key target for therapeutic development.

Localization of ENO1 on the cell membrane can promote tumor development and metastasis. Intracellularly, it can activate glycolytic energy metabolism or act as a regulator in several pathways such as hypoxia signaling, PI3K/AKT/mTOR, Wnt/β-catenin, MAPK, AMPK, and NF-κB. LncRNAs can also regulate the transcriptional and enzymatic activities of ENO1 in tumors. Moreover, ENO1 can be secreted by tumor cells into the tumor microenvironment, where it interacts with other cells, such as tumor-associated macrophages, promoting the release of cytokines or other mediators, which in turn promote the progression of tumors. Therefore, detailed studies of ENO1 transcription, translation, activity, translocation, and roles in cellular signaling pathways are essential to guide effective tumor therapies. The role of ENO1-regulated lactic acid in the tumor microenvironment and the soluble ENO1-mediated interaction between tumor cells and other cells deserve further investigationThe targeted nature of ENO1 renders it an attractive tumor biomarker as well as a therapeutic target. ENO1 is a very suitable cancer biomarker for both prognostic and diagnostic purposes due to its cell surface location. Because of its location in tumor cells, important metabolic roles, and capacity to accelerate the growth of tumors, it can be used to develop a very promising targeted therapeutic strategy to bring hope to cancer patients.