Unlikely role of glycolytic enzyme a-enolase in cancer metastasis and its potential as a prognostic biomarker

Reliance on glycolysis for energy production is considered a hallmark of cancer and the glycolytic enzyme a-enolase is overexpressed in a range of cancer types. However, recent studies have revealed that a-enolase is involved in a variety of unrelated physiological processes and can be found in multiple unexpected cellular locations. This review focuses on the unlikely role of a-enolase as an extracellular plasminogen-binding receptor localised to the plasma membrane. Conversion of plasminogen to plasmin on the surface of cancer cells enhances their ability to invade through stroma by activating collagenases and degrading fibrin as well as extracellular matrix proteins. Increased expression of a-enolase is associated with increased migration and invasion of cancer cells, and decreased metastasis-free survival in patients with several cancer types, including non-small cell lung, pancreatic, breast and colorectal cancers. Due to its overexpression in a range of cancer types and multi-functional roles in key areas of tumour metabolism and metastasis, a-enolase may be useful as a universal cancer prognostic biomarker or therapeutic target.


ENOLASE IS A GLYCOLYTIC ENZYME THAT HAS THREE ISOFORMS
Enolases (EC 4.2.1.11) are metalloenzymes that catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate in the glycolysis pathway [ Figure 1], and catalyse the hydration of phosphoenolpyruvate to 2-phopho-D-glycerate in the reverse anabolic pathway during gluconeogenesis [3] . In mammals, the three genes ENO1, ENO2, ENO3 encode three isoforms, with expression being regulated in a tissue-specific manner. Alpha-enolase (ENO1) is ubiquitously expressed, whereas g-enolase (ENO2) is primarily expressed in neurons and neuroendocrine tissues, and b-enolase (ENO3) in muscle tissues [23] . Active enolase consists of a dimer in which two subunits face each other in an antiparallel formation [24] , and requires two non-covalently bound magnesium ions as cofactors for enzyme activity [25] .

ALPHA-ENOLASE IS A MULTI-FUNCTIONAL PROTEIN
Although many glycolytic enzymes are considered to be housekeeping proteins, a-enolase expression can vary dramatically depending on the stress, metabolic, or pathological state of the cell. A retrospective proteomic meta-analysis identified that a-enolase was the most differentially expressed protein in humans and rodents irrespective of tissue type and pathological condition [26] . Disrupted expression and/or activity of a-enolase has been reported in several pathologies with distinct aetiologies, including Alzheimer's disease, systemic sclerosis, rheumatoid arthritis, bacterial infections and hepatic fibrosis [27][28][29][30][31][32][33][34][35][36][37][38] .
Apart from its role in the glycolytic pathway, recent studies have revealed that a-enolase is a multifunctional protein that controls a variety of cellular processes, including proliferation, survival, migration and invasion. Additionally, using an alternative transcription start codon, the ENO1 gene can produce a 37 kDa protein, c-myc promoter-binding protein (MBP-1). MBP-1 localises to the nucleus, where it acts as a transcription repressor by binding to the c-myc P2 promoter [39] , helping regulate and maintain the function of the glycolysis pathway.

ALPHA-ENOLASE CAN SHUTTLE BETWEEN CELLULAR COMPARTMENTS
Alpha-enolase can be localised to the cytoplasm and plasma membrane, as well as secreted in exosomes, and its location varies with cancer type. For example, in pancreatic, breast and lung tumours, a-enolase is localised to the plasma membrane [22,44,45] , whereas in melanoma, mesothelioma, non-small cell lung, colorectal and prostate cancer a-enolase is also secreted and found in exosomes [43,[46][47][48][49] . Alpha-enolase can shuttle between compartments, performing different functions when in different subcellular locations, such as surface membrane plasminogen binding, controlling the overall metabolic state of the cell, stressrelated or acting as a heat-shock protein, RNA transport, mitochondrial membrane stability, and cell cycle control [50][51][52][53][54][55] .

INCREASED ALPHA-ENOLASE EXPRESSION ENHANCES CELL PROLIFERATION IN A VARIETY OF CANCERS
In most solid tumours, the Warburg effect causes an increase in total glycolysis under both hypoxic and normoxic conditions [ Figure 2]. Enhanced cell proliferation leads to increased anabolic needs, and cancer cells remodel metabolic processes by diverting nutrients to anabolic pathways to satisfy increased cellular energy demands [56] . Therefore, the Warburg effect may provide cancer cells with an advantage when competing with non-cancerous tissues for nutrients. This suggests that increased a-enolase expression will contribute to enhanced proliferation commonly observed in cancer cells.
Indeed, upregulated a-enolase expression has been shown to regulate cell proliferation in various solid tumours in vitro [11,13,[57][58][59][60][61] , and to increase tumour growth in a HCT116 colorectal cancer xenograft model in vivo [11] [ Table 2]. Conversely, silencing of a-enolase in glioma, pancreatic, lung, endometrial, colorectal and breast cancer cells was found to induce cell cycle arrest and senescence, and also to reduce tumour volume in CFPAC-1 pancreatic, MDA-MB-231 breast and U-87MG glioma xenograft models in vivo [6,11,12,62,63] . Furthermore, a-enolase is also implicated in the control of apoptosis and sensitivity to chemotherapeutic agents, as silencing of ENO1 in cancer cells induced apoptosis and increased sensitivity to cisplatin and 5-fluorouracil in vitro [13,62,64] . Unexpectedly, cells respond to a-enolase silencing by inducing catabolic adaptations that lead to restoration of pyruvate, acetyl-CoA bulk and oxidative phosphorylation, and exhibit an increased expression of proteins involved in both oxidative stress-and sirtuin-induced autophagy [62] . Taken together, these studies demonstrate that a-enolase is an important regulator of tumour cell metabolism, proliferation and survival, which by definition make it a perfect target for anticancer therapy.

Cancer
Sample type Detection method Alteration (frequency of alteration) Ref.

ALPHA-ENOLASE IS A SURFACE PLASMINOGEN-BINDING RECEPTOR
In addition to a role in cell proliferation and survival, a-enolase located on the plasma membrane acts as a plasminogen-binding receptor [55] [ Figure 3]. Plasminogen is a zymogen, which is converted to plasmin in the presence of the activators tissue plasminogen activator or urokinase-type plasminogen activator (uPA) [65] . This cell surface interaction concentrates protease activity in the tissue surrounding the cell, protecting plasmin from inactivation by circulating a2-antiplasmin [66] . Plasmin activates collagenases and degrades fibrin and other matrix proteins, resulting in cell migration and invasion into tissue, ultimately underpinning cancer metastasis and relapse.

Role in invasion and migration
Overexpression of a-enolase has been shown to increase the migration and invasion of hepatocellular carcinoma, colorectal and gastric cancer cells in vitro [11,58,60,61,67] and to enhance colorectal cancer metastasis in vivo [11] , demonstrating that it is an important driver of metastasis in multiple cancer types [ Table 3]. Conversely, knockdown or pharmacological inhibition of a-enolase decreased the migration and invasion of glioma, colorectal, pancreatic and endometrial carcinoma in vitro [6,12,63,68,69] , and decreased tumourigenesis and metastasis of endometrial carcinoma in vivo [12] . Furthermore, binding of recombinant a-enolase to the surface of prostate cancer cells was shown to promote cell migration via its plasminogen receptor activity [70] . By contrast, anti-a-enolase monoclonal antibodies inhibited plasminogen-dependent invasion of human pancreatic cancer cells in vitro and metastasis formation in vivo [71] and also lung cancer cell invasion in vitro and growth in vivo [72] [ Table 3]. Overexpression increased cell proliferation and colony formation [59] Breast cancer Downregulation in MDA-MB-231 cells Downregulation decreased cell proliferation and survival in vitro and reduced tumour growth in vivo [62] Colorectal cancer Overexpression and knockdown in HCT116 cells Overexpression promoted cell proliferation and tumour growth in vivo ; Decreased expression decreased cell proliferation and tumour growth in vivo [11] Endometrial carcinoma Knockdown in HEC-1B and Ishikawa Decreased expression reduced cell proliferation in vitro and tumourigenesis in vivo [12] Gastric cancer Knockdown in MGC-803 and MKN45 cells Knockdown led to cell cycle arrest at the G 1 phase and promoted apoptosis, and repressed the rate of cell proliferation and colony formation [13] Knockdown in MKN45 cells Knockdown decreased cell proliferation, induced apoptosis and increased sensitivity to chemotherapeutics [64] Knockdown in AGS cells and overexpression in SGC7901 cells Knockdown decreased proliferation and colony formation, whereas overexpression increased cell proliferation and colony formation [60] Overexpression in AGS cells Overexpression increased cell proliferation and colony formation [61] Glioma

Retinoblastoma Knockdown in Y79 cells and overexpression in Meri-RB1 cells
Knockdown led to cell cycle arrest at the G 1 phase, decreased cell proliferation and increased apoptosis. Overexpression increased cell proliferation. [57]

ALPHA-ENOLASE IS A TUMOUR-ASSOCIATED ANTIGEN
Externalisation of a-enolase by cancer cells exposes it to the immune system as a tumour-associated antigen that has been found to induce autoantibody production in cancer patients, including those with acute and chronic leukaemias, melanoma, and lung, breast, gastric and pancreatic cancers [22,45,[73][74][75][76][77][78][79] . In pancreatic cancer, T cells activated by a-enolase-pulsed dendritic cells lysed pancreatic cancer cells, but not normal human keratinocytes in vitro and inhibited CF-PAC-1 tumour growth in vivo [22] . In oral squamous cell carcinoma, an HLA-DR8-restricted human a-enolase peptide was recognised by CD4 + T cells and produced a cytotoxic response against OSC-20 cells [80] . Additionally, vaccination with ENO1 in Kras G12D / Cre and Kras G12D /Trp53 R172H mice prior to development of pancreatic carcinoma delayed tumour growth and increased survival [81] . Taken together, these studies suggest that immune responses directed against a-enolase may be immunostimulatory and ultimately beneficial to patients.

ALPHA-ENOLASE IS A PROGNOSTIC FACTOR FOR MULTIPLE CANCER TYPES
In addition to being overexpressed in many cancers, a-enolase has been identified as a putative prognostic biomarker in a range of tumour types [ Table 4]. Whilst ENO1 expression was not associated with tumour stage in colorectal cancers, it was significantly correlated with tumour size and presence of distant metastases [11] . Alpha-enolase expression was positively correlated with lymph node status in endometrial and gastric cancer patients [12,13,60] , and increased a-enolase expression in endometrial, gastric, lung, lymphoma and hepatocellular cancer patients was associated with worse overall survival [8,12,13,16,73,82] . Furthermore, increased a-enolase expression was correlated with worse distant metastasis-free survival in breast cancer patients [9] , worse disease-free survival in hepatocellular carcinoma and chordoma [16,83] and worse progression-free survival in lung cancer patients [73] . By contrast, downregulation of a-enolase is a predictor of poor prognosis in clear cell renal cell carcinoma (ccRCC) [84] , demonstrating that a-enolase may control different cellular functions in ccRCC when compared to other cancers.
Autoantibodies generated against a-enolase in its capacity as a tumour-associated antigen represent an additional type of prognostic biomarker that may be assayed in serum. The presence of autoantibodies against a-enolase correlated with longer disease-free survival and overall survival in pancreatic and lung cancer patients [45,[85][86][87] [ Table 4]. Furthermore, compared with healthy individuals, a-enolase antibodies

Cancer Experimental model Effect of modulation of ENO1 expression Ref. Colorectal cancer Overexpression and knockdown in HCT116 cells
Overexpression increased migration and invasion both in vitro and in vivo ; decreased expression decreased migration and invasion both in vitro and in vivo [11] Treatment of HCT116 cells with CS5931 (peptide inhibitor) Treatment with CS5931 decreased cell migration and invasion [69] Endometrial carcinoma Knockdown in HEC-1B and Ishikawa Decreased expression reduced migration and invasion in vitro and metastasis in vivo [12] Gastric cancer Knockdown in AGS cells and overexpression in SGC7901 cells Knockdown decreased migration, and overexpression increased migration [60] Overexpression in AGS cells Overexpression increased migration [ antibody decreased invasion in vitro and in vivo [72] Pancreatic cancer Knockdown in CFPAC-1 cells Knockdown decreased migration and invasion, and reduced adhesion to fibronection and collagen and increased adhesion to vitronectin [68] Anti-human ENO1 antibody Decreased invasion in vitro and metastasis in vivo [71]  are decreased in stage IV lung and breast cancers [88] , and are lower in stage III/IV than in stage I/II lung cancer patients [89] . By contrast, the presence of anti-a-enolase antibodies in sera from chronic lymphocytic leukaemia (CLL) patients is predictive of a shorter time to first treatment [90] , indicating that the presence of a-enolase antibodies are indicative of a disrupted immune system in CLL. Taken together, these studies suggest that autoantibodies against a-enolase are a good prognostic factor in pancreatic, lung and breast cancers, and provide further evidence that targeting a-enolase may be beneficial in solid tumours.

ENOLASE INHIBITORS ARE POTENTIAL ANTICANCER AGENTS
Due to its important cancer-related roles, enolase is one of several glycolytic enzymes being examined as a potential anticancer therapeutic target. Polyamine sulphonamide analogues have proven particularly effective at inhibiting a-enolase activity. Two such compounds have been further developed and shown to Patients with a higher increase in anti-a-enolase had a lower hazard ratio and better PFS [85] Sera from patients with lung cancer (n = 72), benign lung diseases (n = 69), and healthy individuals (n = 70) Autoantibodies were higher in lung cancer sera compared with sera from normal and benign lung disease patients; Autoantibodies were higher in stage I/II than in stage III/IV [89] Lymphoma Peripheral T-cell lymphoma not otherwise classified tissue (n = 87) Increased protein correlated with worse OS [8] Pancreatic cancer Sera from pancreatic ductal adenocarcinoma patients (n = 120) Presence of auto-antibodies correlated with a better clinical outcome [86] Sera and PBMCs from pancreatic ductal adenocarcinoma patients (n = 15) Patients with > 20% peripheral a-enolase-specific T cells or anti-a-enolase antibodies showed a better OS [87] ccRCC Primary ccRCC tissue (n = 360) and TCGA dataset (n = 428) Negative correlation between protein expression, tumour stage and grade. Patients with higher mRNA had lower hazard ratio of recurrence and longer OS [84]  be cytotoxic to KG-1 (AML) cells and to the AML leukaemic stem cell fraction, with minimal effects on normal healthy stem cells [92] . This report highlights that a-enolase is an actionable therapeutic target that may be useful in the treatment of cancer, particularly AML.

CONCLUSION
Alpha-enolase plays a supportive role in cancer progression and has been implicated in three of the hallmarks of cancer: cellular energetics and metabolism; cell proliferation; and invasion and migration.
In cancer cells, a-enolase is overexpressed and localised on the surface, where it acts as a key promotor of metastasis, driving invasion through plasminogen activation and extracellular matrix degradation. In several cancer types, patients develop an immune response against a -enolase, and anti-a-enolase antibodies can be detected in their sera. Increased expression of a-enolase mRNA, proteins or autoantibodies are associated with decreased metastasis-free survival in several cancer types, including non-small cell lung, pancreatic, breast and colorectal cancers. Future examination of the expression and function of a-enolase in cancers may ultimately result in a-enolase becoming a therapeutic target and prognostic biomarker for a range of cancer types.