Tài liệu Báo cáo khoa học: a-enolase: a promising therapeutic and diagnostic tumor target ppt

REVIEW ARTICLE
a
-enolase: a promising therapeutic and diagnostic tumor
target
Michela Capello, Sammy Ferri-Borgogno, Paola Cappello and Francesco Novelli
Department of Medicine and Experimental Oncology, Center for Experimental Research and Medical Studies (CeRMS), San Giovanni Battista
Hospital, University of Turin, Italy
Introduction
Enolase is a metalloenzyme that catalyzes the dehydra-
tion of 2-phospho-d-glycerate to phosphoenolpyruvate
in the second half of the glycolytic pathway. In the
reverse reaction (anabolic pathway), which occurs dur-
ing gluconeogenesis, the enzyme catalyzes the hydra-
tion of phosphoenolpyruvate to 2-phospho-d-glycerate
[1,2]. Enolase is found from archaebacteria to mam-
mals, and its sequence is highly conserved [3]. In mam-
mals, three genes, ENO1, ENO2 and ENO3 encode for
three isoforms of the enzyme, a-enolase (ENOA),
c-enolase and b-enolase, respectively, with high
sequence identity [4–6]. The expression of these iso-
forms is tissue specific: ENOA is present in almost all
adult tissues, b-enolase is expressed in muscle tissues
and c-enolase is found in neurons and neuroendocrine
tissues [1,7–9]. The monomer of ENOA consists of a
smaller N-terminal domain (residues 1–133) and a lar-
ger C-terminal domain (residues 141–431). In eukarya,
enzymatically active enolase consists of a dimeric form
in which two subunits face each other in an antiparal-
lel manner [1,10]; some eubacterial enolases, by con-
trast, are octameric [11]. Enolase can form homo- or
heterodimers, such as aa, ab, bb, ac and cc [1].

Apart from its enzymatic activity, in many prokary-
otic and eukaryotic cells, ENOA is expressed on the
cell surface, where it acts as a plasminogen receptor
promoting cell migration and cancer metastasis [12–
23]. Moreover, ENO1 can be translated into a 37 kDa
protein, c-myc promoter-binding protein (MBP-1), by
using an alternative start codon [24]. MBP-1 lacks the
Keywords
a-enolase; cancer; immune response;
post-translational modifications;
tumor-associated antigen
Correspondence
F. Novelli, Center for Experimental Research
and Medical Studies (CeRMS), San Giovanni
Battista Hospital, Via Cherasco 15, 10126
Turin, Italy
Fax: +39 011 633 6887
Tel: +39 011 633 4463
E-mail:
(Received 5 November 2010, revised 19
January 2011, accepted 21 January 2011)
doi:10.1111/j.1742-4658.2011.08025.x
a-enolase (ENOA) is a metabolic enzyme involved in the synthesis of pyru-
vate. It also acts as a plasminogen receptor and thus mediates activation of
plasmin and extracellular matrix degradation. In tumor cells, EMOA is
upregulated and supports anaerobic proliferation (Warburg effect), it is
expressed at the cell surface, where it promotes cancer invasion, and is sub-
jected to a specific array of post-translational modifications, namely acety-
lation, methylation and phosphorylation. Both ENOA overexpression and
its post-translational modifications could be of diagnostic and prognostic

value in cancer. This review will discuss recent information on the
biochemical, proteomics and immunological characterization of ENOA,
particularly its ability to trigger a specific humoral and cellular immune
response. In our opinion, this information can pave the way for effective
new therapeutic and diagnostic strategies to counteract the growth of the
most aggressive human disease.
Abbreviations
EGFR, epidermal growth factor receptor; ENOA, a-enolase; ERK, extracellular signal-regulated kinase; MBP-1, c-myc promoter-binding
protein; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; PAI-1, plasminogen activator inhibitor-1; PTM, post-
translational modification; TAA, tumor-associated antigen; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator;
uPAR, urokinase-type plasminogen activator receptor.
1064 FEBS Journal 278 (2011) 1064–1074 ª 2011 The Authors Journal compilation ª 2011 FEBS
first 96 residues of ENOA and localizes in the nucleus,
where it binds to the c-myc P2 promoter and acts as a
transcription repressor, leading to tumor suppression
[25–27]. ENOA associates with MBP-1 in the tran-
scriptional regulation of the oncogene c-myc [28].
ENOA is a surface plasminogen-binding
receptor in tumors
In breast, lung and pancreatic neoplasia, ENOA is
localized on the surface of cancer cells [29–31], whereas
in melanoma and nonsmall cell lung carcinoma cells it
can also be secreted by exosomes [32,33]. How ENOA
is displayed on the cell surface remains unknown.
Many glycolytic enzymes and cytosolic proteins that
lack N-terminal signal peptide reach the surface of
eukaryotic cells [34]. In mammal cells, some export
routes of unconventional protein secretion have been
postulated: membrane blebbing, membrane flip-flop,
endosomal recycling or a plasma membrane trans-

porter [35]. One possibility is that phosphoinositides
recruit ENOA and translocate it to the cell surface
[36]. It is not known if surface ENOA is also present
as a monomer. As the monomeric form is catalytically
inefficient it could be available to interact with other
proteins that mediate its transport to the cell surface
[37]. However, in breast cancer cells, surface ENOA
maintains its catalytic activity, suggesting that cell sur-
face localization does not affect this function [31].
Cell surface ENOA is one of the many plasminogen-
binding molecules that include actin [38], gp330 [39],
cytokeratin 8 [40], histidine-proline rich glycoprotein
[41], glyceraldehyde-3-phosphate dehydrogenase [42],
annexin II [43], histone H2B [44] and gangliosides [14].
ENOA and most of these proteins have C-terminal
lysines predominantly responsible for plasminogen acti-
vation [45]. Interaction of the plasminogen lysine-
binding sites with ENOA is dependent upon recognition
of ENOA C-terminal lysines K420, K422 and K434
[14]. In view of the surface potential of the human
ENOA crystal structure, an additional plasminogen
binding site that includes K256 has been proposed [10].
Binding with ENOA lysyl residues leads to activa-
tion of plasminogen to plasmin by the proteolytic
action of either tissue-type (tPA) or urokinase-type
(uPA) plasminogen activators [19,46]. Plasmin is a ser-
ine protease with a broad spectrum substrate, includ-
ing fibrin, extracellular matrix components (laminin,
fibronectin) and proteins involved in extracellular
matrix degradation (matrix metalloproteinases, such as

MMP3) [47–50]. Binding of plasminogen to the cell
surface has profibrinolytic consequences: enhancement
of plasminogen activation, protection of plasmin from
its inhibitor a
2
-antiplasmin and enhancement of the
proteolytic activity of cell-bound plasmin [13,51]. Pro-
teolysis mediated by cell-associated plasmin contributes
to both physiological processes, such as tissue remodel-
ing and embryogenesis, and to pathophysiological
processes, such as cell invasion, metastasis and inflam-
matory response [19,45]. A noteworthy positive corre-
lation exists between elevated levels of plasminogen
activation and malignancy [46,52]. Higher expression
levels of uPA and ⁄ or plasminogen activator inhibitor-1
(PAI-1) in tumor tissues correlate with aggressiveness
and poor prognosis. ENOA takes part, together with
urokinase plasminogen activator receptor (uPAR),
integrins and some cytoskeletal proteins, in a multipro-
tein complex, called metastasome, responsible for
adhesion, migration and proliferation in ovarian can-
cer cells [53]. In human follicular thyroid carcinoma
cells, retinoic acid causes a decrease in ENOA levels
that coincides with their reduced motility [54], and cell
surface ENOA is enhanced in breast cancer cells ren-
dered superinvasive following paclitaxel treatment [55].
In pancreatic cancer patients, deregulated expression
of many proteins involved in the plasminogen pro-fibri-
nolytic cascade (annexin A2, PAI-2, uPA, uPAR, MMP-
1 and MMP-10) correlates with survival [56–59]. In the

same tumor, tPA activates a mitogenic signal mediated
by extracellular signal-regulated kinase (ERK)-1 ⁄ 2
through epidermal growth factor receptor (EGFR) and
annexin A2 [60,61]. These proteins probably form a
complex that also includes ENOA, as it has been pulled
down with annexin A2, cytokeratin 8 and tPA in raft
membrane fractions of pancreatic cancer cells [62].
ENOA is a tumor-associated antigen
(TAA)
TAAs are self-proteins that can trigger multiple spe-
cific immune responses in the autologous host [63].
Activation of the immune system against TAAs occurs
at an early stage of tumorigenesis, as illustrated by the
detection of high titers of autoantibodies in patients
with early-stage cancer [64], and correlates with the
progression of malignant transformation [65]. It is not
entirely clear how TAAs are able to trigger humoral
responses, especially as many of those discovered so
far are intracellular proteins, but are thought to be
altered in a way that renders the proteins immunogenic
[66,67]. Several hypotheses have been proposed: these
self-proteins could be overexpressed, mutated, misfold-
ed, aberrantly degraded or localized so that autoreac-
tive immune responses in cancer patients are induced
[65,68,69]. Moreover TAAs that have undergone post-
translational modifications (PTMs) (e.g. glycosylation,
M. Capello et al. a-enolase in tumor diagnosis and therapy
FEBS Journal 278 (2011) 1064–1074 ª 2011 The Authors Journal compilation ª 2011 FEBS 1065
phosphorylation, acetylation, oxidation and proteolytic
cleavage) may be perceived as foreign by the immune

system [66–68]. The immune response against such
immunogenic epitopes of TAAs induces the production
of autoantibodies as serological biomarkers for cancers
[70]. Both its overexpression in tumors and its ability
to induce a humoral and ⁄ or cellular immune response
in cancer patients classify ENOA as a true TAA.
ENOA expression is increased in
tumors
The overexpression of ENOA is associated with tumor
development through a process known as aerobic gly-
colysis or the Warburg effect [71]. Warburg observed
that cancer cells consume more glucose than normal
cells and generate ATP by converting pyruvate to lac-
tic acid, even in the presence of a normal oxygen sup-
ply [72]. The mechanism of the Warburg effect was
uncertain until the recent identification of upregulation
of glycolytic enzymes by hypoxia-inducible factor.
When a solid tumor exceeds 1 mm
3
, its cells face hyp-
oxic stress due to slow angiogenesis [73,74]. Because
the ENO1 promoter contains a hypoxia responsive ele-
ment [75,76], ENOA is upregulated at the mRNA
and ⁄ or protein level in several tumors, including brain
[77], breast [78–83], cervix [77,84,85], colon [77,86,87],
eye [77], gastric [77,88,89], head and neck [90,91], kid-
ney [77], leukemia [92], liver [77,93,94], lung [77,95–99],
muscle [77], ovary [77,100], pancreas [29,77,101,102],
prostate [77,103], skin [104] and testis [77] (Table 1).
Results from a bioinformatic study support a correla-

tion between ENOA expression and tumorigenicity
[52,77]. Moreover, ENOA’s enzymatic activity may
also be increased in breast tumor tissue, especially in
metastatic sites [82,83]. Increased ENOA expression
can influence chemotherapy treatments, as shown in
estrogen receptor-positive breast tumors, where it
induces tamoxifen resistance [78], and in colorectal car-
cinoma cells, where it is overexpressed after 5-fluoro-
uracil administration [87].
ENOA PTMs in tumors
PTMs are common mechanisms that control signal
transduction, protein-protein interaction and transloca-
tion [105,106]. Reversed-phase liquid chromatography,
nanospray tandem mass spectrometry has been used
to characterize ENOA PTMs in several cancer and
normal cell lines (Table 2) ( />uniprot/P06733) [107–115].
Acetylation, methylation and phosphorylation are
the main PTMs (Table 2). Acetylation was found in
cervix and colon cancer, leukemia, normal pancreatic
ducts and tumoral pancreatic cells. Fourteen acetylated
lysine residues are common to leukemia, pancreatic
cancer and normal pancreas, and one of them is the
only acetylated residue in cervix tumor. Three acetyla-
tions are common to both leukemia and pancreatic
cancer, whereas three are specific for normal and
tumoral pancreatic cells. However, six specific acety-
lated lysines were found in pancreatic cancer cells, and
Table 1. Expression of ENOA, the immune response to it and clinical correlations in cancer.
Cancer ENOA enhanced expression Immune response to ENOA Clinical correlations
Brain m [77]

Breast m (68%), p, e (100%) [78–83] Ab [69,125] DP, DFI, M [69,78]
Cervix m, p [77,84,85]
Colon m, p [77,86,87]
Eye m [77]
Gastric m (73%), p [77,88,89]
Head and neck m (68%), p [90,91] Ab (79%) [91,123,124], T [131,132] OS, PFS [91]
Kidney m [77]
Leukemia p (> 50%) [92] Ab (33–86%) [120,121]
Liver m, p (17–80%) [77,93,94] M [93,94]
Lung m, p (79–100%) [77,95–99] Ab (7–80%) [30,69,96,99,126–129] DP, OS, PFS [69,99]
Muscle m [77]
Ovary m, p [77,100]
Pancreas m (100%), p (82–90%) [29,77,101,102] Ab (62%) [119], T [29] OS, PFS [119]
Prostate m, p (100%) [77,103]
Skin m [104] Ab (38–100%) [104,122]
Testis m [77]
Percentages indicate the reported frequencies of enhanced ENOA mRNA, protein and enzymatic activity or the frequencies of anti-ENOA Ig.
m, mRNA; p, protein; e, enzymatic activity; Ab, antibody production; T, T cell response; DP, disease progression; DFI, disease-free interval;
M, malignancy; OS, overall survival; PFS, progression-free survival.
a-enolase in tumor diagnosis and therapy M. Capello et al.
1066 FEBS Journal 278 (2011) 1064–1074 ª 2011 The Authors Journal compilation ª 2011 FEBS
Table 2. ENOA PTMs in normal and cancer tissues. Asp, aspartate; Glu, glutamate; Lys, lysine; Ser, serine; Thr, threonine; Tyr, tyrosine; numbers refer to the position of each residue in
the ENOA amino acid sequence.
Cell type
Acetylation Methylation Phosphorylation
Reference
Residue Position Residue Position Residue Position
Embryonic kidney Tyr 57 111
Ser 63
Normal pancreas Lys 64, 71, 80, 81, 89, 92, 126, 193,

202, 228, 233, 281, 335, 343,
358, 406, 420
Asp 23, 91, 203, 209, 274, 299, 300,
378
Ser 419 115
Glu 21, 45, 48, 86, 88, 96, 101, 187,
210, 219, 250, 293, 375, 377,
415, 416
Cervix carcinoma Lys 71 Thr 72 112–114
Ser 254, 263
Colon cancer Ser 2 />uniprot/P06733#ref14
Leukemia Lys 5, 60, 64, 71, 80, 81, 89, 126, 193,
199, 221, 228, 233, 256, 281,
285, 343, 406, 420
Ser 37, 40, 281 107–109
Thr 41, 390
Tyr 44, 287
Lung cancer Tyr 44, 287 110
Pancreatic cancer Lys 28, 64, 71, 80, 81, 89, 92, 103,
105, 126, 193, 202, 221, 228,
233, 239, 256, 262, 281, 285,
330, 335, 343, 358, 406, 420
Asp 23, 91, 203, 209, 266, 274, 286,
294, 297, 299, 300, 378, 383
Ser 419 115
Glu 21, 45, 48,86, 88, 96, 101, 167,
187, 210, 219, 222, 225, 250,
293, 352, 375, 377, 414, 415, 416
M. Capello et al. a-enolase in tumor diagnosis and therapy
FEBS Journal 278 (2011) 1064–1074 ª 2011 The Authors Journal compilation ª 2011 FEBS 1067

three in leukemia. The only acetylated serine identified
is specific for colon cancer (Table 2).
Methylation has been assessed in normal and tumor-
al pancreas only. Twenty-four aspartate and glutamate
residues were found in both cell types. However, five
aspartates and five glutamates are specifically methy-
lated only in pancreatic cancer (Table 2).
Phosphorylation is the PTM that displays the most
specific pattern in each cell line. Two serine and one
threonine residues were specifically found in cervix
cancer, one threonine and one serine in embryonic kid-
ney, three serines and two threonines in leukemia;
whereas two tyrosine residues were found in both leu-
kemia and lung cancer and one serine in both tumoral
and normal pancreas.
ENOA in tumor cells is subjected to more acetyla-
tion, methylation and phoshorylation than in normal
tissues, indicating that many PTMs are associated with
cancer development and some are specific for each
kind of tissue or cancer. This can reflect the specific
activation of pro-mitogenic signaling pathways in
tumor cells. In many cases, PTMs regulate the stability
and functions of proteins; for example, in metabolic
enzymes, acetylation acts as an on ⁄ off switch mecha-
nism [116], whereas methylation on carboxylate side-
chains enhances hydrophobicity by increasing the affin-
ity of proteins for phospholipids [115]. We speculate
that PTMs are important mechanisms in the regulation
of ENOA functions, localization and immunogenicity.
ENOA induces a specific immune

response in tumors
Several TAAs induce the production of IgG autoanti-
body in cancer patients via an integrated immune
response triggered by CD4
+
T cells, CD8
+
T cells and B
cells. TAAs released by secretion, shedding or tumor cell
lysis are captured by antigen presenting cells, processed
and presented by either major histocompatibility
complex (MHC) class I or MHC class II molecules for
priming and activation of CD8
+
and CD4
+
T cells,
respectively. Uptake of antigen by B cells also occurs
and is driven by membrane Ig, leading to MHC class II
antigen presentation to CD4
+
T cells. Activated CD4
+
T cells, through the secretion of appropriate cytokines,
trigger B cells to produce IgG against the same TAA
[117], and CD8
+
T cells to differentiate into TAA-spe-
cific cytotoxic T lymphocytes. In vivo maintenance and
survival of TAA-specific cytotoxic T lymphocytes is also

dependent on cytokines released by CD4
+
T cells [118].
This coordinated immune response suggests that IgGs
against TAA are not only a diagnostic tool, but also
allow the selection of TAAs for cancer immunotherapy.
In many cancer patients, including pancreatic [119],
leukemia [120,121], melanoma [104,122], head and neck
[91,123,124], breast [69,125] and lung [30,69,96,99,
126–129], ENOA has been shown to induce autoanti-
body production (Table 1). In pancreatic cancer
patients, autoantibodies to ENOA are directed against
two upregulated isoforms phosphorylated in Ser 419
[115,119] (Table 2). Protein phosphorylation increases
the affinity of peptides for MHC molecules that can be
recognized by T cells [130].
In pancreatic cancer, ENOA elicits a CD4
+
and
CD8
+
T cell response both in vitro and in vivo [29].
Anti-MHC class I Ig inhibited the cytotoxic activity of
ENOA-stimulated CD8
+
T cell against pancreatic
tumor cells, but no MHC class I restricted peptide of
ENOA has been identified so far. Moreover, in pancre-
atic ductal adenocarcinoma patients, production of
anti-ENOA IgG is correlated with the ability of T cells

to be activated in response to the protein [29], thus
confirming the induction of a T and B cell integrated
antitumor activation against ENOA. In oral squamous
cell carcinoma, an HLA-DR8-restricted peptide (amino
acid residues 321–336) of human ENOA recognized by
CD4
+
T cell and able to confer cytotoxic susceptibility
has been identified [131,132].
Clinical correlations
The diagnostic and prognostic value of ENOA expres-
sion and production of autoantibodies to it has been
illustrated in several tumors (Table 1). In breast can-
cer, enhanced ENOA expression is correlated with
greater tumor size, poor nodal status and a shorter dis-
ease-free interval [78]. In head and neck and nonsmall
cell lung cancer, patients with high ENOA expression
had significantly poorer clinical outcomes than low
expressers, including shorter overall- and progression-
free survival [91,99]. In hepatocellular cancer, expres-
sion of ENOA increased with tumor de-differentiation
and correlated positively with venous invasion [93,94].
In breast and lung cancer patients, anti-ENOA
autoantibodies are decreased in the advanced stages of
the disease [69]. In pancreatic cancer, detection of au-
toantibodies against Ser 419 phosphorylated ENOA
usefully complemented the diagnostic performance of
serum CA19.9 levels up to 95%. The presence of this
humoral response was also correlated with a longer
progression-free survival upon gemcitabine treatment

and overall survival, supporting the clinical significance
of phosphorylated ENOA autoantibodies [119].
The concept that autoantibody levels can also function
as markers for the diagnosis and prognosis of cancers
has been extensively pursued [69,133].
a-enolase in tumor diagnosis and therapy M. Capello et al.
1068 FEBS Journal 278 (2011) 1064–1074 ª 2011 The Authors Journal compilation ª 2011 FEBS
Conclusions
Taken as a whole, these findings illustrate the multi-
functional properties of ENOA in tumorigenesis, and
its key implications in cancer proliferation, invasion
and immune response. In cancer cells, ENOA is overex-
pressed and localizes on their surface, where it acts as a
key protein in tumor metastasis, promoting cellular
metabolism in anaerobic conditions and driving tumor
invasion through plasminogen activation and extracel-
lular matrix degradation. It also displays a characteris-
tic pattern of PTMs, namely acetylation, methylation
and phosphorylation, that regulate protein functions
and immunogenicity. In several kinds of tumor,
patients develop an integrated response of CD4
+
,
CD8
+
T cells and B cells against ENOA, together with
anti-ENOA autoantibodies in their sera. Clinical corre-
lations propose ENOA as a novel target for cancer
immunotherapy. In pancreatic cancer, for example, the
pancreas-specific Ser 419 phosphorylated ENOA is

upregulated and induces the production of autoanti-
bodies with diagnostic and prognostic value (Fig. 1).
Acknowledgements
The authors thank Dr W. Zhou for discussion on the
role of post-translational modifications in the regulation
of protein functions and Dr J. Iliffe who critically
reviewed the manuscript. This work was supported in
part by grants from the Associazione Italiana Ricerca
sul Cancro (AIRC); Fondazione San Paolo (Special
Project Oncology); Ministero della Salute: Progetto
strategico, ISS-ACC, Progetto integrato Oncologia;
Regione Piemonte: Ricerca Industriale e Sviluppo
Precompetitivo (BIOPRO and ONCOPROT), Ricerca
Industriale ‘Converging Technologies’ (BIOTHER),
Progetti strategici su tematiche di interesse regionale
o sovra regionale (IMMONC), Ricerca Sanitaria
Finalizzata, Ricerca Sanitaria Applicata; Ribovax
Biotechnologies (Geneva, Switzerland) and Fondazione
Italiana Ricerca sul Cancro (FIRC).
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