Apart from its enzymatic activity, in many prokary-otic and eukaryprokary-otic cells, ENOA is expressed on the cell surface, where it acts as a plasminogen receptor promoting cell migrat
Trang 1a-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
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 eukaryprokary-otic 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: franco.novelli@unito.it
(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.
Trang 2first 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 a2-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-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,
Trang 3phosphorylation, 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 mm3, 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) (http://www.uniprot.org/ 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.
Prostate m, p (100%) [77,103]
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.
Trang 4http://www.uniprot.org/ uniprot/P06733#ref14
Trang 5three 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]
Trang 6Taken 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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