Biochimica et Biophysica Acta 1854 (2015) 687–695

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Biochimica et Biophysica Acta

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Review

Current advances in esophageal cancer proteomics☆

Norihisa Uemura a,1, Tadashi Kondo b,⁎,1
a
Department of Gastroenterological Surgery, Aichi Cancer Center Hospital, 1-1 Kanokoden, chikusa-ku, Nagoya, Aichi 464-8681, Japan
b
Division of Pharmacoproteomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan

a r t i c l e i n f o a b s t r a c t

Article history: We review the current status of proteomics for esophageal cancer (EC) from a clinician's viewpoint. The ultimate
Received 4 June 2014 goal of cancer proteomics is the improvement of clinical outcome. The proteome as a functional translation of the
Received in revised form 4 September 2014 genome is a straightforward representation of genomic mechanisms that trigger carcinogenesis. Cancer proteo-
Accepted 9 September 2014
mics has identified the mechanisms of carcinogenesis and tumor progression, detected biomarker candidates for
Available online 16 September 2014
early diagnosis, and provided novel therapeutic targets for personalized treatments. Our review focuses on three
Keywords:
major topics in EC proteomics: diagnostics, treatment, and molecular mechanisms. We discuss the major
Esophageal cancer histological differences between EC types, i.e., esophageal squamous cell carcinoma and adenocarcinoma, and eval-
Proteomics uate the clinical significance of published proteomics studies, including promising diagnostic biomarkers and novel
Biomarker therapeutic targets, which should be further validated prior to launching clinical trials. Multi-disciplinary collabo-
Neoadjuvant therapy rations between basic scientists, clinicians, and pathologists should be established for inter-institutional validation.
Molecular mechanism In conclusion, EC proteomics has provided significant results, which after thorough validation, should lead to the
development of novel clinical tools and improvement of the clinical outcome for esophageal cancer patients.
This article is part of a Special Issue entitled: Medical Proteomics.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Esophageal cancer (EC) is the fifth for men and the eighth for
women, most common cause of cancer-related death worldwide [1].
Despite the use of modern surgical techniques in combination with
radiotherapy and chemotherapy, early recurrence is common in patients
with advanced disease and the overall 5-year survival rate remains below
40% [2]. In contrast, the 5-year survival rate for patients with early stage
lesions after endoscopic or surgical treatment exceeds 90% [3]. Survival
substantially decreases with the increase of tumor invasion and the pres-
ence of regional lymph node metastases and distant metastases [2]. These
clinical facts clearly indicate that early detection is the key to EC cure.
However, fewer than 30% of EC patients appeared in the hospital at
early stage [4], and early screening modalities are critically needed. For
Abbreviations: 2-DE, Two dimensional gel electrophoresis; 2D-DIGE, Two-dimensional
difference gel electrophoresis; BRE, Brain and reproductive organ-expressed; COX,
Cytochrome c oxidase subunits; CRT, Chemoradiotherapy; EAC, Esophageal adenocarcino-
ma; EC, Esophageal cancer; ESCC, Esophageal squamous cell carcinoma; HER2, Human epi-
dermal growth factor receptor 2; Hsp, Heat shock protein; KLH, Keyhole limpet
hemocyanin; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; MALDI-TOF
MS, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; SELDI-
TOF MS, Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
☆ This article is part of a Special Issue entitled: Medical Proteomics.
⁎ Corresponding author at: Division of Pharmacoproteomics, National Cancer Center
Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Tel.: +81 3 3542
2511; fax: +81 3 3547 5298.
E-mail addresses: nuemura@aichi-cc.jp (N. Uemura), takondo@ncc.go.jp (T. Kondo).
1
Uemura N and Kondo T contributed equally to this study.
the patients with advanced disease, neoadjuvant therapy has been
proved extremely valuable and is established as a standard treatment
[5–11]. However, a significant proportion of treated patients (60–70%)
do not respond well to neoadjuvant regimens and develop severe
adverse effects [10,12]. Thus, risk stratification for neoadjuvant therapy
is required to improve treatment quality for patients with EC. Moreover,
elucidation of molecular mechanisms underlying the resistance to treat-
ment may lead to novel therapeutic strategies and/or agents.
Cancer proteomics has been conducted with the goal to identify
diagnostic biomarkers and improve clinical outcome. The biomarkers
should be used for early cancer detection and effective prediction of
cancer clinical behavior, as well as identification of novel molecular
targets involved in tumorigenesis and disease progression [13]. Cancer
proteomics is a promising approach because proteome is a functional
translation of the genomic aberrations that initiate and promote the dis-
ease. Many lines of evidence indicate the discrepancy between protein
and mRNA expression, and recent studies demonstrated that protein
levels are mainly determined at the translation rather than transcriptional
step [14–16]. Posttranslational modifications, protein interactions,
activity, and localization are also unique data that can be obtained
only by proteomics [17]. Since aberrant protein expression is an impor-
tant feature of cancerous phenotype, the analysis of cancer proteome
can result in the identification of biomarker candidates associated with
clinical characteristics and can further understanding of the molecular
mechanisms underlying the initiation and progression of EC [18].
In the last decade, the advancement in proteomics has led to a
comprehensive understanding of the EC proteome. For example, in

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two-dimensional gel electrophoresis (2-DE), the separation in the first
dimension was improved by the use of immobiline dry strip gels [19],
which enabled the isolation and identification of low-expressed pro-
teins by mass spectrometry. Two-dimensional difference gel electro-
phoresis (2D-DIGE) provided compensation of gel-to-gel variations by
the use of an internal standard sample, while the application of highly
sensitive fluorescent dyes enabled expression profiling of small protein
amounts from laser microdissected tissues [20–22]. The number of
detectable protein spots has increased from hundreds to thousands
through advances in analytical detection of low-molecular-weight pro-
teins [23]. In addition to gel-based techniques, mass spectrometry and
highly sensitive immunodetection methods have also been successfully
applied to EC proteomics [24,25] (Fig. 1).
In the early 2000s, proteomics studies in EC focused on the compar-
ison between tumor and non-tumor tissues, and a considerable number
of proteins with differential expression in esophageal tumors was
detected and functionally examined [26–28]. Although these studies
provided potential keys to the understanding of molecular background
underlying EC development, considerable diversity of biological mech-
anisms involved in cancer initiation and progression, as well as hetero-
geneity of esophageal tumors, made it difficult to link the detected
proteins to specific clinical and pathological data. Then, clinically
oriented proteomics studies, which integrated protein profiling with
clinical and pathological observations of primary tumors from different
patients, were launched as the next step toward clinical application.
Our review focuses on three major topics in EC proteomics: diagnos-
tics, treatment, and molecular mechanisms. We will also consider histo-
logical types of EC, namely esophageal squamous cell carcinoma (ESCC)
and adenocarcinoma (EAC). It has been shown that the predominant
histological type of EC is region-specific. Although ESCC prevails in
most of the world, the incidence of EAC now exceeds that of ESCC in
Australia, the USA, and some Western European countries (e.g., the
UK, Finland, France, and the Netherlands) [29,30]. ESCC and EAC are
Fig. 1. Workflow of proteomics application to biomarker development in EC. Proteins in surgical specimens are extracted and subjected to gel-based and antibody-based proteomics. The
results are verified by immunohistochemistry, and applied to clinical applications [23,24].
also clinically distinct. In a study of 8562 patients who underwent
surgical resection, Merkow et al. found that ESCC histology was the
only factor predictive of pathologic complete response to neoadjuvant
treatment [31], consistent with previous observations showing that his-
tological type might affect response to neoadjuvant chemotherapy and
subsequent prognosis for EC patients [32]. Thus, histologically different
ESCC and EAC should be discriminated in EC proteomics.
Second, we focus on the clinical significance of proteomic studies.
We believe that the ultimate goal of cancer proteomics should be
to bring solutions to clinical problems and provide a better grasp of
situations encountered in clinical practice. Accordingly, we reviewed
previous studies of EC proteome from a clinician's viewpoint.
2. Diagnosis
2.1. Early EC detection
The most sensitive tool for the early detection of EC is
chromoendoscopy with Lugol's dye [33]. During the 1990s, multiple
reports demonstrated that endoscopy provided an easy, inexpensive,
and sensitive means of detecting early and late squamous cell neoplasia.
However, because of an unfavorable balance of risks, benefits, costs, and
psychological burden, the implementation of a national endoscopic
screening program has not been justified [34]. Currently, there is a
need for new, less invasive, and less expensive screening tools, because
most gastroenterology facilities would not be able to support the endo-
scopic screening program [35]. A simple blood test for early EC detection
is superior to endoscopy in terms of cost, testability, and invasiveness. In
current practice, the greatest clinical benefit would be potentially
offered by novel serum biomarkers with high sensitivity and specificity
for EC. With this notion, proteomics studies have been conducted to
develop EC biomarkers that would allow early cancer detection in
blood instead of tissues.
 N. Uemura, T. Kondo / Biochimica et Biophysica Acta 1854 (2015) 687–695 689

2.1.1. ESCC utility should be validated in different populations of EC patients. First,

Fan et al. analyzed serum biomarkers associated with ESCC using the
ClinProt profiling technology based on mass spectrometry [36]. Tubulin
beta chain, filamin A alpha isoform 1, and cytochrome b-c1 complex
subunit 1 were identified as ESCC diagnostic serum biomarkers applica-
ble also to stage I disease (Table 1). Zhang et al. compared protein
expression profiles in pre-surgery and post-surgery sera of 17 ESCC pa-
tients by 2-DE and matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF MS) [26]. One of the differentially
expressed proteins, clusterin, was found to be downregulated in both
cancer tissue and pre-surgery serum, which was validated by RT-PCR
and Western blotting, suggesting that clusterin is a candidate serum
biomarker for ESCC. Liu et al. used 2-DE to examine tumor proteins
that elicit humoral response in ESCC patients [37] and detected autoan-
tibodies to oncogene CDC25B phosphatase in 36.29% of ESCC patients,
8.67% of other cancer patients, and none of the healthy controls. Given
that anti-CDC25B antibodies were found in patients with both early
and late ESCC stages, these antibodies might have clinical utility in
ESCC screening and diagnosis. Fujita et al. examined novel tumor anti-
gens in an ESCC cell line and related autoantibodies in sera of ESCC
patients by proteomics [38]. The levels of an autoantibody against heat
shock protein Hsp70 appeared to be significantly higher in patients
with ESCC than in healthy individuals or patients with gastric or colon
cancer, suggesting that anti-Hsp70 autoantibody may have clinical
utility as a diagnostic marker for ESCC.
2.1.2. EAC
Mechref et al. performed glycosylation profiling of serum samples
from patients with EAC, high-grade dysplasia, Barrett's esophagus, and
healthy individuals and showed that the changes in the relative intensi-
ties of three glycan structures predicted EAC with 94% sensitivity
and more than 60% specificity [39], suggesting that comparative
EAC glycomics can be used for the identification of EAC candidate
biomarkers.
The survival rate of EC patients remains low largely because of the
absence of effective screening markers for early detection. Because
patients with early EC stages benefit from endoscopic treatment or min-
imally invasive esophagectomy as curative resection [40], it is hoped
that novel serum biomarkers for early EC detection, as well as less
invasive and cheaper screening tools, can be developed through multi-
center validation studies.
2.1.3. Perspectives of biomarkers for early detection
Cancer proteomics allows identification of early disease biomarkers
with high sensitivity and specificity; however, the biomarker clinical
the number of cases in the previous studies was limited, and it is ques-
tionable whether high biomarker performance observed in the exam-
ined cohort could be reproduced in the hospitals without further
validation. Second, the reported biomarkers have moderate diagnostic
accuracy, and in screening of a low-prevalence disease, such as EC,
many persons can have false-positive test results. As these patients
will be monitored by expensive and invasive examinations such as
imaging and biopsy, in order to avoid this, the accuracy of biomarker
performance should be additionally validated in a low-prevalence
population. The adverse consequences of high false-positive rate in EC
screening should be thoroughly considered before applying the results
of biomarker studies to early disease detection.
2.2. Prediction of lymph node metastasis
In EC, lymph node metastasis originates from the superficial cancer
and spreads quickly from the neck to the abdomen [41]. The status of
lymph node metastasis is one of the most critical parameters that affects
prognosis and determines treatment strategy. Computed tomography,
magnetic resonance imaging, ultrasonography, endoscopic ultrasonog-
raphy, positron emission tomography, and other methods have been
employed for the diagnostics of nodal metastases. Despite the use of
such modern diagnostic modalities, the diagnosis of lymph node metas-
tasis has insufficient accuracy. Stiles et al. reported that most patients
with cT2-T3N0M0 cancer, a resectable advanced cancer without clinical
lymph node metastases, had pathological lymph node metastases and
despite induction therapy, more than 50% had persistent nodal disease
[42]. The accurate clinical N staging of EC is difficult, and the improve-
ment in diagnostic accuracy could play a crucial role in selecting ade-
quate treatment strategy. The identification of predictive biomarkers
for lymph node metastasis would have important clinical implications
and could resolve particular concerns regarding diagnostics of lymph
node metastasis.
2.2.1. ESCC
Hatakeyama et al. examined the ESCC proteome in 72 laser micro-
dissection samples using two-dimensional difference gel electrophore-
sis (2D-DIGE) and liquid chromatography-tandem mass spectrometry
(LC-MS/MS) [43]. Protein spots with intensities statistically different
between patients without nodal metastasis and patients with more
than five lymph node metastases included cancer-associated factors
such as alpha-actinin 4, hnRNP K, periplakin, squamous cell carcinoma
antigen 1, NudC, and Hsp60; the latter was increased in tumor tissues
almost in parallel with the number of lymph node metastases.

Table 1
Summary of biomarker candidates discovered by proteomics in EC.

Purpose Histology Protein name Reference

Early detection ESCC tubulin beta chain, filamin A alpha isoform 1, and cytochrome b-c1 complex subunit 1 36
clusterin 26
autoantibodies to oncogene CDC25B phosphatase 37
autoantibody against heat shock protein Hsp70 38
EAC glycan structures 39
Prediction of lymph node metastasis ESCC alpha-actinin 4, hnRNP K, periplakin, squamous cell carcinoma antigen 1, NudC, and Hsp60 43
alpha-actinin 4 44
annexin A2, Cdc42 45
EAC Hsp27 family proteins, HER2 46
Prdiction of response to treatments ESCC thioredoxin domain-containing protein 4 precursor and cystathionine gamma-lyase 53
EAC COX protein 54
apolipoprotein A-1, serum amyloid A, and transthyretin 55
Hsp27 56
ESCC and EAC C4a and C3a 57
Prediction of prognosis ESCC transglutaminase 3 23
calreticulin and a 78-kDa glucose-regulated protein 59
EAC Hsp27 and HER2 46
ESCC and EAC apolipoprotein A-I, serum amyloid A, and transthyretin 60
690 N. Uemura, T. Kondo / Biochimica et Biophysica Acta 1854 (2015) 687–695
Fu et al. employed proteomics to analyze protein extracts of 12 ESCC
specimens from patients with distinctly different tumor stages [44].
Using 2D-GE and MALDI-TOF MS, they identified 18 proteins differen-
tially expressed in tumors compared to matched surrounding normal
tissues; among them, the expression of alpha-actinin 4 was shown to
be significantly associated with lymph node metastasis by immunohis-
tochemistry analysis using two-tissue microarray blocks that contained
442 primary tumor samples. Because two independent studies consis-
tently identified alpha-actinin 4, it may be a promising biomarker for
nodal metastasis.
Other proteins related to lymph node metastasis were identified by
Feng et al., who reported that the expression of annexin A2 in ESCCs was
significantly downregulated and that of Cdc42 upregulated compared
with corresponding normal esophagus mucosa; it was also shown that
their levels significantly correlated with nodal metastasis [45]. The
authors proposed that the aberrant expression of annexin A2 and
Cdc42 played a role in the carcinogenesis and metastasis of ESCC.
2.2.2. EAC
Berg et al. analyzed expression profiles of 17 cancer-related signal-
ing proteins in a series of 87 formalin fixed and paraffin-embedded
EAC tissue samples using a reverse phase protein array [46]. They
found a molecular subtype of EAC characterized by low levels of the
Hsp27 family proteins and high expression of the human epidermal
growth factor receptor 2 (HER2) family members. Patients with this
EAC subtype were more likely to have lymph node and distant metasta-
ses and short overall survival. Compared with all other EACs, this EAC
subtype included significantly more cases positive for pN (lymph node
status) and cM (occurrence of distant metastasis): 67% and 15%,
respectively. Another new EAC subtype had the opposite pattern: low
expression of HER2 proteins and high expression of Hsp27 family
members, few pN-positive (21%), and no cM-positive (0%) cases.
2.2.3. Perspectives of biomarkers for lymph node metastasis
Cancer proteomics provided promising predictive biomarkers for
lymph node metastasis. However, the benefit of these biomarkers re-
mains unproven in clinical settings. First, the significance of correlation
between the biomarkers and nodal metastasis should be validated in
larger cohorts in multiple institutions. Second, therapeutic strategies
for the treatment of biomarker-positive but otherwise negative
cases should be established by randomized trials. Nodal metastases un-
detectable by imaging tests are very small, and the presence of such
micrometastases normally has no impact on the prognosis for ESCC pa-
tients [47]. For the patients with image-negative nodal micrometastases
who are positive in biomarker and pathology tests, the prognostic value
of other clinical examinations must be considered when planning for
pre-operative therapy.
2.3. Therapy response prediction
Patients at the early EC stage can be cured by endoscopic or surgical
treatment. However, the patients seen at the hospital usually have a
developed disease, and a number of multimodal approaches to the
treatment of late EC have been developed. Neoadjuvant chemotherapy,
alone or in combination with radiotherapy (CRT), became a standard
approach to locally advanced EC [48]. Although favorable response to
multimodal treatment results in better clinical outcome, randomized
trials of different neoadjuvant regimens for locally advanced cancers
have shown modest increase in survival [5–11]. Only those patients
who achieved complete histopathologic response seem to significantly
benefit from neoadjuvant therapy, whereas most of the treated patients
(60–70%) did not respond well and experienced severe adverse effects
[10,12]; treatment responses may vary even among patients with the
same clinical stage of the disease. The problem is that nonresponsive
patients may lose the option of surgical resection after ineffective
chemotherapy [49], and their prognosis has been found inferior to
that of patients treated by surgery alone [50]. Thus, to avoid potential
morbidity due to ineffective treatment and prevent further disease
progression, it is essential to perform accurate risk stratification of EC
patients. The clinical significance of response predictions for patients
with unresectable EC can be interpreted in a similar fashion.
Nonresponding patients with unresectable EC should receive a different
treatment at early stages. In this context, the identification of predictive
markers is important for the risk stratifications and individualization of
multimodality treatments for patients with advanced EC [51].
2.3.1. ESCC
Hayashida et al. used SELDI-MS to analyze proteomic profiles in
27 serum samples from untreated ESCC patients and predict the efficacy
of preoperative chemoradiotherapy [52]. The authors found that out of
859 protein peaks, a set of four (at 7,420, 9,112, 17,123, and 12,867 m/z)
could be used to distinguish responders from nonresponders with
a sensitivity of 100% and 93.3% in the training and validation sets,
respectively.
Wen et al. investigated the mechanisms of drug resistance in ESCC
by comparing expression profiles of a multidrug-resistant ESCC cell
line EC 109/CDDP with its parental drug-sensitive cell line using
2D-GE and MALDI-TOF MS [53]. The identified 44 differentially
expressed proteins were involved in endoplasmic reticulum stress
response, metabolic processes, DNA replication and repair, nucleotide
binding, calcium binding, and cytoskeleton formation; among them,
thioredoxin domain-containing protein 4 precursor and cystathionine
gamma-lyase were validated by western blot and RT-PCR.
2.3.2. EAC
In a study of 23 EAC pre-therapeutic biopsy samples, Aichler et al.
analyzed proteomic changes associated with response to chemotherapy
by MALDI imaging mass spectrometry and LC-MS/MS, and showed that
clinical response to cisplatin was associated with defects in cancer
cell mitochondrial respiratory chain caused by the loss of specific
cytochrome c oxidase (COX) subunits [54]. These results were further
validated in an independent study showing that reduced COX protein
expression prior to treatment correlated with chemotherapeutic
sensitivity and favorable clinical outcomes, whereas unchanged COX
expression indicated chemoresistance.
Plasma protein profiling in mice with OE19 EAC xenografts
that were treated with clinically relevant chemotherapeutic agents
epirubicin, cisplatin, or 5-fluorouracil resulted in the identification of
apolipoprotein A-1, serum amyloid A, and transthyretin as candidate
biomarkers of response to chemotherapy [55]. However, although the
results were confirmed in clinical samples, the number of patients
with evident pathological response was relatively small; the biomarker
pattern could not be compared with other indicators of clinical response
and required further validation.
To identify predictive biomarkers, Langer et al. analyzed pre-
therapeutic protein expression profiles in tumor biopsy specimens of
34 EAC patients treated with neoadjuvant chemotherapy [56]. The
authors found that the levels of cellular stress response-associated
Hsp27 and Hsp60, glucose-regulated proteins 94 and 78, and a number
of cytoskeletal proteins differed significantly between responders
and nonresponders. Immunohistochemistry and gene expression
analysis confirmed a significant association between low Hsp27 expres-
sion and resistance to neoadjuvant chemotherapy, suggesting that
chemosensitivity may be related to stress-activated inflammatory
mechanisms.
2.3.3. ESCC and EAC
Maher et al. examined proteomic profiles of ESCC and EAC patients
using surface-enhanced laser desorption/ionization time-of-flight
mass spectrometry (SELDI-TOF MS) and ELISA [57]. By comparing
pre-treatment serum samples from 16 poor responders and 15 good re-
sponders, it was found that higher serum levels of complement factors
 N. Uemura, T. Kondo / Biochimica et Biophysica Acta 1854 (2015) 687–695
C4a and C3a were significantly associated with favorable CRT response.
The leave-one-out cross-validation analysis confirmed that C4a and C3a
could predict the response to neoadjuvant CRT with a sensitivity and
specificity of 78.6% and 83.3%, respectively.
2.3.4. Perspectives of treatment response predictive biomarkers
Cancer proteomics has provided promising predictive biomarkers
for multimodal therapies. Therapeutic resistance is multi-factorial and
is associated with several cell signaling mechanisms, including mito-
chondrial redox [53], respiration [54], and cellular stress response path-
ways [56]. Proteomics can be a powerful tool in identifying protein
biomarkers associated with resistance to anticancer therapy [18].
As EC is a clinically diverse disease and treatment modalities are not
fully standardized among hospitals, extensive multi-institutional,
large-scale studies are required to validate the clinical utility of predic-
tive biomarker candidates. It is also urgent to develop novel therapeutic
approaches for patients with biomarker-predicted resistance to
standardized treatment regimens. Thorough investigation of the mech-
anisms underlying functional association of biomarker proteins with
clinical parameters should provide a key to the development of novel
effective clinical applications to treat CRT-resistant EC.
2.4. Prognostic prediction
Prognostic prediction in EC has important clinical implications be-
cause it critically affects decisions regarding the treatment strategy
and follow-up schedule. Patients with good prognosis do not require
intensive adjuvant therapy or routine follow-up, whereas those with
poor prognosis must receive strong adjuvant treatment and be
frequently examined. Thus, an accurate risk stratification of EC patients
is of paramount importance to prevent further disease progression and
reduce morbidity due to ineffective/unnecessary clinical intervention.
2.4.1. ESCC
Uemura et al. compared proteomic profiles of ESCC patients with
favorable and poor prognosis using 2D-DIGE and mass spectrometry
[23] and identified 18 differentially expressed proteins. Of them,
transglutaminase 3 expression was validated in 76 primary tumor sam-
ples by immunohistochemistry and found to be inversely correlated
with shorter patient survival.
In search of ESCC prognostic serum biomarkers using SELDI-TOF MS,
Jiang et al. found no difference between protein profiles patients with
more than 2-year and less than 2-year survival [58]. Furthermore, only
1 protein peak differed significantly between stage III disease patients
stratified by this survival criterion.
However, Du et al., using 2-DE and MALDI-TOF or LC-ESI MS,
found 22 proteins differentially expressed in ESCCs compared to
tumor-adjacent normal esophageal tissues [59] and showed that high
expression of calreticulin and a 78-kDa glucose-regulated protein
were correlated with poor prognosis.
2.4.2. EAC
Berg et al. reported that a particular EAC molecular subtype charac-
terized by low levels of the Hsp27 proteins and high HER2 expression
correlated with nodal and distant metastases, and short overall survival
[46].
2.4.3. ESCC and EAC
Kelly et al. used SELDI-TOF MS to examine EC prognostic biomarkers
in plasma of 20 patients with EAC, 1 with ESCC, 2 with poorly differen-
tiated carcinomas, and 1 with severe dysplasia [60]. Apolipoprotein A-I,
serum amyloid A, and transthyretin in pre-treatment plasma samples
were found to be associated with disease-free survival and overall sur-
vival. The authors also reported the correlation of these three proteins
with response to chemotherapy in a mouse xenograft model [55].
These data suggest that plasma apolipoprotein A-I, serum amyloid A,
691
and transthyretin are promising candidate prognostic biomarkers that
should be validated in EC clinical studies.
2.4.4. Perspectives of prognostic biomarkers
Cancer proteomics provided promising prognostic biomarkers for
EC. In clinical practice, post-operative EC prognosis has been made
based on multiple clinicopathological parameters [61,62]; however,
novel biomarkers will be beneficial in the cases when thus made predic-
tion was not confirmed. Clinicopathological characteristics can directly
reflect EC malignancy, but more accurate prognosis could be obtained
by the combination of traditional clinical parameters with novel
molecular biomarkers identified by proteomics. By conducting sub-
group analysis based on the state of lymph node metastasis, a significant
clinicopathological prognostic factor, Uemura et al. succeeded in discov-
ering a reliable prognostic biomarker [23]. Similarly, Jiang et al. identi-
fied a prognostic biomarker by correlation with EC clinical stage [58].
Patients' stratification according to clinical background would allow
compensation for the heterogeneity of molecular mechanisms involved
in EC and provide a potential to increase the sensitivity and specificity of
EC prognostic biomarkers.
3. Treatment: Novel targets and chemotherapeutic drugs
The prediction of responses to CRT may help reduce the number of
unnecessary treated patients. However, without effective therapies to
be offered to nonresponders, the prediction of CRT outcome would be
tantamount to abandoning nonresponders to their fate. Significant
improvement in overall EC outcome has to be parallel to that for
CRT-resistant population; accordingly, there is an urgent need for new
effective therapeutic approaches for patients unresponsive to conven-
tional treatment. The identification of reliable prognostic biomarkers
through proteomic approaches may identify novel drug targets and
lead to the development of new more effective therapeutic strategies
for CRT-resistant EC patients [63].
3.1. ESCC
Using 2-DE, Fu et al. compared proteomic profiles of ESCC tumors
at different stages and adjacent normal esophageal tissues [44] and
identified two proteins, alpha-actinin 4 and a 67-kDa laminin receptor
associated with ESCC progression (Table 2). The authors suggested
that these candidate biomarkers might be useful for prognostic evalua-
tion, molecular classification, and therapeutic targeting of ESCCs.
3.2. EAC
In the analysis of proteomic changes associated with EAC response
to chemotherapy, Aichler et al. discovered that favorable response of
cancer cells to cisplatin was related to the defects in mitochondrial
respiratory complexes caused by the loss of COX subunits [54]. The
authors noted that the association between cancer cell respiration and
chemosensitivity was consistent with anticancer therapeutics that
target the mitochondrial electron transport chain.
Vona-Davis et al. used 2-DE to examine the response of Barrett's
cancer cells to a novel therapeutic agent keyhole limpet hemocyanin
(KLH) [64], an immunostimulatory oxygen-carrying metalloprotein
from a marine mollusk Megathura crenulata, which inhibits the growth
of Barrett's EC cell lines through both apoptotic and nonapoptotic mech-
anisms [65,66]. Proteomic profiling showed that KLH downregulated
proteins associated with glycolysis and protein synthesis, while upreg-
ulating those related to oxidative stress (the Hsp70 proteins and
UDP-glucose 6-dydrogenase), indicating the molecular mechanisms of
KLH anticancer effects. These results suggest that KLH used as an adju-
vant or topical therapy is a promising drug in the treatment of Barrett's
adenocarcinoma.
692 N. Uemura, T. Kondo / Biochimica et Biophysica Acta 1854 (2015) 687–695

Table 2
Summary of key proteins on treatment and molecular mechanisms discovered by proteomics in EC.

Purpose Histology Protein name Reference

Novel melecular target ESCC alpha-actinin 4 and a 67-kDa laminin receptor 44

EAC COX protein 54
migration-stimulating factor 67
Novel chemotheraupeutic drug EAC keyhole limpet hemocyanin 64
Elucidation of molecular mechanism underlying carcinogenesis EC cell line brain and reproductive organ-expressed protein 80
EAC Rho GDP dissociation inhibitor 2, alpha-enolase, lamin A/C, and 81
nucleoside-diphosphate kinase A
cathepsin D and aldo-ketoreductases 1C2 and 1B10 82
Elucidation of molecular mechanism underlying histological differentiation ESCC stratifin, prohibitin, and squamous cell carcinoma antigen 1 83
annexin A2 84
cathepsin D 85

Hu et al. screened a mouse-derived antibody library generated 4.1. Carcinogenesis

against EC endothelial cells to identify proteins that participate in
tumor angiogenesis [67]. Through selection of monoclonal antibodies To explore carcinogenic mechanisms through protein profiling,
that reacted with EC cell-surface antigens and influenced tumor cell Chen et al. examined soluble proteins differentially expressed in EC
behavior, they identified the antibody against migration-stimulating and non-cancerous cells using 2-DE and MALDI-TOF MS [80]. Among
factor (MSF), which significantly suppressed tumor growth through 20 differentially expressed protein spots, brain and reproductive
inhibition of human tumor-related angiogenesis. These results indicate organ-expressed (BRE) protein was selected and functionally analyzed
that MSF may be a target for the anti-angiogenic treatment of esophageal using small interference RNA (Table 2). BRE silencing upregulated
cancer. tumor-suppressor p53 and downregulated prohibitin, cyclin A, and
CDK2, suggesting that BRE plays an important role in mediating
antiapoptotic and proliferative responses in EC cells.
3.3. Perspectives of novel therapeutics identified by EC proteomics
The insight into potential mechanisms underlying the progression
from Barrett's metaplasia to EAC was gained by Zhao et al. [81], who
Protein profiling resulted in the identification of novel therapeutic
found that Rho GDP dissociation inhibitor 2, alpha-enolase, lamin A/C,
targets, which can lead to a significant improvement of EC prognosis, es-
and nucleoside-diphosphate kinase A were upregulated at both mRNA
pecially for nonresponders diagnosed by predictive protein biomarkers.
and protein levels in EAC compared to Barrett's metaplasia. The results
In other solid tumors, several targeted therapeutic agents, such as
suggest that the identified proteins play a role in EAC development
bevacizumab and cetuximab in colorectal cancer [68], lapatinib and
and may be candidate biomarkers of the progression from Barrett's
trastuzumabin in breast cancer [69,70], and imatinib and sunitinib in
metaplasia to EAC.
gastrointestinal stromal tumor [71,72] have been widely used in clinical
Breton et al. used 2-DE and MALDI-TOF MS to examine proteomic
practice. In EC, the addition of anti-HER2 antibody trastuzumab to the
profiles of 10 esophageal cell lines representing distinct stages in EAC
platinum and fluoropyrimidine doublet regimen is adopted as the stan-
development [82]. Among 33 differentially expressed proteins, the in-
dard care for HER2-positive tumors of the esophagogastric junction [73].
creased levels of cathepsin D and aldo-ketoreductases 1C2 and 1B10
Therapies targeting epidermal growth factor receptor and vascular
in metaplastic and dysplastic cell lines were confirmed by Western blot-
endothelial growth factor remain under active investigation but have
ting and qRT-PCR. The expression patterns of these proteins analyzed
yet to definitively demonstrate clinically significant benefits for EC
from esophageal epithelium from patients with non-erosive and erosive
patients [74]. Multiple molecular targets remain of interest in EC, in-
gastroesophageal reflux disease, Barrett's esophagus, and EAC, suggest
cluding mammalian target of rapamycin, c-MET, insulin-like growth
the contribution to EAC development through effects on apoptosis,
factor, and cytotoxic T-lymphocyte antigen 4 [74]. Clinical application
transport of bile acids, and retinoid metabolism. Further mechanistic
of targeted therapies in EC is less advanced than in other solid tumors,
and clinical investigations are required to fully elucidate the role of
and further proteome studies are required to identify more promising
these molecules in EAC.
molecules.

4.2. Histological differentiation

4. Molecular mechanisms
Using 2-DE and MALDI-TOF MS, Qi et al. examined protein changes
Elucidation of disease molecular mechanisms is directly linked to the
associated with the degree of ESCC differentiation [83] and found that
development of novel diagnostic biomarkers and therapeutic targets
the expression of stratifin, prohibitin, and squamous cell carcinoma
that would bring significant benefits to cancer patients. In the molecular
antigen 1 directly correlated with ESCC differentiation. The same group
mechanistic analysis, proteomics provides tools essential to the discov-
also reported that the expression of annexin A2 was step-wise downreg-
ery of signaling paradigms that trigger tumor initiation and promote
ulated during malignant transformation of epithelial cells [84]: in poorly
cancer progression. The transformation from normal esophageal epithe-
differentiated ESCCs, annexin A2 was either not detected (46%) or had
lium to invasive EC is a multistep process caused by accumulation of
low expression levels (36%). Furthermore, it has been shown that overex-
multiple genetic alterations [75], mainly involving the Wnt, cell cycle,
pression of cathepsin D inversely correlated with ESCC differentiation
and Notch pathways [76]. Despite many genetic studies, the exact mo-
[85]. The significant changes in the expression of these proteins may indi-
lecular mechanisms underlying carcinogenesis and tumor progression
cate that many pathways are involved in ESCC dedifferentiation.
in EC remain unclear [77–79], and comprehensive protein expression
profiling provided by cancer proteomics should contribute to our un-
derstanding of EC biology. Proteomics has identified numerous intrigu- 4.3. Perspectives of proteomics approach to EC molecular mechanisms
ing biomarkers for early cancer diagnostics and prediction of nodal
metastasis, treatment response, and prognosis, which are critically in- Cancer proteomics has resulted in the identification of proteins
volved in each process and require further mechanistic characterization. involved in cancer initiation and progression; further investigation of
 N. Uemura, T. Kondo / Biochimica et Biophysica Acta 1854 (2015) 687–695
the identified molecules may provide deeper understanding of
EC-related mechanisms and contribute to the development of novel
therapeutic strategies. However, the current state of cancer proteomics
indicates fundamental problems and the need for improvement.
Although proteomics provides a unique opportunity of generating com-
prehensive information regarding cellular proteome, it has not been
fully realized in cancer research. At present, cancer proteomics focuses
on a limited portion of proteome, thus excluding some potentially
important molecules, and consequently, it has not yet provided
pathway-wide and molecular family-wide analyses.
Furthermore, in EC, proteomics has been mostly applied to analyze
expression levels, leaving out important proteome features such as
posttranslational modifications, molecular interactions, activity, and
localization, which should be extensively examined in association
with EC clinical and pathological features.
Finally, although cancer proteomics provides high-throughput as-
sessment of protein expression, subsequent functional analysis remains
labor-intense and low-scale. As a consequence, only a small number of
proteins are further investigated, and potentially valuable molecules
may be filtered through functional assessment. All these issues should
be resolved by continuous technological advances.
5. Conclusions
Cancer proteomics has provided a number of promising diagnostic
biomarkers and novel therapeutic targets, as well as intriguing proteins
underlying the molecular mechanisms of carcinogenesis and progres-
sion of EC. Although individual proteins seem to be promising, the num-
ber of clinical samples examined by proteomics has been limited, and
multi-institutional validation studies are required to launch clinical
trials of biomarkers and therapeutic targets. In parallel with confirming
proteomic data, biomarker-based therapeutic strategies integrating the
diverse clinical features of ES should be developed. Current studies have
shed a light on limited aspects of the EC proteome, and comprehensive
analysis will be possible with further technological progress. For exam-
ple, novel antibody production engineering systems that allow generate
appropriate antibody for immunohistochemistry will facilitate the
discovery of biomarker candidates as well as the validation study. In
addition to improving the performance of proteomic modalities, inter-
disciplinary collaboration is essential to obtain research data that will
benefit patients with EC.
Conflicts of interest
The authors report no conflicts of interest.
Role of the funding source
No funding was received for this review.
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