LETTER TO THE EDITOR

Deregulated expression of annexin-A2 and galectin-3 is associatedwith metastasis in gastric cancer patients

Mariana Ferreira Leal • Danielle Queiroz Calcagno • Janete Chung •

Vanessa Morais de Freitas • Samia Demachki • Paulo Pimentel Assumpcao •

Roger Chammas • Rommel Rodrıguez Burbano • Marılia Cardoso Smith

Received: 24 January 2014 / Accepted: 17 June 2014

� Springer-Verlag Italia 2014

Abstract Gastric cancer (GC) is the second highest cause

of cancer mortality worldwide. However, nowadays, most

of the studies aiming to understand the gastric carcino-

genesis analyzed tumors of individuals from Asian popu-

lation and, thus, may not reflect the distinct biological and

clinical behaviors among GC processes. Since several

membrane proteins have been implicated in carcinogenesis,

we aimed to evaluate ANXA2 and GAL3 role in gastric

tumors and GC cell lines of individuals from northern

Brazil. The cellular localization of ANXA2 and GAL3 in

the GC cell lines was evaluated by immunofluorescence.

Gene expression was evaluated by real-time reverse-tran-

scription PCR and protein expression by Western blot in

gastric adenocarcinomas and non-neoplastic gastric

samples, as well as in GC cell lines. ANXA2 and GAL3

were presented as dots in the plasma membrane and

cytoplasm in ACP02 and ACP03 cell lines. ANXA2 mRNA

expression was up-regulated in 32.14 % of gastric tumors

compared to non-neoplastic tissues. ANXA2 up-regulation

was associated with the metastasis process in vivo and with

cell line invasive behavior. GAL3 protein expression was

at least 1.5-fold reduced in 50 % of gastric tumors. The

reduced GAL3 expression was associated with the presence

of distant metastasis and with a higher invasive phenotype

in vitro. Our study shows that ANXA2 and GAL3 dereg-

ulated expression was associated with an invasive pheno-

type in GC cell lines and may contribute to metastasis in

GC patients. Therefore, these proteins may have potential

prognostic relevance for GC of individuals from northern

Brazil.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10238-014-0299-0) contains supplementarymaterial, which is available to authorized users.

M. F. Leal (&) � D. Q. Calcagno � M. C. Smith

Disciplina de Genetica, Departamento de Morfologia e Genetica,

Universidade Federal de Sao Paulo, R. Botucatu, 740, Sao Paulo,

SP CEP 04023-900, Brazil

e-mail: mariana.morf@epm.br

M. F. Leal

Departamento de Ortopedia e Traumatologia, Universidade

Federal de Sao Paulo, Sao Paulo, SP 04038-031, Brazil

D. Q. Calcagno � S. Demachki � P. P. Assumpcao

Nucleo de Pesquisa em Oncologia, Hospital Universitario

Joao de Barros Barreto, Universidade Federal do Para,

Belem, PA 60673-000, Brazil

J. Chung

Departamento de Microbiologia, Imunologia e Parasitologia,

Universidade Federal de Sao Paulo, Sao Paulo, SP 04023-062,

Brazil

V. M. de Freitas

Laboratorio de Biologia da Matriz Extracelular, Departamento

de Biologia Celular e do Desenvolvimento, Instituto de Ciencias

Biomedicas, Universidade de Sao Paulo, Sao Paulo,

SP 05508-000, Brazil

R. Chammas

Laboratorio de Oncologia Experimental, Departamento de

Radiologia, Faculdade de Medicina, Universidade de Sao Paulo,

Sao Paulo, SP 01246-903, Brazil

R. Chammas

Centro de Investigacao Translacional em Oncologia, Instituto do

Cancer do Estado de Sao Paulo, Sao Paulo, SP 01246-000, Brazil

R. R. Burbano

Laboratorio de Citogenetica Humana, Instituto de Ciencias

Biologicas, Universidade Federal do Para, Belem,

PA 66073-000, Brazil

123

Clin Exp Med

DOI 10.1007/s10238-014-0299-0

Keywords Membrane proteins � Proteomic � Gastric

cancer � Annexin 2 � Galectin-3

Dear editor,

Gastric cancer (GC) is the fourth most frequent cancer type

and the second highest cause of cancer mortality world-

wide [1]. A better understanding of the biology of this

neoplasia progression is crucial to reduce the mortality

ratio with the development of novel therapeutic strategies.

However, nowadays, most of the studies aiming to under-

stand the gastric carcinogenesis analyzed tumors of indi-

viduals from Asian population and, thus, may not reflect

the distinct biological and clinical behaviors among GC

processes. GC is marked by global variations in incidence,

etiology, natural course, and management [2]. Although,

about 90 % of stomach tumors are adenocarcinomas [3],

several factors lead to biological and clinical GC subsets,

including the ethnicity of the afflicted population (differing

levels of susceptibility and aggressiveness of the tumors)

[2].

Membranes have a critical role in cell structure by

providing a physical barrier between the cell, the envi-

ronment, and several subcellular compartments. Several

other membrane-associated proteins such as small GTP-

ases, kinases, and catenins are implicated in carcinogenesis

[4]. In the present study, we analyzed the expression of two

proteins that may be localized at cell membranes: annexin-

A2 (ANXA2) and galectin-3 (GAL3). To elucidate whether

ANXA2 and GAL3 have a key role in gastric carcino-

genesis, we evaluated their expression in clinical samples

and GC cell lines, and we also investigated the possible

associations with clinicopathological characteristics and/or

invasion and migration capability of the cell lines.

Here, we evaluated the gene and protein expression in

gastric adenocarcinomas and non-neoplastic gastric sam-

ples from our previous proteomic study [5]. These samples

were obtained from individual of Para’s State, northern

Brazil, where the human population is composed of inter-

ethnic crosses between three main origin groups: European

(mainly represented by the Portuguese), Africans, and

Amerindians [6]. In addition, we also performed the

expression analysis in the ACP02 and ACP03 GC cell

lines, which are also derived from tumor samples of indi-

viduals from northern Brazil [7]. These cell lines retain,

in vitro, genetic alterations present in their parental primary

tumors [7, 8]. In addition, these cell lines have differential

invasive capacity. Although ACP03 cells have higher

migration capability, ACP02 cells is more invasive than

ACP03 cells [9]. Moreover, only ACP03 is able to start a

tumorigenesis process in immunosuppressed non-human

primates [10]. Therefore, these cell lines represent inter-

esting models of gastric carcinogenesis in our population

and may help in the identification of GC diagnostic and

prognostic biomarkers.

First, we evaluated the membrane proteome profile from

a pool of ACP02 and ACP03 GC cell lines to screen the

membrane proteins that may have a role in gastric carci-

nogenesis (Online resource 1: Supplementary methods). In

total, we identified 2,608 proteins in cell membrane enri-

ched fractions. Of these 2,608 proteins, about 90 % (2,305

proteins) were previously described as membrane proteins

(Online Resource 2). Of the membrane proteins, 558

(24.21 %) were previously assigned to the plasma mem-

brane, 1,484 (64.38 %) are located or associated with

organelle membranes, and 345 (14.97 % of the membrane

proteins) were described as endoplasmic reticulum or

Golgi apparatus membrane proteins (Online Resource 2).

Of the plasma membrane proteins, 37 were described in the

only one previous study that performed a comprehensive

analysis of membrane proteome of GC cell lines (Online

Resource 2), in which most of the evaluated cell lines were

derived from tumor samples of Asiatic individuals [11].

We highlight the plasma membrane proteins since they are

readily accessible and well suited as diagnostic and thera-

peutic targets. Extensive classification of malignancies

based on cell surface proteins is often hampered by the lack

of known disease-specific cell surface markers and the lack

of well-characterized antibodies against these markers [12].

One hundred proteins were previously detected as dif-

ferential expressed between GC and non-tumoral samples

in individuals from northern Brazil [5], including 27 pro-

teins assigned to the plasma membrane (Online Resource

2). Among the proteins that may be localized at cell

membranes, ANXA2 and GAL3 are described as cancer

biomarkers according to the human protein atlas database

(http://www.proteinatlas.org/) [13]. Using proteomic

approaches, our group previously reported that ANXA2

seems to be up-regulated in gastric tumors, especially of

individuals with lymph node metastasis, by proteome

analysis [5]. On the other hand, our previously proteomic

analyses had suggested that GAL3 expression might be

reduced in primary gastric tumors. However, their role in

gastric carcinogenesis remains to be understood.

The cellular location of ANXA2 and GAL3 in ACP02

and ACP03 was evaluated by indirect immunofluorescence

according to Calcagno et al. [9]. For this analysis, cells

were incubated with the primary antibodies anti-ANXA2

(1:50; sc-28385, Santa Cruz Biotechnology, USA) or anti-

GAL3 (1:50; clone M3/38, American Type Culture Col-

lection, USA) in PBS overnight at 4 �C. The immunolo-

calization showed that ANXA2 (Fig. 1a, b) and GAL3

(Fig. 1c, d) were presented as dots in the plasma membrane

and cytoplasm in ACP02 and ACP03 cell lines, which is in

agreement with our first screen by membrane proteomic

analysis.

Clin Exp Med

123

ANXA2 mRNA and protein expression and GAL3

expression were evaluated as previously described by our

group [5, 14, 15]. For Western blotting analysis, we used

primary antibodies to anti-ANXA2 (overnight at 4 �C,

1:300, sc-28385, Santa Cruz Biotechnology, USA), anti-

GAL3 (1 h at room temperature, 1:50, clone M3/38,

American Type Culture Collection, USA), and the refer-

ence control (anti-b-actin; Ac-74, 1:3,000, Sigma-Aldrich,

USA). In addition, the ANXA2 mRNA expression was

analyzed by real-time quantitative reverse-transcription

PCR (qRT-PCR) using TaqMan assays for the target gene

(ANXA2: Hs03044144_m1; Applied Biosystems, USA) and

reference (ACTB: Hs03023943_g1; GAPDH: Hs99999

905_m1; Applied Biosystems, USA).

In the present study, we observed that 9 tumors

(32.14 %) presented more than 1.5-fold increased ANXA2

mRNA expression and four presented at least 1.5-fold

reduction (14.28 %; Fig. 2a). However, only one tumor

(5.26 %) presented more than 1.5-fold increased protein

expression and 2 (10.52 %) presented at least 1.5-fold

reduced expression (Fig. 2b, c). No correlation was

observed between ANXA2 mRNA and protein expression

(q = 0.257; P = 0.319, by Spearman’s test). The ANXA2

overexpression was previously reported in several neopla-

sias, including in gastric carcinoma [16], which also cor-

roborates our previous proteomic study [5]. The lack of

correlation between ANXA2 protein expression by Western

blotting and its mRNA expression, as well our previous

proteomic study (data not shown), may be due to the pre-

sence of different isoforms in the gastric tumors that are not

distinguished by the antibody applied. Therefore, different

ANXA2 protein isoforms may have different roles in the

gastric carcinogenesis.

The increased ANXA2 mRNA level was associated with

the presence of lymph node metastasis [P = 0.016, by

Mann–Whitney test; effect size (r) = -0.452; Table 1].

Moreover, ANXA2 mRNA expression was 1.65-fold higher

in ACP02 as compared to ACP03. One previous study

evaluated ANXA2 expression in gastric tumors by immu-

nohistochemistry analysis and reported that ANXA2

overexpression was more frequently found in tumors with

intestinal type, lymph node, and venous invasion [16],

which is in part in agreement with our mRNA expression

analysis and our previous proteomic study in gastric tumors

[5]. ANXA2 is a Ca2?-dependent phospholipid binding

protein that also associates with actin filaments and medi-

ates membrane–membrane and membrane–cytoskeletal

interactions, playing a role in regulating the actin [17–19].

ANXA2 may also act in the regulation of Rho membrane

associations that impact Rho-dependent signaling pathways

and related actin cytoskeletal remodeling during cell

migration [20]. Therefore, ANXA2 may play a role in an

invasive state of gastric tumors.

It is important to highlight that no significant increase

was observed on the protein level in the ACP02 cell line

compared to ACP03 (ratio of ACP02/ACP03 was 0.88) and

Fig. 1 Immunolocalization of

ANXA2 and GAL3 in gastric

cancer cell lines. a ACP02-

presenting ANXA2 expression;

b ACP03-presenting ANXA2

expression; c ACP02 presenting

a GAL3-labeling diffuse all

over cell body; d ACP03

presenting a GAL3-labeling

diffuse all over cell body.

Arrows show suggestive

membrane labeling in both cell

lines. Green target antibody;

blue nuclei (DAPI) (color figure

online)

Clin Exp Med

123

we did not found an association between ANXA2 protein

expression and metastasis by Western blotting analysis

(P [ 0.05, by Mann–Whitney test; Table 1). Therefore,

further investigations are necessary to evaluate which

ANXA2 protein isoforms may be implicated in gastric cell

migration.

GAL3 is an endogenous galactoside-binding lectin

which may be involved in tumor cell adhesion and prolif-

eration. However, the role of GAL3 in gastric carcino-

genesis is still ambiguous. Some previous studies reported

that positive immunoreactivity of GAL3 was frequently

observed in GC [21–23]. However, reduced GAL3

expression was reported in one GC study by immunohis-

tochemistry [24]. In the present study, we quantified GAL3

expression and we observed that GAL3 was at least 1.5-

fold reduced in 50 % and increased in 9.1 % of GC sam-

ples compared to their paired non-neoplastic gastric tissue

(Fig. 2c, e). Reduced GAL3 expression was also previously

described in several human neoplasias such as prostate [25,

26], ovarian [27], colon [28], head and neck squamous cell

carcinoma [29], and breast cancers [30].

The reduced GAL3 expression was associated with the

presence of known distant metastasis (P = 0.038, by

Mann–Whitney test; r = -0.443; Table 1). In addition,

GAL3 protein expression was 2.05-fold higher in ACP03

than ACP02, a more invasive cell. Our results corrobo-

rates Okada et al.’s study in which reduced GAL3

expression was associated with poor prognosis of GC

patients by immunohistochemistry analysis [24].

Decreased GAL3 expression was also associated to poorer

prognosis in several human cancers [25–30]. It has been

proposed that GAL3 increases cell–cell and cell–extra-

cellular matrix adhesive interactions at the primary

tumors, disfavoring the separation of individual cancer

cells and metastatic dissemination. Therefore, decreased

GAL3 expression would lead to reduced adhesiveness

between tumor cells and facilitate cancer cell invasion

[31], which is in agreement with the observations in the

present study.

Here, a correlation was observed between GAL3 and

ANXA2 protein expression (q = 0.605; P = 0.006, by

Spearman’s test). On the other hand, no significant

Fig. 2 ANXA2 and GAL3 expression in gastric carcinogenesis.

a Relative quantification of ANXA2 mRNA expression in gastric

tumors normalized by matched non-neoplastic gastric tissue; b repre-

sentative Western blotting using anti-ANXA2 and anti-ACTB

antibodies in pairs of gastric tumors and non-neoplastic gastric tissue

samples from the three studied patient; c Western blotting using anti-

GAL3 and anti-ACTB antibodies; d ratio of ANXA2 protein

expression between tumor and matched non-neoplastic gastric tissues

by Western blotting analysis; e ratio of GAL3 protein expression

between tumor and matched non-neoplastic gastric tissues by Western

blotting. RQ relative quantification; T tumor gastric sample; N non-

neoplastic gastric samples

Clin Exp Med

123

correlation was observed between GAL3 protein and

ANXA2 mRNA expression (q = -0.049; P = 0.852, by

Spearman’s test). The lack of correlation may in part due to

the large interindividual differences observed in our samples

(Fig. 2).

In conclusion, our study shows that ANXA2 and GAL3

deregulated expression was associated with an invasive

phenotype in GC cell lines and may contribute to metas-

tasis in GC patients. Therefore, these proteins may have

potential prognostic relevance for GC of individuals from

northern Brazil.

Acknowledgments This study was supported by Conselho Nacional

de Desenvolvimento Cientıfico e Tecnologico (CNPq; RC, MACS

and RRB) and Fundacao de Amparo a Pesquisa do Estado de Sao

Paulo (FAPESP; MFL, JC and DQC) as grants and fellowship awards.

We acknowledge the Mass Spectrometry Laboratory at Brazilian

Biosciences National Laboratory, CNPEM-ABTLuS, Campinas,

Brazil and Dr. Adriana F. Paes Leme and technicians for their

assistance with the mass spectrometric analyses.

Conflict of interest The authors declare that they have no conflict

of interest.

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