significance of aeg-1 expression in correlation with vegf, microvessel density and...

Journal of Surgical Oncology 2011;103:184–192

Significance of AEG-1 Expression in Correlation With VEGF, Microvessel Density

and Clinicopathological Characteristics in Triple-Negative Breast Cancer

CONG LI, PhD,1 RUI LI, MD,2 HONGTAO SONG, PhD,1 DONG WANG, PhD,2 TIAN FENG, PhD,5

XIAOGUANG YU, PhD,5* YULAN ZHAO, PhD,1 JUNJUN LIU, PhD,1 XIAOYU YU, PhD,1 YANBO WANG, MD,4

AND JINGSHU GENG, PhD1,3**

1Department of Pathology, the Affiliated Tumor Hospital of Harbin Medical University, Harbin 150040, China2Department of Surgery, People’s Hospital, Peking University, Beijing 100044, China3Department of Medical Genetics, Harbin Medical University, Harbin 150086, China

4Department of Breast Surgery, the Affiliated Tumor Hospital of Harbin Medical University, Harbin 150040, China5Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Harbin Medical University, Harbin 150081, China

Purpose: Our study is to examine astrocyte-elevated gene-1 (AEG-1) expression in triple-negative breast cancer and to determine whether it is

associated with vascular endothelial growth factor (VEGF), microvessel density (MVD), clinicopathological parameters and poor survival.

Methods: Specimens from 125 patients with triple-negative breast cancers were investigated by immunohistochemistry for MVD, AEG-1 and

VEGF expression. Correlations between the expression of AEG-1, VEGF, MVD, and various clinicopathological factors including survival status

were studied.

Results: AEG-1 and VEGF were highly expressed in 56.8% and 52.8% of triple-negative breast cancer patients, respectively. The intensity of

AEG-1 was gradually up-regulated from VEGF�MVD�low, VEGF�high, or MVD�high to VEGF�MVD�high tissues using Western blot

analysis. Statistically significant correlation was found among AEG-1 and VEGF, and MVD. Moreover, AEG-1 expression was correlated with

clinical stage, lymphatic venous invasion, lymph nodal metastasis, tumor size, Ki67, and recurrence. Patients with AEG-1 high-expression

showed far lower disease-free survival (DFS) and overall survival (OS) rates than those with AEG-1 low-expression. For VEGF and MVD, there

were similar results in these patients. Only AEG-1 expression and tumor size were independent prognostic factors for both DFS and OS by

multivariate analysis. However, the prognostic impact of tumor size was not as strong as that of AEG-1.

Conclusions: AEG-1 expression may be related with tumor angiogenesis and progression and is a valuable prognostic factor in patients with

triple-negative breast cancer.

J. Surg. Oncol. 2011;103:184–192. � 2010 Wiley-Liss, Inc.

KEY WORDS: triple-negative breast cancer; prognosis; angiogenesis; astrocyte-elevated gene-1; vascular endothelialgrowth factor

INTRODUCTION

Breast cancer is the second leading cause of cancer related death

among women in the Western world [1]. It is a heterogeneous disease

for which there is a variety of biology, natural history, and treatment

options. The triple-negative breast cancer, denoted by lack of

expression of all three protein receptors (ER-PR-HER2-), has recently

emerged as a distinct subtype [2]. This type of breast cancer, which

carries a poor prognosis, is insensitive to most available hormonal or

targeted therapeutic agents [3,4]. Therefore, it is important for the

prevention of triple-negative breast cancer to find reliable biomarkers

that may be used to individualize the patient prognosis.

In recent years, emphasis has increasingly been placed on pro-

and anti-angiogenic factors in malignant tumor, because unbalance

between them results in angiogenesis. Angiogenesis, the process of

forming neovascularization from existing vascular networks, is a

fundamental event in the development and maintenance of solid tumors

and their metastasis [5,6]. Current studies have demonstrated that the

remodeling for breast cancer to grow beyond a certain size requires

constant vascular growth [7]. Nowadays, a multitude of both pro- and

anti-angiogenic factors have been described [8–11], but there are no

studies investigating the expression of these factors in the triple-

negative breast cancer tissues.

Astrocyte-elevated gene-1 (AEG-1), also known as metadherin

(MTDH) [12], is initially identified as a human immunodeficiency

virus (HIV)-1 and tumor necrosis factor (TNF)-a-inducible gene in

primary human fetal astrocytes [13]. Ectopic expression of AEG-1 as a

prominent contributor to cancer progression can promote anchorage-

independent growth and invasion of non-tumorigenic melanocytes and

astrocytes [14]. Partly due to the phenomenon that the 8q22 gain

functions independently in poor prognosis in breast cancer, human

AEG-1 gene is located at chromosome 8q22 [12,15]. Its genomic

amplification has also been found in diverse cancers including breast

cancer in comparison with their normal counterparts [16,17]. AEG-1 is

Abbreviations: AEG-1, astrocyte-elevated gene-1; HIV-1, human immu-nodeficiency virus type-1; LVS, lymphatic and venous; MVD, microvesseldensity; PI3K, phosphoinositide 3-kinase; TNF-a, tumor necrosis factor-a;VEGF, vascular endothelial growth factor.

Grant sponsor: Natural Science Foundation of Heilongjiang ProvinceD2009–21; Grant sponsor: Innovation Fund for Excellent Graduates ofHarbin Medical University HCXB2010018; HCXS2010001.

*Correspondence to: Xiaoguang Yu, Department of Biochemistry andMolecular Biology, College of Basic Medical Science, Harbin MedicalUniversity, Harbin, 150081, China. E-mail: [email protected]

**Correspondence to: Jingshu Geng, Department of Pathology, theAffiliated Tumor Hospital of Harbin Medical University, Harbin 150040,China. Fax No.: 86-451-86298232. E-mail: [email protected]

Received 15 July 2010; Accepted 22 September 2010

DOI 10.1002/jso.21788

Published online 23 November 2010 in Wiley Online Library(wileyonlinelibrary.com).

� 2010 Wiley-Liss, Inc.

a downstream target of Harvey-Ras (Ha-ras) and plays an important

role in Ha-ras mediated oncogenesis and angiogenesis through the

phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway [18].

The other signaling pathway activated by AEG-1 is nuclear factor k-B

(NF-kB) via IkBa degradation and p65 translocation [19,20]. AEG-1

also enhances DNA binding and transcriptional activities of NF-kB in

Hela cells [19,20]. NF-kB regulates the expression of various

molecules such as matrix metalloproteinase (MMP), inflammatory

cytokines, chemokines, and cell adhesion proteins, all of which initiate

cancer cell invasion and angiogenesis [19,20]. Recent studies have

demonstrated that AEG-1 up-regulation in epithelial cells inhibits

apoptosis and increases the invasiveness of malignant cells, favoring

tumorigenesis, neovascularization, and metastasis [21]. The lung-

homing domain of AEG-1 can mediate breast cancer cells metastasis to

lung by tumor cell adhesion to lung vasculature both in vivo and

in vitro [12,15]. In addition, AEG-1 is frequently over-expressed in

highly proliferative breast cancer and high-grade lesions [22,23]. Its

expression status is strikingly correlated with the progression and poor

prognosis of various cancers, including breast cancer [23–26].

Immunohistochemical analysis has revealed that enhanced expression

of AEG-1 in tumor sections can augment expression of specific

angiogenesis molecules including angiopoietin-1 (Ang1), MMP-2, and

TNF-a, further supporting a potential role of AEG-1 in tumor

angiogenesis [20]. This study also identifies that AEG-1 is indeed an

oncogene and a direct regulator of angiogenesis by up-regulating key

components such as vascular endothelial growth factor (VEGF) in the

process of blood vessel formation [20,27]. VEGF is a specific

mitogenic factor for endothelial cells and promotes tumor angiogenesis

[28]. This glycoprotein facilities the permeability of the endothelium

of blood vessels, which in turn allows for cancerous cells to penetrate

the vessel walls and begin the process of metastasis, even in very

distant organs [29–31]. Previous studies have also indicated that

VEGF expression in tumors is a predictive factor of an increased risk of

metastatic diseases and poor survival in breast cancer [32–34]. An

enhanced understanding of AEG-1 and VEGF involved in angiogen-

esis of triple-negative breast cancer will pave the way for the

development of more potent and selective inhibitors of breast cancer.

To the best of our knowledge, our study is the first to immuno-

histochemically determine AEG-1 expression in triple-negative breast

cancer and to correlate its expression with VEGF, MVD, clinico-

pathological features, and survival in a large number of patients with

triple-negative breast cancer.

MATERIALS AND METHODS

Patients and Tissue Specimens

This study used archival material from the Department of Pathology

at the Affiliated Tumor Hospital of Harbin Medical University. The

invasive breast cancer tissue specimens were obtained from patients

undergoing primary mastectomies at our institution from December 1,

2003 to January 25, 2005. According to clinical requirement, our

department also routinely stained Ki67 and p53 for all invasive breast

cancer patients. In this study, the positive expression of Ki67 and p53

occurred in 110/125(88.0%) and 69/125(55.2%) patients with a

diagnosis of primary triple-negative breast cancer, respectively. In

addition, all three molecular markers (ER, PR, HER-2/neu) of these

patients were available. Excluded were patients who presented with

recurrent tumor, non triple-negative breast tumors, non-invasive tumor,

metastatic disease at presentation, bilateral tumors, other previous

tumor, or those who had previously received neo-adjuvant treatment.

Tumor size at the largest diameter of the invasive carcinoma was

measured in millimeters by the pathologist.

In all cases, the histological diagnosis was confirmed by

corresponding paraffin-embedded materials, and when necessary,

panels of immunohistochemical study were carried out, according to

the most recent World Health Organization (WHO) classification.

Breast cancers were routinely divided into ductal carcinoma, lobular

carcinoma and others (medullary carcinoma, mucinous carcinoma).

The tumor grade of invasive carcinoma was classified according to the

Scarff–Bloom–Richardson system [35]. Based on the frequency of

cell mitosis, tubule formation, and nuclear pleomorphism, invasive

carcinoma was graded as grade 1 (low), 2 (moderate) or 3 (high grade).

The presence of lymph node metastases was reviewed for each patient.

The tumor TNM (tumors, nodes, metastases) stage was done according

to the Handbook of Cancer Stages, Edition 6, AJCC: 25 patients (20%)

stage I, 71 patients (56.8%) at stage II, 29 patients (23.2%) at stage III

[36]. The median age of the patients was 52 years (range, 29–

72 years). WHO Criteria for determining menopause include any of

the following: prior bilateral oophorectomy; age 60 years; age

<60 years and amenorrhea for 12 or more months in the absence of

chemotherapy, Toremifene, tamoxifen, or ovarian suppression and

FSH and estradiol in the post-menopausal range (WHO 1996). Forty-

six cases were menopause and 79 cases were non-menopause. All

protocols were reviewed and approved by the Ethical Committee of

Harbin Medical University, Harbin, China. All patients gave informed

consent to the diagnostic procedures and the proposed treatment.

Follow-Up

Clinical and pathological records of all patients on the study were

reviewed periodically. Patients were followed regularly for 5 years at

the Affiliated Tumor Hospital of Harbin Medical University. Clinical

records were obtained from the departments providing follow-up care

to six patients on the study who moved to other parts of the country. All

patients were followed until death or the study closing date (April 25,

2010). Disease-free survival (DFS) and overall survival (OS) were the

two assessments used for prognostic analyses.

Triple-Negative Breast Carcinoma Tissues

The data on ER, PR, and HER-2/neu were obtained through

standard clinical testing. Immunohistochemical markers were assayed

in paraffin-embedded, formation-fixed tissue stained with hematoxylin

and eosin using antibodies to the proteins ER, PR, and HER-2/neu

(Dako, Glostrup, Denmark). For ER and PR, receptor positivity was

based on more than 10% of cells testing positive. HER-2/neu was

assessed through immunohistochemistry and scored on a qualitative

scale from 0 to 3þ, based on interpretation of staining, with 0 and 1þclassified as negative, and 3þ as positive [37]. HER-2/neu scores of 2þwere considered positive for the current study because this was the

accepted classification scheme at the time of clinical treatment. Each

case was analyzed by two independent scientists. It should be noted

that we only selected those with HER-2/neu scores of 0 as HER-2/neu

negative for accuracy in this study.

Immunohistochemical Staining

Immunohistochemical staining was performed in all tissue samples,

using UltrasensitiveTM S-P kit and diaminobenzidine (Maixin-Bio Co.,

Fuzhou, China), as recommended by the manufacturers. Briefly,

sections (4 mm, thickness) were deparaffinized with xylene, rehydrated

through graded alcohol and rinsed in phosphate-buffered saline (PBS).

Antigen retrieval was performed by placing the slides in boiling citric

acid buffer at pH 6.0 for 5 min. Endogenous peroxidase activity was

blocked with 3% hydrogen peroxide in methanol for 20 min.

Incubation with 10% normal goat serum in PBS was performed for

10 min to eliminate non-specific staining. Incubation with primary

antibody was carried out next, followed by incubation with secondary

antibody (according to the primary antibody). Sections were stained

Journal of Surgical Oncology

The Role of AEG-1 in Breast Cancer 185

with 0.02% DAB and 0.02% H2O2 in 0.05 M Tris–HCl buffer for

10 min. Finally, sections were lightly counterstained with 10% Mayer

hematoxylin, dehydrated, mounted, and observed. Immunostaining

was performed with a rabbit polyclonal immunoglobulin (Ig) G

specific for AEG-1 (dilution 1:400; Proteintech Group Inc., Carpin-

teria, CA), rabbit polyclonal IgG specific for VEGF (dilution 1:200;

Santa Cruz Biotechnology, Carpinteria, CA), and mouse monoclonal

IgG specific for CD34 (dilution 1:50; DakoCytomation, Carpinteria,

CA). Histofine Simple Stain Max PO (Multi, Tokyo, Japan) was used

as a secondary antibody (Nichirei, Tokyo, Japan). The negative

controls were substituting mouse (for mAb) or rabbit (for poly Ab)

non-immune IgG for the primary antibody and omitting the primary

antibody in the staining protocol. The positive controls were the

prostate cancer with positive expressions of AEG-1, VEGF, and CD34.

Evaluation of AEG-1 Immunohistochemistry

AEG-1 and VEGF staining were mainly localized in the cytoplasm

in the vast majority of cancer tissues. Staining for AEG-1 and VEGF

was assessed in a series of randomly selected 10 high-power fields, and

they were believed to be representative of the average in tumors at

400� magnification. The sections were scored by combining the

proportion and intensity of positively stained tumor cells [23,24,38].

The proportion of positively stained tumor cells was scored as follows:

0 (no positive tumor cells), 1 (<10% positive tumor cells), 2 (10–50%

positive tumor cells), and 3 (>50% positive tumor cells). Staining

intensity was classified according to the following criteria: 0 (no

staining); 1 (weak staining¼ light yellow), 2 (moderate staining¼yellow brown), and 3 (strong staining¼ brown).

Staining index (SI) was calculated as the staining intensity

score� the proportion score. Using this method, we evaluated the

expressions of AEG-1 and VEGF in triple-negative breast cancer

specimens by determining the SI, with scores 0, 1, 2, 3, 4, 6, or 9. AEG-

1 and VEGF cut-off values were based on measuring heterogeneity by

the log-rank test with regard to OS. The SI score of 4 (a cut-off point)

was used to distinguish between low and high expression of AEG-1

and VEGF.

Assessment of the staining was scored independently by two

investigators (Q.J.X: Qiuju Xu; J.B.: Jing Bai) without knowledge of

the clinicopathological findings. The scoring staining and allocation of

tumors by the two investigators were similar. Cases with discrepancies

were re-reviewed simultaneously by the original two pathologists and a

senior pathologist until a consensus was reached.

Western Blotting

Total tissue proteins from frozen triple-negative breast cancer

tissues that had different expression levels of VEGF and MVD were

extracted by suspending in a lysis buffer consisting of 20 mM Tris–

HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and

protease inhibitors. These tissues were from tissue sample reservoir in

our department, which was established this year. The selection of these

tissues was similar to the criterion of selected specimens from 125

patients with triple-negative breast cancers. Total tissue proteins from

frozen triple-negative breast cancer tissues (n¼ 12) that had different

expression levels of VEGF and MVD were harvested from patients

with triple-negative breast cancer. These frozen tissues were from

tissue sample reservoir in our department. The selection of these

tissues was similar to the criterion of selected specimens from 125

patients with triple-negative breast cancers. Then, we classified

VEGF�MVD�low tissues (n¼ 3), VEGF�high tissues (n¼ 3),

MVD�high tissues (n¼ 3) and VEGF�MVD�high tissues (n¼ 3)

for our further Western blot analysis. Using the bicinchoninic acid

protein assay kit, protein concentrations were quantified, and 30 mg of

protein per sample was separated onto a denaturing polyacrylamide gel

containing SDS and transferred to a methanol-activated nitrocellulose

filter membrane (Bio-Rad, Carpinteria, CA). Before immunodetection,

membranes were blocked within 5% non-fat dry milk. Primary

antibodies, anti-AEG-1 (1:400; rabbit polyclonal; Proteintech Group),

anti-VEGF (1:200; Santa Cruz Biotechnology), and anti-CD34 (1:50;

DakoCytomation), were diluted in the buffer and incubated at 48Covernight. After being subsequently washed with TBST, membranes

were incubated with secondary antibody (horseradish peroxidase

conjugated anti-rabbit) for 1 h at room temperature. The experiment

was repeated in triplicate. The bands were detected by enhanced

chemiluminescence detection reagents (Applygen Technologies Inc.,

Beijing, China).

Quantification of Microvasculature Density

Microvascular profiles identified by CD34 staining were performed

as described by Weidner et al. [39][40]. Any brown stained cells or cell

clusters that had been clearly separated from adjacent microvessels

were considered a single, countable microvessel. Undefined endothe-

lial cells that appeared to be fragments were not counted as micro-

vessels. Branching structures were counted as single vessel unless there

was a break in continuity of the structure. A visible vascular lumen was

not required to count as a microvessel [41]. Subsequent to immuno-

staining, the entire section was scanned at low power (40� and 100�)

to identify the hot spots, which represented the highest vascular density

(so called ‘‘hot spot’’). The microvessel count was determined by

counting the number of CD34-positive vessels in three separate hot

spots at a still higher magnification (200�, 0.4-mm2 per field area).

The mean value of the CD34-positive vessel counts in the three

selected hot spots per section was then calculated. They were then

taken as the MVD of the tumor. The median MVD (value 76) of all

patients was used to classify patients in high and low MVD groups. All

counts were performed by a pathologist who did not know the clinical

data. Figure 2E showed a representative field of high MVD in triple-

negative breast cancers. For comparison, Figure 2F depicted a

representative field of triple-negative breast cancers with low MVD.

Statistical Analysis

All data were analyzed by statistics software (SPSS 13.0 for

Windows; SPSS, Inc., Chicago, IL). Analyzed variables included age,

tumor size, histological type, depth of invasion, lymph node status,

venous and lymphatic invasion, TNM stage, and expressions of Ki67,

p53, AEG-1, VEGF, and CD34. The correlation between expressions

of Ki67, p53, AEG-1, VEGF, and CD34, and the other variables was

assessed with the chi-square and Fisher exact tests. Bivariate

correlations between two independent variables were analyzed by

calculating the Spearman’s correlation coefficients. Survival analysis

was performed using the Kaplan–Meier method and compared by the

log-rank test. Prognostic relevance was evaluated by multivariate Cox

regression analysis. P< 0.05 was considered as significant.

RESULTS

Expression of AEG-1, VEGF, and MVD in

Triple-Negative Breast Cancer

The intensity of AEG-1 expression was gradually elevated from

VEGF�MVD�low tissues to VEGF�high or MVD�high tissues

using Western blot analysis. In addition, AEG-1 expression in

VEGF�high and MVD�high tumors was lower than that in the

high-expressed tumors for both (Fig. 1).

Immunoreactivity for both AEG-1 and VEGF proteins was shown

as brown color and presented diffusely or focally in the cytosolic and


186 Li et al.

perinuclear regions of triple-negative breast cancer and some

inflammatory cells (Fig. 2). The expression of AEG-1 was high in 71

(56.8%) and low in 54 (43.2%) of paraffin-embedded, archived triple-

negative breast cancer tissues (Fig. 2). When VEGF staining was

analyzed, it was found that 66 (almost half of the cases) were

VEGF�high-expression; the remaining 59 were VEGF�low-expres-

sion. The median MVD (value 76) of all patients was used as the cut-

off point to separate tumors with high versus low microvessel density

(MVD). Fifty-nine samples were high MVD (MVD >76); the other 66

were low MVD.

Relationships Between Immunoreactivity of AEG-1,

VEGF and MVD, and Clinicopathological Factors

The association between clinicopathological variables and the

expression of the investigated proteins was provided in Table I. AEG-1

expression was significantly correlated with Ki67 expression, differ-

entiated carcinomas, advanced cases, metastatic lymph nodes, and

positive venous invasion. The expression of VEGF was closely related

to depth of invasion, Ki67 expression, metastatic lymph nodes,

lymphatic and venous invasion, and TNM stage. The MVD score was

significantly high in patients with deep primary tumor invasion, higher

Ki67 expression, metastatic lymph nodes, increased tumor size, and

positive venous and lymphatic invasion.

Correlation of AEG-1 to VEGF and MVD

In the current study, MVD was significantly correlated with the

expression of AEG-1 in tumor tissue, that was, the stronger the

expression of AEG-1, the higher the MVD (Table II). The finding

suggested that AEG-1 was related to MVD and angiogenesis.

Consistent with other studies, MVD count also showed correlation

with VEGF expression in our study (Table II). In addition, Table I

showed that AEG-1, VEGF, and MVD were closely related to tumor

growth.

Effect of AEG-1, VEGF, and MVD on Patients’ Survival

The Kaplan–Meier 5-year survival curves stratified for AEG-1

expression, VEGF, and MVD were provided in Figure 3. High

expression for both AEG-1 and VEGF was clearly associated with poor

DFS (56.1% vs. 83.3% and 59.1% vs. 78.0%, respectively) and OS

(60.6% vs. 87.0% and 62.3% vs. 83.4%, respectively) rate. Patients

with high MVD score had worse DFS (57.6% vs. 77.2%) and OS

(62.7% vs. 80.3%) rates.

Univariate and Multivariate Analysis for Prognosis of

Patients With Triple-Negative Breast Cancer

Both univariate and multivariate survival analysis was used to

evaluate the effect of AEG-1, VEGF, MVD, and clinicopathological

characteristics (including age, clinical stage, tumor size, LVS invasion,

lymph node metastasis and so on) on prognosis. Univariate Cox

regression analysis identified that tumor size, LVS invasion, TNM

stage, MVD, p53, Ki67, AEG-1, and VEGF expression were

prognostic factors influencing both 5-year DFS and OS (Table III).

By multivariate analysis, we further examined prognostic parameters

of triple-negative breast cancers that were significant in univariate

analysis. Only tumor size and AEG-1 expression were independent

prognostic factors influencing both 5-year DFS and OS (Table IV).

Lymphatic venous invasion retained its significance as an independent

prognostic factor for DFS (Table IV). Lymph node status remained as

an independent significant prognostic factor for OS (Table IV).

DISSCUSSION

Angiogenesis occurs at the earliest stages of breast cancer patients

[42]. Tumor growth, invasiveness, and metastasis are critically

dependent on the ability of malignant cells to induce the growth of

new capillary blood vessels [30]. Weidner and coworkers were the first

to show that quantifying tumor neovascularization yielded prognos-

tically useful information in patients with invasive breast cancer

[28,43]. In addition, Fuhrmann et al. [44] have demonstrated that

VEGF concentration and the degree of tumor vascularization in breast

cancer may represent a good indicator for the assessment of

angiogenesis in solid tumors. Emdad and Brown as well as their

colleagues have recently proposed that AEG-1 can regulate angiogen-

esis and function as an oncogene [12,21]. Besides, AEG-1 over-

expression occurs in more than 40% of breast cancer patients and

promotes metastatic seeding and chemoresistance of breast cancer

[15]. In this research, we clarified AEG-1 expression levels in triple-

negative breast cancer and determined if they correlated with VEGF,

MVD, clinicopathological characteristics, and survival in the triple-

negative breast cancer, which was not previously reported.

We observed that 71 of 125 patients (56.8%) were high AEG-1

expression, and 66 of 125 patients (52.8%) were high VEGF

expression. Our results agreed with previous studies in breast cancer

showing that AEG-1 was predominantly expressed in the cytoplasm of


Fig. 1. Western blotting shows AEG-1 protein levels in triple-negative breast cancers that have different expression levels of VEGFand MVD. A: The intensity of AEG-1 expression was gradually up-regulated from VEGF�MVD�low, VEGF�high or MVD�high toVEGF�MVD�high tissues. Moreover, the intensity of AEG-1expression in VEGF�high and MVD�high tumors was lower thanthat in the high-expressed tumors for both. Expression levels werenormalized with b-actin. B: AEG-1 expression in the individual tissuesamples was calculated as AEG-1 expression relative to b-actinexpression. Data are mean� SD from three independent experiments.Western blotting analysis revealed the variation to be consistent with itsappearance in the gel images (P< 0.001).[Color figure can be viewedin the online issue, available at wileyonlinelibrary.com]


breast cancer cells [15,23,24]. Employing Western blot, we found that

the intensity of AEG-1 expression is significantly higher in

VEGF�MVD�high cases than that in VEGF�MVD�low cases.

The AEG-1 protein levels of VEGF�high and MVD�high tumors

were higher than those of VEGF�MVD�low tumors. A statistically

significant correlation was found between high AEG-1 expression and

tumor size in our research. A similar relationship was found between

the number of newly formed vessels and the size of triple-negative

breast cancers, confirming Folkman’s theory that tumor size was

restricted to a few cubic millimeters in the absence of neovasculariza-

tion [40]. With clinical experience, the size of a malignant tumor

represented a classic prognostic indicator in breast cancer patients [45].

The results of our study revealed that, along with the size of the tumor,

increased AEG-1 expression was an independent prognostic factor

influencing both 5-year DFS and OS. Though tumor size was an

additional predictor of DFS and OS, the prognostic impact was

statistically inferior to AEG-1 expression in the univariate and

multivariate analysis. Moreover, we demonstrated that AEG-1-high

patients had lower OS and DFS rates as compared with AEG-1-low

patients. From these results, it can be seen that AEG-1 may become a

valuable predictor for prognosis and survival among triple-negative

breast cancer patients.

Our clinical studies demonstrated positive correlation of AEG-1

expression with increased MVD, greater metastatic potential and a

poor outcome in triple-negative breast cancer patients. High AEG-1

expression was also previously found in patients with breast cancer

[24]. However, the relationship between AEG-1 expression and MVD

in breast cancer has not been previously characterized. Tumor

angiogenesis in invasive breast cancer was quantitated by MVD,

which was also a crucial factor for the assessment of this tumor

prognosis [28]. There was confirmed evidence that antibody against

CD34 in the assessment of MVD was particularly reliable, so we

adopted CD34 immunostaining to assess MVD in our study [39,40].

High MVD was associated with elevated risk of metastasis and shorter

survival in solid tumors including breast cancer [28]. Based on these

reports, we investigated the association of AEG-1 expression with

MVD and looked at its correlation with prognosis in breast cancer. We

revealed that AEG-1 expression was significantly correlated with

MVD and disease prognosis. On the basis of above studies, we indicate

that AEG-1 may play a crucial part in angiogenesis, which is an

essential component in breast cancer development, growth, and

progression to metastasis. Statistic analysis of the relationship between

AEG-1 staining and the clinical characteristics of patients presented a

significant correlation of AEG-1 expression with clinical stage, lymph


Fig. 2. Immunohistochemical staining of AEG-1, VEGF, and CD34 in triple-negative breast cancer tissues. A: high AEG-1 expression (B) lowAEG-1 expression (C) high VEGF expression (D) low VEGF expression (E) CD34-positive endothelial cells of blood vessels in high MVD area,the mean MVD was 84.25 in this sample (F) CD34-positive endothelial cells of blood vessels in low MVD area, the mean MVD was 45.66 in thissample. Original magnification, 200� (A–F).[Color figure can be viewed in the online issue, available at wileyonlinelibrary.com]

188 Li et al.

node metastasis, and LVS invasion, further supporting a potential role

of AEG-1 in tumor angiogenesis [40]. Since a high MVD represents a

negative prognostic factor for the specific survival of breast cancer

patients [28,46], the adverse clinical course of our case may be due to

the high vascular supply, determined by an intense angiogenesis

correlated to the high VEGF and AEG-1 levels. In the classically

prognostic markers of breast cancer (such as p53 and Ki-67) [2,41],

only Ki-67 showed statistical significance related to AEG-1 expres-

sion. This may be because AEG-1 is commonly over-expressed in

highly proliferative lesions of breast cancer [22,23]. Ki-67 and p53

expression showed correlation with survival in breast cancer only by

univariate analysis, but they were not an independent prognostic factor.

It could be related to other significant independent factors such as

tumor size and lymph-node status. It is suggested that the diagnosis of

triple-negative breast cancer can be improved by the measurement of

AEG-1 level in combination with any other biomarkers such as Ki67,

p53, and VEGF [2,23,30,35,41]. However, further efforts are needed to

identify whether combined use of these prognostic markers of breast

cancer would be more valuable in improving prediction efficiency.

Previous study demonstrated the significance of the AEG-1-

dependent pathway in tumor-associated angiogenesis and tumor

growth [21]. They identified VEGF as the downstream molecule that

responded to AEG-1, which consequently could contribute as a

promoter of tumor growth and tumor-associated angiogenesis [21]. It is

not until the primary tumor becomes vascularized that tumor cells

come into the systemic circulation [42]. Tumor cells were able to enter

this circulation due to the increased diameter and an incomplete

basement membrane of the newly formed tumor with irregular shape

and spontaneous hemorrhage, thrombosis, or both [40]. VEGF as a


TABLE I. Correlations of AEG-1, VEGF, and CD34 Expression and Clinicopathologic Factors

Variable Patients

AEG-1

P*

VEGF

P*

MVD

P*Low High Low High Low High

Age (years)

<50 73 34 39 0.363 32 41 0.376 36 37 0.359

�50 52 20 32 27 25 30 22

Menopausal status

Pre-menopausal 79 38 41 0.150 36 43 0.636 40 39 0.529

Post-menopausal 46 16 30 23 23 26 20

p53

Negative 56 19 37 0.059 27 29 0.838 33 23 0.216

Positive 69 35 34 32 37 33 36

Ki67

Negative 15 3 12 0.023 5 10 0.038 6 9 0.045

Positive 110 51 59 54 56 60 50

Pathology type

IDC 105 48 57 0.196 52 53 0.236 57 48 0.450

ILC others 20 6 14 7 13 9 11

Histology grade

I 19 10 9 0.372 14 5 0.012 11 8 0.632

II III 106 44 62 45 61 55 51

pTNM stage

I 25 18 7 0.001 18 7 0.005 17 8 0.090

II III 100 36 64 41 59 49 51

Tumor size

T1 35 22 13 0.005 23 12 0.009 24 11 0.028

T2 T3 90 32 58 36 54 42 48

Lymph node status

N0 61 35 26 0.012 37 24 0.003 39 22 0.035

N1 N2 N3 64 19 45 22 42 27 37

LVS invasion

No 104 51 53 0.003 54 50 0.018 60 44 0.015

Yes 21 3 18 5 16 6 15

Recurrence metastasis

No 85 45 40 0.001 46 39 0.024 51 34 0.019

Yes 40 9 31 13 27 15 25

AEG-1, astrocyte elevated gene-1; VEGF, vascular endothelial growth factor; MVD, microvessel density; LVS invasion, lymphatic and venous invasion; TNM,

tumor, node, metastasis system.*Spearman rank correlation test P-value.

TABLE II. Spearman Correlation Analysis of AEG-1 and VEGF

Expressions and Microvessel Density

AEG-1 expression

VEGF expression

PLow (n¼ 59) High (n¼ 66)

Low (n¼ 54) 42 12 <0.001

High (n¼ 71) 17 54

MVD

AEG-1 expression P

Low (n¼ 54) High (n¼ 71)

Low (n¼ 66) 37 29 0.002

High (n¼ 59) 17 42

VEGF expression

MVD P

Low (n¼ 66) High (n¼ 59)

Low (n¼ 59) 44 15 <0.001

High (n¼ 66) 22 44



TABLE III. Univariate Analysis of Clinicopathological Factors for 5-Year DFS and OS of 125 Patients With Triple-Negative Breast Cancer

Variables Risk ratio

DFS

P Risk ratio

OS

P95%CI 95%CI

Age (years)

<50/�50 1.054 0.563–1.972 0.870 1.354 0.698–2.628 0.370

Menopausal status

Pre-menopausal/post-menopausal 0.938 0.490–1.797 0.847 1.167 0.593–2.295 0.654

p53

Negative/positive 2.527 1.281–0.987 0.015 3.539 1.275–8.051 0.020

Ki67

Negative/positive 2.318 1.432–7.987 0.026 3.156 2.485–6.531 0.038

Pathology type

IDC/ILC others 0.926 0.389–2.205 0.862 0.875 0.339–2.255 0.782

Histology grade

I/II III 2.652 0.818–8.606 0.104 3.498 0.839–14.584 0.086

pTNM stage

I/II III 3.810 1.174–12.364 0.026 4.972 1.193–20.730 0.028

Tumor size

T1/T2 T3 4.441 1.579–12.487 0.005 5.128 1.569–16.759 0.007

Lymph node status

N0/N1 N2 N3 4.308 2.048–9.064 0.0263 4.800 2.094–11.003 0.013

LVS invasion

No/yes 3.872 2.036–7.363 0.015 3.184 1.583–6.407 0.001

AEG-1

Low/high 3.299 1.569–6.935 0.002 3.761 1.642–8.617 <0.001

VEGF

Low/high 2.148 1.108–4.166 0.024 2.525 1.212–5.258 0.013

MVD

Low/high 2.090 1.101–3.966 0.024 2.052 1.034–4.075 0.040

Fig. 3. A–C: Five-year DFS, and (D–F) OS curves of patients with triple-negative breast cancer according to the AEG-1, VEGF, and CD34immunostaining.[Color figure can be viewed in the online issue, available at wileyonlinelibrary.com]

190 Li et al.

positive regulator has been proved to be exerted an important role in

this so-called angiogenic switch relying on a net balance of positive

and negative angiogenic factors in breast cancer [47]. Many

investigations have shown a significant relationship between VEGF

and MVD expressions in patients with breast cancer and have proposed

the use of both these indicators to assess prognosis in these patients

[47][48]. It has also been confirmed that the treatment of breast cancer

can be improved by the monoclonal antibody bevacizumab (Avastin,

Genetech, Inc.South San Francisco, CA) targeting the VEGF [49].

Consistent with these results, we identified the close correlation of

VEGF with AEG-1 expression in the patients with triple-negative

breast cancer. Furthermore, VEGF expression was significantly

associated with progression and reduced survival time of these patients

in our study, which also proved the effectiveness of VEGF as

prognostic factors. Collectively, these findings indicate that AEG-1

inhibitors together with some agents, which block the AEG-

1�VEGF�dependent pathway will become useful therapeutic tools

against triple-negative breast cancers.

In conclusion, our data imply that AEG-1 is an important mediator

of angiogenesis and is a valuable prognostic factor in patients with

triple-negative breast cancer. These results are consistent with the

previous reports of the roles of AEG-1 in tumor progression [11–27],

including breast cancer, and further support a relationship between

high expression of AEG-1 protein and unfavorable biological behavior

in breast cancer. Additional prospective studies must be performed to

confirm its role as a useful marker of breast cancer initiation, develop-

ment and progression. Indeed, further investigations are needed to

determine whether AEG-1 protein may be used as a therapeutic target

to identify the patients who gain benefit from anti-angiogenic agents

and to monitor the efficacy of these treatment regimens.

ACKNOWLEDGMENTS

We would like to thank Aiwen Hou for her support and help in the

preparation of this manuscript. We thank Jing Bai and Qiuju Xu for

their technical assistance. We thank Di Feng, Yan Li and Yuan Zhang

for their contribution to experiments and data. This research is funded

by Natural Science Foundation of Heilongjiang Province (No.D2009–

21) and Innovation Fund for Excellent Graduates of Harbin Medical

University (No. HCXB2010018 and HCXS2010001).

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TABLE IV. Multivariate Analysis of Clinicopathological Factors for 5-Year DFS and OS of 125 Patients With

Triple-Negative Breast Cancer

Variables Risk ratio

DFS

P Risk ratio

OS

P95%CI 95%CI

AEG-1

Low/high 3.032 1.388–6.622 0.014 3.484 1.505–8.062 <0.001

Tumor size

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LVS invasion

No/yes 2.162 1.090–4.288 0.027

Lymph node status

N0/N1 N2 N3 3.346 1.014–11.045 0.047


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significance of aeg-1 expression in correlation with vegf, microvessel density and...

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