Download Astrocyte Elevated Gene-1 (AEG

Astrocyte Elevated Gene-1 induces breast cancer proliferation and invasion through
upregulating HER2/neu expression
Xin Zhang1*, Ning Zhang2 and Meixin Zhang
1Department
of chemotherapy,
2Department
of Breast surgery, Qilu Hospital,
Shandong University School of Medicine, Jinan, P. R. China, 3Shandong Provincial
Family Planning Institute of Science and Technology, Jinan, P. R. China
*Correspondence to: Xin Zhang, Department of Chemotherapy, Qilu Hospital,
Shandong University School of Medicine, Jinan 250012, P.R. China. Tel:
0531-82169114; Fax: 0531-86927544; Email: [email protected]
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Abstract
Astrocyte elevated gene 1 (AEG-1), primarily identified as a late response gene induced
by HIV-1 infection, plays multiple roles in the process of oncogenesis. This novel gene
has been demonstrated to be involved in the several potent carcinogenic pathways,
including PI3K/Akt pathway, NF-κB pathway, and Wnt/β-catenin pathway. Although the
function of AEG-1 has been intensively investigated in recent years, the molecular
mechanism underlying its oncogenic role is largely unknown. In order to explore the
potential function of AEG-1 in breast cancer development and progression, we
ectopically overexpressed AEG-1 in breast cancer MCF-7 cells and studied its biological
effects on the proliferation and invasion of MCF-7 cells. With MTT and invasion assays,
we found that overexpression of the AEG-1 promoted the proliferation and invasion
ability of breast cancer cells, and upregulated the expression of HER2/neu, a crucial
oncogene involving in breast cancer carcinogenesis. We concluded that AEG-1 might
facilitate the proliferation and invasion of breast cancer cells by upregulating HER2/neu
expression, which provides a potential target for breast cancer therapy.
Key words
AEG-1; HER2/neu; invasion; breast cancer
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Background
Breast cancer has been ranked No. 1 in the list of female malignant diseases
around the world. Each year there are approximately 400,000 deaths because of
breast cancer [1]. It has been universally accepted that breast cancer is caused by
alterations of oncogenes, tumor-suppressor genes, microRNA genes and others [2].
Activation of oncogenes can facilitate cell growth, thus breaking the balance between
cellular proliferation and apoptosis. Novel oncogenes can become potential candidates
for targeted therapy based on their roles in cancer initiation and progression.
Astrocyte elevated gene-1 (AEG-1) [3], also known as Metadherin (MTDH) [4], and
Lysine-rich CEACAM-1-associated protein (Lyric) [5, 6], was initially identified as a novel
response gene induced by HIV-1 infection and tumor necrosis factor-α in the primary
human fetal astrocytes [3, 7]. AEG-1 encodes a single-pass transmembrane protein
with a calculated molecular mass of 64 KDa [7], and is overexpressed in different
cancers, including breast cancer, melanoma, esophageal squamous cell carcinoma,
neuroblastoma, and prostate cancer [8].
As a potent mediator in the development and progression of malignancies, AEG-1
is involved in multiple pathological steps, including malignant transformation,
chemoresistance acquisition, angiogenesis, and metastasis. Accumulating evidence has
also established its role in various signal pathways. Ha-ras can induce the expression of
AEG-1 though the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway [9].
Knockdown of AEG-1 expression inhibits cancer progression by regulating the FOXO3a
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activity which is mediated by reduced Akt activity [10, 11]. Furthermore,
AEG-1-induced activation of the NF-κB pathway is considered to be a key molecular
mechanism of oncogenesis [12, 13]. AEG-1 has also been found to mediate
hepatocellular carcinoma progression by activating Wnt/β-catenin signal pathway [14].
In brief, AEG-1 may play a multi-facet role during cancer development and progression.
It is still unclear about the complicated network involved in the AEG-1-induced
proliferation and metastasis.
In our previous studies, we discovered several variants of AEG-1 which could
influence the susceptibility to breast cancer development [15]. We have also found
that AEG-1 promoted epithelial–mesenchymal transition in breast cancer cells and
enhanced their aggressive behavior. Overexpression of AEG-1 could lead to
upregulation of mesenchymal markers, downregulation of epithelial markers, and
nuclear accumulation of beta-catenin [16]. In addition, we observed a potential
correlation between AEG-1 expression and HER2/neu status in ductal carcinoma in situ
(DCIS) with Immunohistochemistry [17]. Herein, we hypothesized that AEG-1 might
induce breast cancer proliferation and metastasis by upregulating HER2/neu
expression.
MATERIALS AND METHODS
Materials
Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco-BRL
(Rockville, MD). Fetal bovine serum (FBS) was supplied by Haoyang biological
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manufacture Co., Ltd (Tianjin, China). Anti-HER2/neu (1:2000) antibodies were
purchased from Dako Corp. (Carpinteria, CA). Anti-mouse IgG horseradish peroxidase
(HRP) antibody (1:4000) was from ZhongShan Goldenbridge (Beijing, China).
Pro-lighting HRP agent for western blotting detection was from Tiangen Biotech CO.,
LTD (Beijing, China). All the other chemicals were from Merck (Darmstadt, Germany)
and Sigma-Aldrich (St. Louis, MO) unless described specifically.
Cell culture
Breast cancer cell line MCF-7 was obtained from American Type Culture Collection
(ATCC, Manassas, VA), and the cells were routinely cultured in DMEM/high glucose
medium supplemented with 10% FBS, 100 U/ml penicillin, and 100μg/ml streptomycin,
under the condition of 5% CO2 at 37 ºC.
Plasmid construction and transfection
The plasmid construction was performed as described previously [18, 19]. In brief,
a PCR cloning strategy was used to amplify the complete open reading frame region of
the AEG-1 cDNA with primers incorporated with appropriate restriction sites. The PCR
product was cloned into the multiple cloning site of the pcDNA3.1 vector (Invitrogen,
Carlsbad, CA) and the plasmid was verified by sequencing both strands. Transfection of
MCF-7 cells using lipofectamine 2000 transfection reagent (Invitrogen) was performed
according to the manufacturer’s protocol. Overexpression of AEG-1 was confirmed
with Western blot analysis and -real-time PCR. The mock vector of pcDNA3.1 was used
as a control.
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Real-time PCR analysis
Total RNA was extracted with TRIZOL reagents (Invitrogen) according to the
manufacturer’s protocol. Real-time PCR was performed using the SYBR green PCR mix
with Applied Biosystems StepOne Plus Real-Time PCR System. The expression of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous
control. The gene expression DCt values of the mRNA from each sample were
calculated by normalizing with endogenous control GAPDH. The experiment was
repeated thrice.
Western blot analysis
Protein of the breast cancer cells was collected with RIPA lysis buffer (Sigma, St
Louis, MO) in the presence of protease inhibitors. Equal amount of protein was loaded
onto the SDS-PAGE gels and the protein was then transferred to a PVDF membrane.
The membrane was incubated with the primary anti-HER2/neu antibody overnight at 4
ºC and then with the secondary antibody. Signal was detected with enhanced
chemiluminescence. Beta-actin was used as the control. The representative data of the
three independent experiments were shown in the figure.
Cell viability assay
Cell
viability
was
determined
with
the
3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) assay. Cells were incubated in 96-well plates for
0 to 5 days. After that, 20 μl of MTT (5 mg/ml in PBS) was added to each well, and cells
were further incubated for 4 h at 37ºC. The supernatants were then removed and 100
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μl of dimethyl sulfoxide was added to dissolve the precipitant in each well. The plates
were gently shaken for 10 minutes and the absorbance values were measured at 570
nm with a Microplate Reader (Bio-Rad, Hercules, CA) [20].
Invasion assay
Invasion assay was performed in 24-well transwell system (8-μm pore size with
polycarbonate membrane; Corning Incorporated, Corning, NY) coated with matrigel
(BD Biosciences, Bedford, MA). Five hundred µl complete medium with 20% FBS was
added to the lower well of each chamber and 1105 MCF-7 cells (suspended in 100 μl
of serum free medium) were loaded onto the upper wells. Each cell group was plated
in triplicates. After incubation for 18 h, the upper surface of the membrane was swiped
with cotton swabs to remove the noninvasive cells. Cells adhering to the lower surface
of the membrane were fixed in methanol for 15 minutes and stained with Giemsa. The
number of the cells invaded successfully was acquired in six random fields under
microscopy.
Statistical analysis
The software SPSS V16.0 was used for statistical analysis. Student’s t-test was used
to analyze the statistical difference between any two sets of data. P < 0.05 was
accepted as significant. The data were expressed as mean ± standard deviation (SD).
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RESULTS
AEG-1 overexpressed in breast cancer cell line
To overexpress AEG-1 in breast cancer cell line, the pcDNA3.1-AEG-1 plasmid was
transfected into the MCF-7 cells to generate the AEG-1 expressing cells (MCF-7 AEG-1
cells). The overexpression of AEG-1 in MCF-7 AEG-1 cells was confirmed by both
real-time PCR and western blot analysis. Figure 1 shows that the AEG-1 expression in
the MCF-7 AEG-1 cells was significantly higher than that in the control cell line
transfected with the mock vector at both mRNA and protein levels.
AEG-1 overexpression enhanced the proliferation and invasion of breast
cancer cells in vitro.
Because AEG-1 has been reported to be associated with the development of
breast cancer and prognosis of breast cancer patients [15, 18], we tested the effects of
AEG-1 overexpression on the aggressiveness of breast cancer cells. MTT assay was
used to investigate the effect of overexpressed AEG-1 on the proliferation of breast
cancer cells. As shown in Figure 2, MCF-7 cells transfected with the pcDNA3.1-AEG-1
plasmid displayed increased proliferation ability in a time-dependent manner (P =
0.006).
Invasiveness is one of the most important processes during cancer metastasis. To
investigate whether AEG-1 could enhance the invasion ability of breast cancer cells,
invasion assay with a transwell system was applied to test the effect of AEG-1 on the
MCF-7 cell invasiveness. Figure 3 demonstrates that the MCF-7 cells overexpressing
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AEG-1 exhibited significantly increased ability of invasion, compared with the control
cells. These data strongly suggest that AEG-1 plays an important role in the regulation
of the invasiveness of breast cancer cells.
AEG-1 upregulated HER2/neu expression
Because the proliferation and invasion ability of breast cancer cells is biologically
and clinically linked to the expression of HER2/neu, we therefore examined whether
AEG-1 could induce breast cancer proliferation and invasion by upregulating HER2/neu
expression. Western blot analysis was done to determine the expression level of
HER2/neu in breast cancer cells expressing AEG-1, and as shown in Figure 4,
overexpressed AEG-1 increased the protein expression of HER2/neu in MCF-7 breast
cancer cells (lane 2).
DISCUSSION
Recently, AEG-1 has become the focus with accumulating evidence indicating its
multi-facet roles in the modulation of cancer development and progression [21].
However, the molecular mechanism underlying its role in the carcinogenesis and
cancer progression requires further clarification. In the present study, we introduced
AEG-1 gene into the MCF-7 cells with transfection techniques. The MTT assay
demonstrated the role of AEG-1 in promoting the proliferation of breast cancer MCF-7
cell. Meanwhile, overexpression of AEG-1 could lead to increased invasion ability of
MCF-7, which was in accordance with the results in previous studies [18]. Because
gene amplification and/or overexpression of the HER2/neu tyrosine kinase are
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associated with poor prognosis in breast cancer [22], we decided to analyze the
HER2/neu protein expression with Western blotting. In the present study, for the first
time, we reported that HER2/neu could be upregulated by overexpression of AEG-1 in
MCF-7 cells. Taken together, AEG-1 might enhance proliferation and invasion ability of
breast cancer cells by upregulating HER2/neu expression.
HER2/neu gene encodes a glycoprotein receptor with intrinsic tyrosine kinase
activity [23]. Gene amplification contributes to the overexpression of HER2/neu,
resulting in accumulation of HER2/neu product in cancer cells. Subsequently, HER2/neu
overexpression induces increased HER2/neu heterodimerization with EGFR and HER3,
interferes the endocytic degradation of EGFR, and results in increased EGFR membrane
expression. Increased HER2/neu-EGFR dimers therefore enhance the proliferative and
invasive functions of breast cancer cells [24]. Different research groups have reported
that HER2/neu gene is overexpressed in 10% to 40% of breast cancers and its
overexpression is associated with a more aggressive phenotype of breast cancer [25].
Breast cancer, as one of the most common female malignancies, causes over one
million new diagnoses each year [1]. Recently, combination of surgery, chemotherapy,
and radiotherapy has significantly improved the prognosis of patients with breast
cancer. Because HER2/neu receptor can regulate many key processes in breast cancer
and it has a low expression level in normal adult tissues, HER2/neu is an ideal target for
therapy [22]. Trastuzumab is a humanized monoclonal antibody that binds to the
extracellular domain of the HER2/neu receptor. It has been suggested that the binding
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of trastuzumab with HER2/neu receptor induces the cell cycle arrest at G1 phase and
sequentially inhibits the cell proliferation [26]. In addition, trastuzumab blocks
receptor dimerization as well as angiogenesis [27].
Understanding of the molecular mechanism contributing to the proliferation and
invasiveness of cancer cells is crucial to develop novel therapeutic strategies against
breast cancer. In our study, we found that AEG-1 could upregulate the HER2/neu
expression and thus enhance the proliferation and invasion ability of MCF-7 cells,
suggesting AEG-1 might serve as a novel target for therapy in breast cancer [28].
However, the interaction mode how AEG-1 regulates HER2/neu expression and the
specific signal pathways needs further investigation.
Recently, AEG-1 has been reported to be highly expressed in 56.8 % of
triple-negative breast cancers and a significant correlation has been found between
AEG-1 and vascular endothelial growth factor (VEGF) [29]. The findings may be
explained by the identification of VEGF as the downstream molecule that responds to
AEG-1. AEG-1 and VEGF pathway may contribute as a promoter to tumor growth and
tumor-associated angiogenesis [30]. Thus, we proposed that AEG-1 could function as
an oncogene depending on other pathways when HER2/neu is inactivated.
In summary, our study showed that AEG-1 upregulated the expression of
HER2/neu, suggesting a new mechanism underlying the proliferation and invasiveness
of breast cancer cells [28]. Our results might provide a new way to elucidate the
oncogenic role of AEG-1 and a new strategy for therapeutic development in the future.
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Competing interests
All the authors declared no conflict of interest.
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Figure legends
Figure 1. Overexpression of AEG-1 in breast cancer MCF-7 cells. (A) Western blot
analysis showed that AEG-1 was overexpressed (lane 2). β-actin was used as the
control. The results represented one of the three independent experiments. (B)
Overexpression of the AEG-1 in the transfected cells was also confirmed by real-time
PCR. The data obtained from triplicate experiments were expressed as mean ± SD.
Figure 2. MTT analysis demonstrated that AEG-1 promoted MCF-7 cell proliferation in
vitro. AEG-1 significantly enhanced cell viability in a time-dependent manner. The
experiments were performed in triplicate and the data were presented as mean ± SD.
Figure 3. Overexpression of AEG-1 enhanced the invasion ability of MCF-7 cells.
Invasion assays of the MCF-7 cells transfected with the control vector (A) and with the
pcDNA3.1-AEG-1 plasmid (B). (C) Quantitative analysis of the invasion assay data. The
data were presented as mean ± SD.
Figure 4. HER2/neu was upregulated by AEG-1 in MCF-7 cells at the protein level as
determined with Western blot analysis (lane 2). The representative data of three
independent experiments were shown in the figure.
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