Download Ang IIAT1R Increases Cell Migration Through PI3K/AKT and NFB

ORIGINAL RESEARCH ARTICLE
Journal of
Ang II–AT1R Increases Cell
Migration Through PI3K/AKT and
NF-kB Pathways in Breast Cancer
Cellular
Physiology
YANBIN ZHAO,1 HONGBIN WANG,2 XIULI LI,1 MENGRU CAO,1 HAILING LU,1
QINGWEI MENG,1 HUI PANG,1 HAILIN LI,3 CHRISTINA NADOLNY,4 XIAOQUN DONG,4**
1
AND LI CAI *
1
Department of Internal Medical Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang Province, China
2
Department of Surgical Oncology, Harbin Medical University Cancer Hospital, Harbin, Heilongjiang Province, China
3
Hongqi Hospital, Mudanjiang, Heilongjiang Province, China
4
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, Rhode
Island
Angiotensin II (Ang II), a biologically active peptide of the renin–angiotensin system (RAS), plays an important role in promoting cell
migration via Angiotensin II type 1 receptor (AT1R). In this study, we examined the mechanisms by which Ang II affected cell migration in
AT1R-positive MDA-MB-231 human breast cancer cells. Ang II increased cell migration and expression of matrix metalloproteinase
(MMP)-2,-9 in a dose-dependent manner. Ang II-mediated cell migration was reduced by specific blocking of MMP-2 and MMP-9, as well as
with pretreatment with inhibitors of AT1R, phosphatidylinositol 3-kinase (PI3K), Akt, and NF-kB. Similarly, Ang II-mediated expression of
MMP-2,-9 was downregulated by pretreatment with inhibitors of AT1R and PI3K. In addition, Ang II treatment significantly induced
phosphorylation of PI3K, Akt, and resulted in increased NF-kB activity. These findings suggest that Ang II activates the AT1R/PI3K/Akt
pathway, which further activates IKKa/b and NF-kB, resulting in enhanced expression of MMP-2,-9 and migration in human breast cancer
cells. Therefore, targeting Ang II/AT1R/PI3K/Akt/NF-kB signaling could be a novel anti-metastatic therapy for breast cancer.
J. Cell. Physiol. 229: 1855–1862, 2014. © 2014 Wiley Periodicals, Inc.
Angiotensin II (Ang II), a biologically active peptide of the
renin–angiotensin system (RAS), plays a fundamental role in
controlling blood pressure, tissue remodeling, and renal
homeostasis (Dzau, 1988). Recently, Ang II has been reported to
play critical roles in stimulating cell proliferation and angiogenesis
of various tumors, including gastric cancer (Kinoshita et al., 2009),
breast cancer (Chen et al., 2013), ovarian cancer (Suganuma
et al., 2005), prostate (Uemura et al., 2011), and bladder cancer
(Shirotake et al., 2011). Ang II transmits biological signals to target
cells through specific G-protein-coupled Angiotensin II type 1
receptor (AT1R). Expression of AT1R has been detected in a
variety of tumors, and is associated with a poor prognosis (Ino
et al., 2006; Arrieta et al., 2008; De Ronde et al., 2013). Moreover,
previous literature has shown that Ang II can modulate tumor cell
migration and invasion (Luo et al., 2011; Dominska et al., 2012).
Likewise, AT1R blockers (ARBs) can efficiently reduce tumor
growth, angiogenesis, and metastasis in mouse experimental
models (Deshayes and Nahmias, 2005; George et al., 2010; Wasa
et al., 2011).
Breast cancer is one of the most common malignances and
the leading cause of cancer death in women worldwide, with an
estimated 1.4 million new cases and 458,000 deaths in 2008
(Ferlay et al., 2008). The mortality for female breast cancer has
increased by 20.1% during the last decade in China (Guo
et al., 2012). The occurrence of distant metastasis leads to a
reduced survival of patients with breast cancer. Cancer
metastasis is a multiple-step process, where cancer cells first
detach from the primary tumor, invade surrounding tissues and
intravasate into blood and/or lymphatic systems, extravasate
from the vasculature, and subsequently settle and colonize at
the target organs.
Matrix metalloproteinases (MMPs) belong to a zincdependent endopeptidases family and play a key role in tumor
metastasis. MMPs act as enzymes that degrade extracellular
matrix (ECM) and destroy the basement of membranes (Zitka
© 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .
et al., 2010). So far, most investigators have focused on the
expression profile of MMP-2 (gelatinase A) and MMP-9
(gelatinase B). These MMPs are upregulated in various
malignancies, and high levels are linked to metastasis
(Kohrmann et al., 2009). Generally, MMP-2 is overexpressed
constitutively in highly metastatic tumors, whereas MMP-9 is
induced by stimulating factors such as inflammatory cytokines
and epidermal growth factor (EGF) (Turpeenniemi-
Abbreviations: Ang II, Angiotensin II; AT1R, Angiotensin II type 1
receptor; ARB, AT1R blocker; ECM, extracellular matrix; MMP,
matrix metalloproteinase; PI3K, phosphatidylinositol 3-kinase;
RAS, renin–angiotensin system.
Yanbin Zhao and Hongbin Wang contributed equally to this work.
The authors declared that they have no conflicts of interest.
Contract grant sponsor: Educational Commission of Heilongjiang
Province of China;
Contract grant number: 12521246.
*Correspondence to: Li Cai, Department of Internal Medical
Oncology, Harbin Medical University Cancer Hospital, Harbin
150040, Heilongjiang Province, China.
E-mail: [email protected]
**Correspondence to: Xiaoqun Dong, Department of Biomedical
and Pharmaceutical Sciences, College of Pharmacy, The University
of Rhode Island, Pharmacy Building, 7 Greenhouse Road, Kingston,
RI 02881.
E-mail: [email protected]
Manuscript Received: 5 January 2014
Manuscript Accepted: 28 March 2014
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 1 April 2014.
DOI: 10.1002/jcp.24639
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ZHAO ET AL.
Hujanen, 2005; Rydlova et al., 2008; Roy et al., 2009).
Therefore, MMP-2 and MMP-9 may play a more important role
in cancer cell migration and invasion. MMPs expression is
regulated by transcriptional factors (like NF-kB) through
upstream pathways including PI3K–Akt pathways (Westermarck and Kahari, 1999), which can regulate a variety of
cellular functions, including proliferation, apoptosis, and
migration (Castaneda et al., 2010).
Recently, studies have shown that Ang II may act directly on
breast cancer cells to accelerate the development of metastasis
in vivo. Microarray studies revealed an increase in MMP-2, MMP9, and ICAM-1 expression levels in response to Ang II
(Rodrigues-Ferreira et al., 2012). Ang II can stimulate the
expression of MMP-2 via activation of AT1R, but not MMP-9, in
B16F10 melanoma cells (Akhavan et al., 2011). These results
suggest that Ang II facilitates migration and metastasis in tumor
cells, and MMP-2 or MMP-9 is involved in Ang II-induced
migration. However, the mechanisms of MMP-2,-9 induced by
Ang II in human breast cancer cells are not well defined. In this
study, we demonstrate that Ang II/AT1R signaling promotes
migration and enhanced expression of MMP-2 and MMP-9 in
breast cancer cells. In addition, we found that phosphatidylinositol 3-kinase (PI3K), Akt, IKKa/b, and NF-kB signaling
pathways are involved in Ang II-mediated migration. These data
suggest that Ang II/AT1R signaling through activation of the
PI3K/Akt/NF-kB pathway plays a critical role in migration of
breast cancer, and may guide AT1R-targeted therapy to treat
human breast cancer.
Materials and Methods
Reagents and antibodies
Human Ang II was purchased from AnaSpec (San Jose). AT1R
antagonist losartan (DuP 753) was purchased from DuPont Merck
(Hangzhou, China). AT2R antagonist PD123319 and NF-kB
inhibitor pyrrolidine dithiocarbamate (PDTC) were purchased
from Sigma–Aldrich (St. Louis). LY294002, p-p85, Akt, p-AktThr308, p-Akt-Ser473, IkB, p-IkB, IKKa/b antibodies were
purchased from Cell Signaling Technology (Boston). Akt inhibitor,
MK-2206, was purchased from Selleck Chemicals (Houston).
MMP-2 siRNA, MMP-9 siRNA, IKKa-siRNA and IKKb-siRNA
were purchased from Santa Cruz (CA).
Cell lines and culture
The MDA-MB-231 cell line was a generous gift from Heilongjiang
Cancer Institute (Harbin, China). Cells were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum and
50 U/ml penicillin/50 mg/ml streptomycin, then incubated at 37°C
in a humidified atmosphere of 5% CO2. Experimental cells were
grown for an additional 24 h with either serum-free medium
(vehicle), Ang II (1–1,000 nM) alone, or a combination of Ang II
with Losartan (105 M), PD123319 (105 M), LY294002 (50 mM),
Akt inhibitor (0.5 mM). All dilutions were made in serum-free
medium. These cells were then used for subsequent assays.
Cell migration assays
Cell migration was evaluated by a Boyden chamber assay. Prior to
migration assay, cells were pretreated for 30 min with inhibitors
including Losartan, LY294002, Akt inhibitor, PDTC, or vehicle
control (0.1% DMSO). These concentrations of inhibitors did not
affect the survival of MDA-MB-231 cells as shown by cell viability
assay (data not shown). Approximately 1 104 cells in 200 ml of
serum-free medium were placed in the upper chamber, and 300 ml
of the same medium containing Ang II (1–1,000 nM) was placed in
the lower chamber. The cells were incubated for 24 h at 37°C in
5% CO2, then fixed in methanol for 15 min and stained with 0.05%
crystal violet in PBS for 15 min. Cells on the upper side of the filters
were removed with cotton-tipped swabs, and the filters were
JOURNAL OF CELLULAR PHYSIOLOGY
washed with PBS. Cells on the underside of the filters were
examined and counted under a microscope. Each experiment was
repeated at least three times. The number of invading cells in each
experiment was adjusted by the cell viability assay to correct for
proliferation effects of Ang II treatment (corrected migrating cell
number ¼ counted migrating cell number/percentage of viable
cells) (Pilarsky et al., 1998).
Zymography analysis
To analyze metalloprotease enzymatic activity, supernatants
collected from MDA-MB-231 cell cultures were mixed with
sample buffer without reducing agent or heating. Samples were
loaded onto 10% SDS–PAGE gel containing 1 mg/ml gelatin and
electrophoresed under constant voltage. Afterward, the gel was
washed with 2.5% Triton X-100 to remove SDS, rinsed with
50 mM Tris–HCl, pH 7.5, and then incubated overnight at room
temperature with developing buffer (50 mM Tris–HCl, pH 7.5,
5 mM CaCl2, 1 mM ZnCl2, 0.02% thimerosal, and 1% Triton
X-100). Zymographic activity was revealed by staining with
1% Coomassie Blue.
Real-time RT-PCR
Total RNA was extracted from cells with TRIzol reagent
(Invitrogen, San Diego, CA) according to the manufacturer’s
instructions, and quantified by measuring the absorbance at 260 nm.
Gene expressions were detected by quantitative real-time RT-PCR
(qRT-PCR) using the standard SYBR Green RT-PCR kit (Takara,
Dalian, China) according to the manufacturer’s instructions. The
cDNA was synthesized using the Revert Aid First-Strand cDNA
Synthesis kit (Takara) according to the manufacturer’s instructions.
Primer sequences were designed with Origene Technologies, Inc.
(Beijing, China). The primers used were as follows:
MMP-2—sense: 50 -TCTTCAAGGACCGGTTCATTTG-30 ;
antisense: 50 -GATGCTTCCAAACTTCACGCTC-30 ;
MMP-9—sense: 50 -ACCCTGCCAGTTTCCATTCATC-30 ;
antisense: 50 -CCGGCACTGAGGAATGATCTAAG-30 ;
b-actin—sense: 50 -CTACAATGAGCTGCGTGTGGC-30
antisense: 50 -CAGGTCCAGACGCAGGATGGC-30 ;
PCR conditions consisted of pre-denaturation at 94°C for
2 min, followed by 35 cycles of denaturation at 94°C for 30 sec,
annealing at 60°C for 30 sec and extension at 72°C for 1 min and a
final extension at 72°C for 7 min.
Data were analyzed by 244Ct.
SiRNA transfection
The siRNA against human MMP-2 (sc-29398), MMP-9 (sc-29400),
IKKa (sc-29365), IKKb (sc-35644), and control siRNA (sc-37007)
were purchased from Santa Cruz Biotechnology (USA). For
experiments using targeted siRNA transfection, each consisted of
a scrambled sequence that did not lead to the specific degradation
of any known cellular mRNA. Cells were transfected with siRNAs
(100 nM) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions.
Western blot analysis
MDA-MB-231 cells were lysed 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. After centrifugation at
12,000g for 5 min at 4°C, the supernatant was obtained. The
supernatant was used as a total cell lysate and analyzed for
protein concentration by the Bradford method (BioRad,
Hercules, CA). Equal amounts of cellular proteins (30 mg/lane)
were separated by SDS–PAGE and transferred to a
nitrocellulose membrane. After blocking with 1% skimmed milk
in Tris-buffered saline/Tween-20 overnight at 4°C, the blots
Ang II–AT1R AND BREAST CANCER
were incubated with rabbit anti-human p-p85, p85, Akt, p-AktThr308, p-Akt-Ser473, IkB, p-IkB, IKKa/b, p65, or p-p65 at a
dilution of 1:600 for 2 h at room temperature. The blots were
subsequently washed three times (10 min for each wash) with
Tris-buffered saline/Tween-20 and then treated with the
appropriate alkaline phosphatase-conjugated anti-rabbit
secondary antibody (dilution 1:5,000; Promega, Madison) for 1 h
at room temperature. The bands were visualized using BCIP/
NBT (Promega) coloration method.
Statistics
Data are expressed as the mean SD. Statistical analysis was
carried out using the Student’s t-test or ANOVA. A P-value < 0.05
was considered statistically significant. Each experiment was
repeated three times with similar results.
Results
Ang II-induced MDA-MB-231 cell migration is mediated
through AT1R
In order to determine the dose-dependent effects of Ang II
on cell migration, MDA-MB-231 cells were treated with Ang
II (1–1,000 nM) for 24 h using the Transwell assay. Ang IIinduced cell migration in a dose-dependent manner, with a
maximal response obtained at 100 nM Ang II at 24 h
(172.76 13.24%, P < 0.01, Fig. 1a). In order to determine if
Ang II receptors were involved in Ang II-induced cell
migration, MDA-MB-231 cells were treated with Losartan
(105 M, an AT1R antagonist) or PD123319 (105 M, an
AT2R antagonist) for 30 min prior to Ang II treatment.
Losartan significantly inhibited Ang II-induced cell migration
effects (125.06 8.52%, P < 0.01), whereas PD123319 had no
effect (169.51 18.17%, P > 0.05, Fig. 1b). These data suggest
that Ang II may induce cancer cell migration via AT1R.
Therefore, cells were not pretreated with PD123319 in
subsequent experiments.
Involvement of MMP-2,-9 in Ang II-induced MDA-MB-231
cell migration
MMP-2 and MMP-9 have been shown to play important roles in
cancer cell migration. To explore the involvement of MMP-2,-9
in Ang II-induced breast cancer cell migration, qPCR, Western
blot, and zymography assays were carried out to analyze
expression and activity. Treatment of cells with Ang II
(1–1,000 nM) significantly increased transcriptional expression
of MMP-2,-9 in a dose-dependent manner, peaking at 100 nM
Ang II at 24 h measured by qPCR (Fig. 2a,b, MMP-2: 100 nM,
2.8 0.16%, P < 0.05; MMP-9: 100 nM, 2.95 0.12%, P < 0.05).
Ang II also increased protein expression of MMP-2,-9 in
MDA-MB-231 cells in a dose-dependent manner, peaking at
100 nM Ang II at 24 h (Fig. 2c). Expression and enzyme activity
of MMP-2,-9 were also increased in the supernatant (Fig. 2d).
Furthermore, MMP-2,-9 expression and enzyme activity were
abolished by Losartan (105 M, an AT1R antagonist) (Fig. 2a–d).
To further confirm that Ang II-induced MDA-MB-231 cell
migration is associated with upregulation of MMP-2 and
MMP-9, cells were transfected with siRNAs against MMP-2 or
MMP-9. RT-PCR, Western blot confirmed the efficiency of
the MMP-2 or MMP-9 siRNA (Fig. 2e). The Transwell migration
assay demonstrated that Ang II-induced migration was
significantly attenuated by knockdown of MMP-2 or MMP-9
compared to control cells (MMP-2: 131.27 10.85%, P < 0.01;
MMP-9: 138.61 15.48%, P < 0.01, Fig. 2f). These results
confirm that Ang II may regulate the expression of MMP-2,-9
via AT1R, and MMP-2,-9 may be involved in Ang II-induced
MDA-MB-231 cell migration.
PI3K/Akt signaling pathway is involved in Ang II-mediated
MMP-2,-9 up-regulation and migration of breast cancer
cells
To further study the mechanisms underlying the migrationpromoting effects of Ang II, we investigated the signaling
Fig. 1. Ang II enhanced migration of MDA-MB-231 cells. MDA-MB-231 cells were incubated with Ang II (1–1,000 nM), and in vitro migration
activity was measured using the Transwell system. a: Ang II-induced cell migration was dose-dependent. b: Cells were pretreated with
Losartan (105 mM, an AT1R antagonist) or PD123319 (105 M, an AT2R antagonist) for 30 min followed by stimulation with Ang II (100 nM).
The in vitro migration activity measured after 24 h showed that Losartan inhibited cell migration. Results were expressed as the mean SE
for three replicate wells (*P < 0.01).
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Fig. 2. Ang II increased the expression and enzymatic activity of MMP2 and MMP9 in MDA-MB-231 cells. a,b: Cells were incubated with 1–
1,000 nM Ang II for 24 h; cell lysates were collected; MMP2 and MMP9 mRNA levels were determined using qPCR. c: Cells were incubated with
1–1,000 nM Ang II for 24 h; supernatants and cell lysates were then collected. MMP2 and MMP9 protein levels in cell lysates were determined
by Western blot analysis. d: Enzymatic activity of MMP2 and MMP9 in cell lysates and supernatants was measured using zymography. The
mRNA, protein levels, and enzymatic activity increased in a dose-dependent manner. Cells were pretreated with Losartan (105 mM, an AT1R
antagonist) for 30 min followed by stimulation with Ang II (100 nM) for 24 h. Losartan significantly reduced the expression at both mRNA and
protein levels, as well as inhibited enzymatic activity of MMP2 and MMP9. e,f: Cells were transfected with siRNAs specific blocking MMP2 and
MMP9 for 24 h; in vitro migration was measured with the Transwell assay after 24 h. Transfection with MMP2 and MMP9 siRNAs significantly
reduced Ang II-induced cell migration compared to the control and Ang II-treated group (*P < 0.01).
JOURNAL OF CELLULAR PHYSIOLOGY
Ang II–AT1R AND BREAST CANCER
molecules downstream of AT1R. MDA-MB-231 cells were
treated with LY294002 (50 mM, a PI3-kinase inhibitor) for
30 min prior to Ang II treatment. LY294002 significantly
reduced the level of Ang II-mediated cell migration
(124.91 10.79%, P < 0.01, Fig. 3a), as well as Ang II-induced
MMP-2,-9 protein expression (Fig. 3b). As p85 is the regulatory
subunit of PI3K, we then examined whether Ang II-induced
PI3K activation. As shown in Figure 3c, Ang II led to a significant
increase in phosphorylation of p85 in a dose-dependent
manner, peaking at 100 nM Ang II in breast cancer cells.
Next, we examined the role of Akt in Ang II-induced cancer
migration and upregulation of MMP-2,-9. Pretreatment of cells
Fig. 3. The PI3K/Akt signaling pathway is involved in Ang II-mediated upregulation of MMP2/MMP9 and cell migration of breast cancer. a,b:
MDA-MB-231 cells were treated with LY294002 (50 mM, a PI3-kinase inhibitor) or Akt inhibitor for 30 min prior to the Ang II treatment.
LY294002 significantly inhibited Ang II-mediated cell migration as well as reduced the expression of MMP2 and MMP9. c: Ang II led to a
significant increase in phosphorylation of p85 in a dose-dependent manner peaking at 100 nM Ang II in breast cancer cells. d: Ang II stimulated
the phosphorylation of Akt-Thr308 and Akt-Ser473 in a dose-dependent manner. Ang II (100 nM) caused maximal phosphorylation of Akt in
MDA-MB-231 cells (*P < 0.01).
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with Akt inhibitor (0.5 mM) antagonized Ang II-induced
migration and expression of MMP-2,-9 in cells
(129.93 14.39%, P < 0.01, Fig. 3a,b). We also investigated
whether Akt-Ser473 and Akt-Thr308 were phosphorylated in
response to Ang II treatment. We found Ang II stimulated the
phosphorylation of both Akt-Thr308 and Akt-Ser473 in a dosedependent manner. Ang II (100 nM) caused maximal
phosphorylation of Akt in MDA-MB-231 cells (Fig. 3d).
Based on these results, Ang II may act through the AT1R/
PI3K/Akt signaling pathway to enhance MMP-2,-9 expression
and cell migration in human breast cancer cells.
NF-kB signaling is involved in Ang II-mediated MMP-2,-9
upregulation and cell migration
The promoter region of MMP2 contains binding sites for
NF-kB, which is a pathway that can be activated by PI3K/AKT
(Kane et al., 1999; Westermarck and Kahari, 1999). To
determine if NF-kB activation is involved in Ang II-induced cell
migration and MMP-2,-9 upregulation in breast cancer, we used
the NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC).
Figure 4a shows that pretreatment with PDTC (10 mM)
inhibited Ang II-induced cell migration (139.23 17.40%,
Fig. 4. NF-kB signaling is involved in Ang II-mediated MMP-2,-9 upregulation and cell migration. a: Pretreatment with PDTC (10 mM), IKKa,
or IKKb siRNA inhibited Ang II-induced cell migration (*P < 0.05). b: Treatment of cells with PDTC also antagonized Ang II-induced
expression of MMP-2,9. c: Ang II-induced IKKa/b phosphorylation in a dose-dependent manner. d: Ang II also caused IkB phosphorylation in a
dose-dependent manner. e: Treatment of cells with Ang II for various time intervals resulted in p65 Ser536 phosphorylation. f: Hypothesized
mechanism of Ang II-induced cell migration in breast cancer (*P < 0.01).
JOURNAL OF CELLULAR PHYSIOLOGY
Ang II–AT1R AND BREAST CANCER
P < 0.01). Treatment of cells with PDTC also antagonized Ang
II-induced expression of MMP-2,-9 (Fig. 4b). We further
analyzed whether Ang II induces MMP-2, MMP-9 expression
through activation of NF-kB upstream molecules IKKab and
p65 through phosphorylation. Phosphorylation of IKKa/b and
p65 was examined by Western blot. Stimulation of cells with
Ang II-induced IKKa/b phosphorylation in a dose-dependent
manner (Fig. 4c). To further confirm that Ang II-induced
MDA-MB-231 cell migration is associated with upregulation of
IKKa and IKKb, cells were transfected with siRNAs against
IKKa and IKKb. RT-PCR, Western blot confirmed the
efficiency of the IKKa or IKKb siRNA (Fig. 4a). Transfection
with IKKa–siRNA or IKKb–siRNA significantly inhibited Ang
II-induced cancer cell migration (IKKa–siRNA:
141.2 13.79%, P < 0.01; IKKb–siRNA: 137.5 16.95%,
P < 0.01, Fig. 4a). The data suggest that IKKa/b activation is
involved in Ang II-induced cell migration of breast cancer. Ang II
also caused IkB phosphorylation in a dose-dependent manner
(Fig. 4d). Furthermore, it has previously been shown that
p65 Ser536 phosphorylation increases NF-kB transactivation.
Treatment of cells with various doses of Ang II resulted in
p65 Ser536 phosphorylation (Fig. 4e). These results indicate
that NF-kB activation is important for Ang II-induced cancer
cell migration and the expression of MMP-2,-9.
Discussion
Previous studies have shown that local RAS exists in various
malignant tumor tissues, especially in breast cancer. Ang II
promotes tumor cell proliferation, anti-apoptosis, migration,
and angiogenesis. Ang II promotes the migration of androgenindependent prostate cancer cells, but not androgendependent cells (Dominska et al., 2012). Ang II also plays an
important role in intrahepatic cholangiocarcinoma (ICC) cell
migration by mediating epithelial–mesenchymal transition
(EMT) (Okamoto et al., 2012). Ang II enhances migratory
capability and vimentin expression, reduces E-cadherin
expression, and stimulates translocation of b-catenin from
cytoplasm into the nucleus in ICC cells (Okamoto et al., 2012).
In breast cancer, Ang II can upregulate integrin b1 expression
(Berry et al., 2000). An in vivo model of metastasis in breast
cancer cells (D3H2LN) revealed that Ang II accelerates the
formation of metastatic foci at secondary sites (RodriguesFerreira et al., 2012). It is also reported that Ang II reduces cell
adhesion through AT1R by downregulation of integrins (a3 and
b1) (Puddefoot et al., 2006). In this study, we proposed that the
activation of the AT1R/PI3K/Akt/NF-kB cascade is involved in
Ang II-induced cell migration of breast cancer.
First, we demonstrated that Ang II significantly enhanced
migration of AT1R-positive MDA-MB-231 cells in a dosedependent manner. Moreover, to determine whether the
receptor is responsible for Ang II-mediated motility, we used
receptor-type-specific inhibitors. We found the AT1R
antagonist, but not AT2R antagonist, completely suppressed
Ang II-enhanced migration of MDA-MB-231 cells. These
findings suggest that Ang II-mediated tumor cell migration
requires the activation of AT1R; thus, selective AT1R blockade
therapy could efficiently inhibit the migration of AT1R
expressing tumor cells in response to Ang II.
We next investigated potential pathways that participate in
Ang II-enhanced cell migration. Enzymatic degradation of ECM
is one of the crucial steps in cancer invasion and metastasis.
MMPs, a family of more than 20 extracellular enzymes, play an
important role in metastasis because of their proteolytic
activities, which assist in degradation of ECM and the basement
membrane (Egeblad and Werb, 2002; Kerkela and SaarialhoKere, 2003; Tsai et al., 2010). Cytokines, growth factors,
chemokines, and MMPs regulate tumor cell migration and
invasion through autocrine and paracrine pathways
JOURNAL OF CELLULAR PHYSIOLOGY
(Woodhouse et al., 1997). Ang II can induce MMP2 expression
in gastric cancer cells (Huang et al., 2009). Ang II can stimulate
the expression of MMP-2 and MMP-13 through activation of
AT1R, but not MMP-9, in B16F10 melanoma cells (Akhavan
et al., 2011). In breast tumor D3H2LN cells, Ang II upregulates
MMP-2/MMP-9 and ICAM1, which are involved in cell adhesion,
migration, and invasion (Castaneda et al., 2010). In this study,
we found that Ang II enhanced the expression and enzymatic
activity of MMP-2 and MMP-9 in human MDA-MB-231 cells. In
addition, specific blocking of MMP-2 and MMP-9 by siRNA
significantly suppressed Ang II-induced cell migration.
Therefore, MMP-2 and MMP-9 are proposed to be Ang IIresponsive mediators that can cause the degradation of ECM
and lead to subsequent events of cancer migration and
metastasis.
Stimulation of AT1R has been shown to trigger the
activation of PI3k/Akt (Takahashi et al., 1999), which plays a
major role in a wide range of cellular processes, including
motility, cell-cycle progression, anti-apoptosis, and angiogenesis. PI3K phosphorylates the 30 -OH group of the inositol
ring in inositol phospholipids after activation by a variety
of extracellular stimuli. Previous findings support a dosedependent effect of Ang II on cancer cell proliferation and
anti-apoptosis through the PI3K/Akt pathway (Zhao et al.,
2008, 2010). Furthermore, Ang II mediated cell migration in
choriocarcinoma cells can be abolished by a selective AT1R
antagonist and a PI3K inhibitor (Ishimatsu et al., 2006). In
MDA-MB-231 cells, pretreatment with PI3K inhibitor
LY294002 antagonized Ang II-induced cell migration and
MMP-2,-9 upregulation. Moreover, Ang II increased phosphorylation of the p85 subunit in a dose-dependent manner.
The most important downstream effector of PI3K is Akt.
The induction of Akt activity is primarily under the control of
phosphoinositide products of PI3K, PIP2, and PIP3, which bind
to the pleckstrin homology domain of membrane-associated
Akt. Subsequently, full activation of Akt requires
phosphorylation of two amino acids in Akt, one within the
activation loop (Thr308) and one at the C-terminus
hydrophobic domain (Ser473) (Datta et al., 1999; Kandel and
Hay, 1999). In this study, Ang II increased the protein levels of
p-Thr308 and p-Ser473 in MDA-MB-231 cells in a dosedependent manner. Moreover, Akt inhibitor blocked Ang IImediated cell migration and expression of MMP-2,-9. Our data
indicate that PI3K/Akt may play an important role in
upregulating MMP-2,-9 and promoting cell migration of human
breast cancer.
The PI3K/AKT signaling pathway influences NF-kB activity
by promoting IKK activation as well as subsequent
phosphorylation and degradation of IkB, which activates the
RelA/p65 subunit of NF-kB in a number of systems (Yin
et al., 2006; Kloo et al., 2011). NF-kB regulates genes that are
responsible for cell growth, anti-apoptosis, and migration. Ang
II has been reported to prevent cisplatin-induced apoptosis
through NF-kB activation in pancreatic cancer cells (Amaya
et al., 2004). In this study, we demonstrated that Ang II-induced
migration was inhibited by PDTC, an NF-kB inhibitor,
indicating that activation of NF-kB might be involved in the
induction of cell migration stimulated by Ang II. We also found
that exposure of MDA-MB-231 cells to Ang II resulted in
increased phosphorylation of IKK, IkB, and p65. The promoter
region of MMP2 contains binding sites for transcription factors
(NF-kB, AP-1, and SP-1) (Sato and Takino, 2010). We found
that NF-kB inhibitors, as well as IKKa and IKKb siRNA,
reduced Ang II-induced cell migration and expression of
MMP-2,-9. Therefore, the NF-kB-binding sites may be critical
for Ang II-induced MMP-2,-9 expression and cell migration in
human breast cancer. However, we did not examine the role of
other transcriptional factors, including CREB and p53, which
also bind to the MMP2 promoter region. Whether other
1861
1862
ZHAO ET AL.
transcription factors are involved in Ang II-induced MMP-2
expression in breast cancer needs further examination.
Because the prognosis of breast cancer patients with distant
metastasis is very poor, detection and prevention of metastasis
is essential to improve clinical outcome of those patients. Our
study revealed that Ang II increased expression of MMP-2,-9
and enzymatic activities as well as enhanced migration of human
breast cancer cells via the AT1R/PI3K/Akt/NF-kB pathway.
These findings will help us to understand the mechanisms of
human breast cancer metastasis and may lead to the
development of effective therapies in the future.
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