Role of ERK in Modulating MMP 2
and MMP 9 with Respect to Tumour Invasiveness in Human Cancer Cell Line MCF-7
and MDA-MB-231
Shuvojit
Moulik, Sekhar Pal, Jaydip Biswas, Amitava Chatterjee
Shuvojit
Moulik, Sekhar Pal, Amitava Chatterjee, Department of Receptor Biology &
Tumor Metastasis, Chittaranjan National Cancer Institute, 37, S.P Mukherjee
Road, Kolkata: 700026, India
Jaydip
Biswas, Department of Surgery, Chittaranjan National Cancer Institute, 37, S.P
Mukherjee Road, Kolkata: 700026, India
Correspondence
to: Amitava Chatterjee, Department of Receptor Biology & Tumor Metastasis,
Chittaranjan National Cancer Institute, 37, S.P Mukherjee Road, Kolkata:
700026, India
Email: amitavachatterjee24@gmail.com
Telephone: +91-9830128320
Received: September 26, 2013
Revised: November 4, 2013
Accepted:
November 11, 2013
Published
online: Febuary 18, 2014
ABSTRACT
Extracellular signal regulated kinase (ERK) is a key molecule located in
the Mitogen activated protein kinase (MAPK) pathway responsible for several
cell process in cancer including cell invasiveness which primarily involved
breakdown of extracellular matrix. Matrix metalloproteinase facilities cell
invasiveness by degrading Gelatinase which are again differentially regulated
by Integrins, a family of receptors. Our study analyses the role of ERK in
modulating cell invasiveness. Aims:
the role of ERK1/2 in modulating MMP2 and MMP9 and thereby cell
migration and invasion. Materials & Methods: The study involves
downregulation of ERK through potent inhibitors and ERK1/2 siRNA and its effect
on MMP2 and MMP9 and Integrin alpha 5 and beta 1 molecules. Western Blot,
Gelatin Zymography, Cell invasion and Cell Migration assays are the primarily
used to interpret the effect. Result: There was a considerable change in MMP 9
and MMP 2 after ERK inhibition which was also reflected in the downstream
process of the ERK pathway and Integrins alpha 5 and Beta 1. Conclusion: There
might be a direct correlation of ERK activity and MMP2 and MMP 9 activity with
targeted modulation of ERK. Specific inhibition of ERK also might lead to
integrin regulation.
© 2014 ACT. All rights reserved.
Key words: ERK1/2; MMP9; MMP 2; cell invasion;
MAPK pathway
Moulik S, Pal S,
Biswas J, Chatterjee A. Role of ERK in Modulating MMP 2 and MMP 9 with Respect
to Tumour Invasiveness in Human Cancer Cell Line MCF-7 and MDA-MB-231. Journal
of Tumor 2014; 2(2): 87-98 Available from: URL:
http://www.ghrnet.org/index.php/JT/article/view/602
INTRODUCTION
Among the signaling
pathways which integrates external clues in Cancer, Mitogen-activated protein
kinase (MAPK) cascade is a key intracellular signaling pathway that regulates
diverse cellular functions including cell proliferation, cell cycle regulation,
cell survival, angiogenesis and cell migration[1,2]. Disruption of
the integrity of the basement membrane is a key histological marker of tumor’s
transition to invasive carcinoma. The basement membrane forms a cellular
support for tumors and is made up of a complex mix of Extracellular matrix
(ECM) proteins, including laminin, collagens and proteoglycans. MAPKs have been
shown to be involved in regulating the proteolytic enzymes that degrade the
basement membrane. In metastatic cells ERK activity was shown to be higher than
non-metastatic cancer cells. In response to external stimuli phosphorylated ERK
translocates from cytoplasm to the nucleus and in the nucleus ERK activates a
host of transcriptional factors including activating protein-1(AP-1), as a
result of the induction of the c-fos gene, via phosphorylation of the ELK-1
ternary complex factor by phospho-ERK1/2. In tumors sustained MAPK activation
could lead to enhance induction of proteolytic enzymes in the surrounding
environment, leading to destruction of ECM, a key histological marker of
invasive carcinoma. Elevated expression of MMP9 is associated with increased
metastatic potential in many cancer types including breast, melanoma, prostrate
and brain cancers. Matrix metalloproteinase-9 (MMP-9) represents the largest
and most complex member among the MMPs. MMP-9 has a gelatin binding fibronectin
domain, composed of three fibronectin repeats, inserted between the active site
domain and Zn2+ binding domain. Among the known substrates for MMP-9
are gelatin, elastin, fibronectin, proteoglycans, laminin and type IV, V, VII
& X collagens. On the other hand the major substrate for MMP2, is type IV
collagen. It has also been implicated in the degradation of type V, VII and X
collagens, gelatins, elastin and fibronectin. Beside degradation of ECM, tumor
cell invasion and migration rely on the receptor dependent attachment and
release from the matrix and other cells. MMP activity directly modulates the
cell-cell and cell matrix attachment and regulates the process of cell invasion
and migration. Integrins play central role in anchorage dependent growth,
apoptosis, differentiation, and migration. Integrins differentially regulate
the production of MMPs. In human melanoma cell lines, α5β1 integrin and αVβ3 integrin modulates
release of MMP-2 and subsequent invasive behaviour. There is evidence that the
sustained activation of the Ras/Raf/MEK/ERK pathway is involved in the
expression of β3 integrins[3,4].
Recent studies have shown that significantly increased active ERK was found in
primary breast tumors compared with their adjacent matched normal breast
tissues and this increase ERK activity correlates with enhanced nodal
metastasis. Hence in this study the molecular mechanism of regulation and
deregulation of ERK was analysed with respect to MMP 2 and MMP 9 as a factor
for tumor invasiveness which is an essential requirement considering the
complexity lying beneath[5]. The study necessarily try to illustrate
that if ERK gets modulated the activity of MMP 2 and MMP 9 gets effected
directly. The work is conducted over a series of experiments where Fibronectin
induced human breast cell lines MCF-7 and MDA-MB-231 is used as a model. If the
results are properly transcribed there is huge significance with respect to the
molecules involved and the probable
direct co-relation between ERK and MMP2/9 can be used for therapeutic
prognosis of breast cancer.
MATERIALS
AND METHODS
Minimal
Essential Medium (MEM), fetal bovine serum (FBS), fibronectin (440
kDa), Protease Inhibitor Cocktail Tablets (complete, mini, EDTA-free), Protein
G agarose were purchased from Roche, Germany. Gelatin Sepharose 4B beads was
purchased from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. West Femto
substrate for Western Blot was acquired from Thermo scientific Pierce,
Rockford, IL USA. All primary antibodies (monoclonal and polyclonal), secondary
antibodies, ERK 1 and ERK2 siRNA and negative (scramble) control siRNA were
purchased from Santa Cruz Biotechnologies, Santa Cruz, CA, USA.ERK inhibitor
(PD98059), MEK inhibitor (U0126) were purchased from Promega, Madison, WI, USA.
Lipofectamine 2000 was purchased from Invitrogen, Life Technologies (USA).
Costar Transwell Plate was purchased from Corning Incorporated, Corning, New
York, USA. Human breast cancer cell lines MCF-7, MDA-MB-231, were obtained from
NCCS, Pune.
Cell culture
Cells were grown and
maintained in required medium (MEM, DMEM and RPMI) containing 10% FBS in a CO2
incubator at 37℃. For all the experiments cells were
grown as a monolayer on culture plates in absence and in presence and absence
of fibronectin (20 µg/ml) for 2 hours (for MCF-7) and 8 hours (for MDA-MB-231).
Cell Extraction
Cells were initially
grown in required medium supplemented with 10% FBS. The cells were collected
from culture flasks by trypsinisation and homogenized in Cell Extraction Buffer
(Tris-37.5 mM, NaCl-75mM, Triton-X-0.5% and pH adjusted to 7.5) at 15 minutes
of intervals for 1-1.5 hours at 4℃. It will then be centrifuged at 15,000 r.p.m for 10 mins at 4℃. The supernatant was collected and the pellet was
discarded and the protein estimated by Lowry’s Method. (It can also be stored
at -86℃ till use).
Zymography:
Cells were initially
grown in required medium supplemented with 10% FBS in petridishes, treated for
the stipulated time period in SERUM FREE CULTURE MEDIUM (SFCM) for 24hours and
the respective SFCMs was collected. The culture supernatant was collected by
centrifugation at 3000 r.p.m for 5 minutes. The matrix metalloproteinase was
separated from SFCM using Gelatin Sepharose 4B beads (75 L) for overnight at 4℃. The beads was washed x3 with Tris-buffered saline with
Tween-20 (TBST) and suspended in 50 Lof 1X sample buffer (0.075 gm Tris, 0.2 gm SDS in 10 ml water, pH
6.8). The suspension was incubated for 30 minutes at 37℃ and then centrifuged at 3000 r.p.m. for 3 minutes. The
supernatant was then subjected to zymography on 7.5% SDS-PAGE co-polymerized with
0.1% gelatin. Gel was washed in
2.5% Triton-X-100 for 30 minutes to remove SDS and wasthen incubated overnight
in reaction buffer (50 mM Tris-HCl pH 7, 4.5 mM CaCl2, 0.2 M NaCl). After
incubation, the gel was stained with 0.5% Coomassie Blue in 30% methanol and
10% glacial acetic acid. The bands was visualized by distaining the gel with
water.
Western Blot Assay
Cells were initially
grown in required medium supplemented with 10 % FBS in petridishes. The
respective cells were collected. Cell extraction was carried out using the
standard extraction protocol and the protein content of the extracts was
estimated by Lowry’s method. Equal amount of protein wastaken and after boiling
with 0.1 volumes b-mercaptoethanol for 5-8 minutes at 80-90ºC is finally subjected
to electrophoresis on 10 % SDS-PAGE. The proteins was transferred on to PVDF
membranes by Western Blot at 300 mA for 3 hours. The membranes was blocked with
1% BSA and subsequently washed x3 with TBST. The immunoblots was reacted with
required primary antibody. The blots was developed using respective alkaline
phosphatase coupled second antibodies. The color was developed using ECL as
substrate.
Cell Invasion assay
24 well transwell
plate (Corning) with 12 inserts were taken and the lower chamber of each well
was poured with 600 mL MEM SFCM. Control and fibronectin treated cells (100,000 cells/insert) were seeded
in triplicate on membrane in the upper chamber of the insert. Cells were then
allowed to grow for 24 and 48 h. After 24 and 48 h of incubation, media was
pipetted out from membrane. SFCMs from lower chambers were collected and
centrifuged at 3000 rpm for 3 min. The membranes of the inserts were washed
thrice with PBS. Cells were then fixed with 4% formaldehyde solution, followed by washing with PBS. Cells
were then stained with Gill’s hematoxylin for 10 min. Membranes were then
washed thoroughly in running water. The upper side of the membranes was scraped
with buds; membranes were then cut and mounted with glycerol. The cells
migrated through the membrane pore were observed under microscope.
Wound Healing Assay
Cells were grown as
a monolayer on culture plates in absence and in presence and absence of
fibronectin (20 µg/mL). The monolayer was scratched with a sterile pipette tip,
followed by washing with SFCM to remove cellular debris. Cell migration across
the wound was observed by microscopy and documented by photography at 0 hr, 6
hrs and 24 hrs, 48 hrs.
siRNA transfection
Single transfection procedure. Cells were plates 24 hrs prior to
transfection. The cells were incubated at 37℃ for 24 hrs under 5% CO2.After incubation, the plate was
with PBS (once), and layer with 1.75 mL OptiMEM. The following mix was prepared
and incubated at room temperature for 5 minutes.
Tube 1: siRNA: 100 nM (Stock conc: is 100 µM,
siRNA: 2uL ); OptiMEM: 198 µL
Tube 2: Oligofectamine: 4 µL; OptiMEM : 46 µL
The content of both the
tubes were mixed and incubated for further 30 minutes. siRNA-lipid mix on the
cells (dropwise with constant swirling) was layered. The plate was incubated at
37℃ for 4 hrs. Fresh media was added after 4
hours incubation and incubate further 24 hrs. Cells were analyzed depending
upon the experiment.
Double transfection (combinatorial
transfection) procedure
For experiment with
two round of siRNA transfection, second round of transfection was done in the
similar manner as described above after 24 hours of first round of
Transfection. Scramble control for siRNA was similarly prepared.
RESULTS
Effect of fibronectin on activity and
expression of MMP2, MMP-9 and ERK1/2
Figure 1 shows the
effect of Fibronectin on activity and expression of MMP2 and MMP9 and also
depicts the expression of pERK1/2 and ERK1/2. There was appreciable increase in
the activity of MMP2 as well as MMP9 with the introduction of 20 ug Fibronectin
in MCF-7 cells (Figure 1A). Activity of MMP9 also got increased in MDA-MB-231
cells. The total protein levels of MMP9/MMP2 didn’t change at all in both the
cell lines. An appreciable increase of pERK1/2 was noted and both the cell
lines, though the increase was higher in MDA-MB-231 cells with respect to MCF-7
cells. The total (dephospho) protein levels of ERK1/2 in both the cell lines
had hardly any effect of Fibronectin treatment. This activation corresponded with
the activity of MMP2 and MMP9 in both the cell lines and hence showed an
indication of a possible correlation between the two events. The internal control Beta tubulin didn’t
show any change. (we did this experiment to study possible interrelationship of
ERK ½ and MMP-2/9 activity).
Effect of MEK/ERK inhibitor PD98059 on
activity and expression of MMP 2/MMP9 and ERK1/2
Figure 2 depicts the
effect of MEK/ERK inhibitor PD98059 on activity and expression of MMP2 and MMP9
as well as ERK1/2. There was a sharp decrease of both MMP2 and MMP9 in Zymogram
of MCF-7 cells. This also corresponded with the inhibition of pERK1/2 levels in
MCF-7 cells. In MDA-MB-231 cells PD98059 effectively inhibited phosphorylation
of ERK1/2. There was also a decrease in activity of MMP9 in MDA-MB-231 cells. The
levels of total protein of MMP9 and MMP2 in both the cell lines didn’t show any
change with the introduction of the inhibitor. Dephospho form of ERK1/2 was
almost unaffected by inhibitor treatment. (we did this experiment to see the
inhibition of ERK ½ on MMP-2 /9 activity).
Effect of MEK/ERK inhibitor U0126 on
activity and expression of MMP 2/MMP9 and ERK1/2
Figure 3 portrays
the expression and activity of ERK1/2 in relation to MMP2 and MMP9 after
treatment with inhibitor U0126. There was noticeable decrease of pERK1 with the
treatment of the inhibitor, though the dephospho protein didn’t show any
visible change (Figure 3A). Both MMP2 and MMP9 showed reduction in their
activity in degrading Gelatin. In MDA-MB-231 cells there was a sharp decrease
of pERK1/2 levels which corresponded to the appreciable decrease of MMP9 activity. The
total protein levels of MMP2 and MMP9 didn’t change (we did this study to see
the effect of ERK½ inhibition on MMP-2/9 activity).
Effect of ERK1/2 siRNA on activity of
MMP 2/MMP9 and activity and expression of ERK1/2
Figure 4
demonstrates the effect of specific inhibition of ERK1/2 by sequential double
transfection of ERK1 and ERK2 siRNA on activity of MMP2 and MMP9. In MCF-7
cells there was an 80% (approx) decrease in the phosphorylation levels of
ERK1/2 along with the sharp decrease of the dephospho levels. The MMP9 and MMP2
activity levels showed a sharp decrease after siRNA treatment. In MDA-MB-231
cells along with the decrease of ERK1 levels, the activity of MMP9 also reduced
to basal levels. Thus it might be said that in both the cell lines there was a
possible correlation between ERK and MMP9 and MMP2. The scramble control didn’t
show any change throughout. (we did this experiment to study specific ERK 1 or
ERK 2 on MMP-2/9 activity).
Effect of ERK1/2 inhibition on activity
and expression of JAK2, STAT3 and PI-3K
To investigate the
effect of ERK1/2 inhibition on other signaling molecules the levels of (Janus
kinase) JAK2, (Signal transducer and activator of transcription) STAT3 and
(Phosphoinositide-3 kinase) PI3K were checked in Figure 5. In MCF-7 cells there
was no visible effect of ERK inhibition by inhibitor or siRNA on activity or
expression of JAK2 (Figure 5A). The results were also similar in MDA-MB-231
cells. The expression of pSTAT3 in MCF-7 cells showed a decrease when PD98059
was introduced, after a sharp increase in Fibronectin (Fn) treated cells. With
U0126 treatment this decrease was similar which further went down significantly
with siRNA treatment. In MDA-MB-231 cells the results were similar with a sharp
escalation of STAT3 activity in Fn lane, which went down when MEK/ERK
inhibitors were introduced. With the introduction of ERK1/2 siRNA, the pSTAT3
levels went further down suggesting a role of activated STAT3 in this pathway. The
expression of STAT3 however remain unchanged in both the cell lines. The
phospho PI3K slightly increased with Fn treatment but decreased with
administration of PD98059. When MEK/ERK inhibitor U0126 was treated, MCF-7
cells showed a slight decrease in PI3K activity. But the levels didn’t change
with ERK1/2 siRNA treatment. The expression of PI3K didn’t alter in the total
protein form. In MDA-MB-231 cells the activity of PI3K remain almost similar
and only changed when cells were treated with U0126. (we did this experiment to
see the effect of ERK ½ inhibition on other signalling pathway).
Effect of ERK1/2 inhibition on cell
migration
Figure 6 shows the
effect of ERk1/2 inhibition on cell migration with the upper lanes showing
MCF-7 cells and the lower lanes showing MDA-MB-231 cells. In MCF-7 cells there
was increase of cell migration in cells treated with PD98059, which slowed down
with U0126 treatment and was almost comparable with control cells with ERK1/2
siRNA treatment. In MDA-MB-231 cells both the inhibitors slowed down the rate
of migration, but the combinatorial transfection of ERK1/2 siRNA showed a
remarkable slowdown of migrating cells. This gives an idea of the importance of
ERK1 modulation along with ERK2. ( we did this experiment to study the study
the inhibition of ERK ½ on cell migration & invasion).
Effect of ERK1/2 inhibition on cell
invasion
The rate of cell
invasion after ERK1/2 inhibition is shown in Figure 7. The invasion rate of
MCF-7 cells (Figure 7A) went higher with Fibronectin treatment, but decelerated
with PD98059. The invasion rate of MCF-7 cells went further down with U0126
treatment and became similar as control cells with ERK1/2 siRNA treatment. The
results were similar in MDA-MB-231 cells but with siRNA treatments it showed
the highest effect on the invasion rate of MDA-MB-231 cells.
Effect of ERK1/2 inhibition on activity
and expression of RKIP, MEK1/2 and cFos
To further
investigate the role of ERK1/2 the activity and expression of RKIP (Raf kinase
inhibitor protein), MEK1/2 and cFos were checked after ERK1/2 modulation
(Figure 8). In MCF-7 cells with the introduction of Fibronectin the activity of
RKIP reduced considerably which slightly increased with inhibitor treatment
(PD98059 and U0126) and went further up with ERK1/2 siRNA treatment. The
expression of RKIP also followed the same pattern with siRNA treated cells
having the highest band intensities. The results of pRKIP in MDA-MB-231 cells
were similar. The total protein levels of RKIP almost remained unaltered with
only Fibronectin treated cells showing a sharp decrease in the band
intensities. The activity of MEK1/2 in MCF-7 cells remained unaltered althrough
the lanes with only an appreciable increase in Fn treated cells. The total
protein levels remained unaltered. In MDA-MB-231 cells Fn treated cells showed
increased levels of pMEK1/2, which remained constant with the inhibitors and
siRNA treatments. There was hardly any change in the expression of MEK1/2. In
MCF-7 cells with only ERK1/2 siRNA treatment the levels of pcFos went down
considerably which was otherwise constant. The total protein levels of cFos
remained unaltered. When MDA-MB-231 cells were treated with Fibronectin, there
was a sharp increase of pcFos activity which didn’t change with PD98059
treatment. With the administration of MEK/ERK inhibitor U0126, the levels of
pcFos went down and further decreased with ERK1/2 siRNA. The expression of cFos
showed an appreciable decrease with U0126 treatment while the band intensities
went down further with the administration of ERK1/2 siRNA.
Effect of ERK1/2 inhibition on the
expression of Integrin alpha 5, Integrin Beta 1, FAK and Paxillin
Figure 9 shows the
effect of ERK1/2 inhibition on the expression of integrin alpha v, integrin
beta1, FAK (Focal adhesion kinase) and paxillin. In MCF-7 cells alpha 5
increased with Fn treatment, remained constant with PD98059 and U0126 treatment
but showed a considerable change in band intensities with ERK1/2 siRNA
treatment. In MDA-MB-231 cells (Figure 9 B) there was a sharper decrease of
alpha 5 levels with siRNA treatment. In MCF-7 cells Fn treated cells showed an
increase in beta1 band intensities, which went down with the administration of
U0126 and reached basal level with ERK1/2 siRNA. Beta1 showed a sharp fall with
ERK1/2 siRNA, but otherwise remained constant in MDA-MB-231 cells. This suggest
ERK1/2 and Integrin alpha5 beta1 might have direct interaction between the two.
In both the cell lines the activity of FAK went up with the treatment of
Fibronectin, didn’t change with the administration of PD98059 or U0126 and had
an appreciable decrease with transfection of ERK1/2 siRNA. The expression of
FAK had hardly any visible change in both the cell lines with only one
exception in MCF-7 cells where the total protein level went down by 30% in
U0126 treated lanes. The expression of paxillin in MCF-7 cells remain unaltered
throughout the lanes only decreasing sharply in siRNA transfected cells. In
Figure 9B the levels of paxillin increased in Fn induced cells but went down
when inhibitor treatment (PD98059/U0126) was introduced and reduced to trace
amounts with ERK1/2 siRNA transfection. (we did this study to see the effect of
ERK ½ inhibition on FAK and paxillin).
DISCUSSION
The role of ERK1/2
in cell invasiveness has been elucidated in many recent evidences. Cell
invasiveness primarily involves the breakdown of extra cellular matrix. The
family of zinc dependent endopeptidases, which are involved in the breakdown of
extra cellular matrix, normal tissue remodeling and repair process are known as
matrix metalloproteinase (MMP). In cancer they play a critical role to
facilitate cell migration by removing physical barriers promoting angiogenesis.
Among MMPs, MMP2 and MMP9 (gelatinase A and gelatinase B) have found to be
related in major cases of tumor invasion and metastasis with their ability to
degrade collagen type IV, a major component basement membrane[9,10].
Integrins are the principal cell surface adhesion receptors, which mediates
cell adhesion to the ECM, transduce intra cellular signal and thus regulates
proliferation, survival and motility of cells. Integrins differentially
regulate the production of MMPs[6]. Many recent studies in our lab
have shown the cross talk between integrins and MMPs. These cross talks were
best-shown using ligand dependent activation of MMP9 and MMP2. These ligands
include fibronectin, laminin, victronectin which are substrates of MMPs[6,7,8].
Many recent evidences have been linked with the activation of ERK1/2 pathway
with MMPs. Specific ERK inhibition could inhibit MMP2 and MMP9 expression in
human gallbladder cell line[9]. In this present study we aimed to
look at the role of ERK1/2 in modulating MMP2 and MMP9 in a fibronectin induced
system. The study also takes a sneak pick in the possible signaling molecules
contributing in the modulation with respect to cell invasion and migration.
The cell line MCF-7 and
MDA-MB-231 cells were chosen very carefully as the expression of ERK1/2 in both
the cell lines were prominent enough in fibronectin induced system. In presence
of 20 ug fibronectin there was an appreciable activation of pERK1/2 in MCF-7
and MDA-MB-231 cells in 2 hours and 8 hours respectively. The concentration and
the activation time were chosen after series of time and dose dependent
experiments with fibronectin. This activation corresponded with the activity of
MMP2 and MMP9 in both the cell lines and hence showed an indication of a
possible correlation between the two events.
ERK1/2 holds a tight
selectivity on their activation mechanism and only get phosphorylated by MEK1/2
making them excellent targets for the development of therapeutic agents against
cancer. PD98059, one of the non-ATP competitive inhibitors of MEK1/2 had an
appreciable effect on the activation of ERK1/2 along with down regulation of
MMP2 and MMP9. This suggested a possible role of ERK1/2 in the modulation of
MMP. Interestingly, the total protein levels seem unaffected indicating the
importance of activated protein in transducing signals in the cell.
To investigate further
effects of ERK1/2 inhibition, another specific selective inhibitor U0126 was
introduced. The results of MCF-7 and MDA-MB-231 cells again indicated a direct
correlation between ERK1/2 inhibitor and gelatin degrading activity of both
MMP2 and MMP9. While PD98059 treated MDA-MB-231 cells showed an appreciable
decrease in MMP9 activity the levels went further down in U0126 treated cells
signifying the selective role of the inhibitory potency of this non-competitive
therapeutic compound.
Though the inhibitors were
potent to inhibit ERK phosphorylation in our study, the results of
invitrokinase assays in other studies using purified proteins suggested that
PD98059 and U0126 inhibited the phosphorylation and activation of MEK1/2 but
not the ability of MEK1/2 to phosphorylate ERK1/2. Hence we opted for more
targeted approach by small interfering RNA. With sequential double transfection
method ERK1 siRNA and ERK2 siRNA was transfected in both the cell lines. The
results again confirmed the role of ERK inhibition in diminishing MMP9 and MMP2
activity. These results were in accordance with various other recent evidences,
which suggests that specific blockade of ERK pathway inhibits invasiveness of
tumor cells by down regulation of matrix metalloproteinase[4]. Our
study also indicated that this regulation was probably dependent on the
activated protein as the total protein levels of ERK1/2 showed very less
variation throughout.
To understand the effects of
ERK inhibitors and siRNA on other signaling pathways and investigate more about
the role of ERK signaling, specific signaling molecules from different
signaling pathways were checked. Recent studies have shown the co-operation of
ERK1/2 pathway with JAK/STAT pathways involving JAK2 and STAT3 molecules. In
our Fn induced system JAK2 didn’t show much change in band intensities until
ERK1 siRNA was introduced, which might have effected JAK2 activation. The
results of STAT3 activity were in accordance with recent studies[10,11],
which were inhibited by treatment by MEK/ERK inhibitors. The decrease levels of
pSTAT3 with siRNA transfection also indicated the selective regulation of
JAK/STAT pathway, which might have added to the post-transcriptional modulation
of MMP2/MMP9 activity. The sudden decrease of PI3K levels in the activated form
with the treatment of PD98059 might indicate the non-specificity of the
inhibitor with respect to ERK1/2 inhibition.
The phenotypical assays were
performed to see the direct efficacy of ERK1/2 modulation on cell migration and
cell invasion. The result corresponded with MMP activity showing clear
indication of increased cell migration and invasion rate with ERK1/2 activation
and the subsequent down regulation with ERK1/2 inhibition. The striking
decrease in the rate of cell invasion as well as cell migration with ERK1/2
siRNA transfection again implied the importance of targeted inhibition.
Raf-1 kinase inhibitor
protein (RKIP) was originally identified physiologic inhibitor of MAPK/ERK
pathway, but was later also recognized as clinically relevant metastasis
suppressor genes[5]. In many recent discoveries it has been shown
that RKIP protein expression is down regulated in metastatic cancer cells of
breast, prostate and melanoma origin[10,12,13]. We examined RKIP
expression and activity in our model system and found that, there was a sharp
decrease of RKIP/pRKIP levels which slowly increased with inhibitor treatments
and became comparable to the control cells (in both the cell lines). This
suggests that the inhibition of ERK1/2 (by inhibitors/siRNA) might have induced
a feedback loop to normalize RKIP levels. The possibilities of feedback
mechanism are well discussed in recent evidences[14,15]. Needless to
mention here that RKIP is an upstream molecule in the MAPK pathway. We wanted
to check the levels of sole activator of ERK1/2 that is MEK1/2. The immunoblots
showing the expression of MEK1/2 didn’t show any visible change though the
activated MEK1/2 (pMEK1/2) increased with Fn treatment, implying that Fn can
activate MEK1/2 and subsequently ERK1/2. The obvious decrease of pMEK1/2 levels
with MEK/ERK inhibitor was normalized with ERK1/2 siRNA transfection showing
the specificity of the knockdown without affecting any other molecule. One
possible mechanism by which sustained MAPK activation could result in MMP9 or
MMP2 induction is through regulation of an essential transcription factor cFos[16].
Evidences suggest that phosphorylation of cFos by ERK1/2 enhances MMP9
expression (Chen et al). There are evidences that MMP2/MMP9 down
regulation in human leukemia cells is mediated by ERK-cFos pathway[17].
When we checked the expression and activity of cFos, the results indicated that
both the expression and the activity dropped down to basal level with siRNA
treatment suggesting the reason behind negative regulation of MMP9/MMP2. One of
the possible mechanisms of regulating MMP9 involves transfection factor AP-1
that has a consensus sequence in MMP9 promoter region and can be regulated
directly or indirectly by MAPK pathway through cFos and ELK1[18]. Though
MMP2 doesn’t share the conserved proximal AP1 site but recent studies[19]
reported regulation of MMP2 by functional AP1 binding site.
Integrins which control a variety
of signal transduction pathways functional to cell survival, cell proliferation
and differentiation are known to have altered expression levels in many types
of cancer. The majority of Integrins are multiple ligand binding apart from the
only receptor alpha5 beta1 which has a unique ligand specificity of
Fibronectin. Implication of this receptor in malignant progression is well
reported. Evidences suggest Integrin alpha5 beta1 can control invasion of human
breast carcinoma cells by direct and indirect modulation of MMP2 activity[20].
alpha5 beta1 are also known to be functional in Fn induced MMP9 expression and
activity when MCF-7 cells were probed for alpha5, there was a significant
decrease with ERK1/2 siRNA transfection which otherwise got higher with
Fibronectin treatment. This suggested a correlation between ERK1/2 and alpha5.
Integrin beta1 also behaved similarly. The decrease in the levels of integrins
(alpha5 beta1) in MDA-MB-231 cells were even steeper than MCF-7 indicating a
strong dependency on ERK inhibition. This was in accordance with works, which
reported a blockade in the expression of integrins with MEK/ERK inhibition[21].
The work also reported alpha5 beta1 Integrin expression with sustained
activation of ERK pathway, which might also holds true in our study where
activation of ERK1/2 by Fibronectin enhanced Integrin expression. This
correlation between ERK1/2 and Integrin alpha5 beta1 might involve direct
interaction between the two like in case of Integrin beta6, which directly
associates with ERK2[22]. Focal adhesion kinase (FAK) is a major
non-receptor kinase activated after Integrin mediated adhesion to ECM proteins
like Fibronectin. FAK lies at the crossroad of ERK1/2, PI3K/ AKT (protein
kinase B) and JAK/STAT pathways. Paxillin actually connects the ERK and FAK
pathways. The binding of ERK2 paxillin induces the binding of FAK to paxillin
resulting in further downstream activation[23]. In our study when we
probed for paxillin in both the cell lines, there was an increase with Fn
treatment, which went down to basal level with specific ERK inhibition by siRNA
supporting the correlation of ERK1/2 with paxillin, The decrease in paxillin
levels corresponded with the decrease in the activity of FAK which might
indicate a dependence on ERK pathway with paxillin which coordinates the focal
adhesion turnover and migration functioning as a regulated scaffold for a
localize connection between ERK/MEPK and FAK signaling pathways.
Our study, which
investigates the possible role of ERK1/2 in modulating MMP2 and MMP9, suggests
that there might be a direct correlation between ERK1/2 activity and MMP2 and
MMP9 activity which might involve STAT3 and cFos. This is the key point of this
study. The study also throws a light on the involvement of other signaling
molecules like MEK1/2, RKIP in this respect. MMP2 and MMP9, which were taken as
an indicator of tumor invasiveness along with other phenotypical parameters
like cell invasion and cell migration were shown to be regulated by ERK1/2
modulation. Interestingly this regulations were mostly through the activated
form of the concerned proteins and dependent on specific or targeted inhibition
of ERK1/2. Another key point the study elucidates is the role of ERK1/2 in
regulating alpha5 beta1 integrins, which differentially regulates MMP2 and MMP9
adding the importance of ERK signaling in cell proliferation, differentiation
and survival. With additional transcriptional study on these molecules, which
was beyond the scope of our work, novel mechanisms can be identified which
might help in targeted multi-therapeutic approaches for cancer prognosis and
survival.
ACKNOWLEDGMENTS
We acknowledge
Department of Science and Technology,New Delhi (DST, SR/SO/HS-93/2010) for
financial assistance and Director, Chittaranjan National Cancer Institute
(CNCI) foracademic and
infrastructural facilities.
CONFLICT
OF INTERESTS
There are no
conflicts of interest with regard to the present study.
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Peer reviewer: Zhenyi Ma, Professor,
Department of Biochemistry and Molecular Biology, School of Bascic Midical Sciences,
Tianjin Medical University, Room 402 South Building, 22 Qixiangtai Road, Heping
District, Tianjin, 300070, China.
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