Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis
et al. (2014) Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic
Cancer Growth and Metastasis. PLoS ONE 9(5): e97016. doi:10.1371/journal.pone.0097016
Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis
Ramadevi Subramani 0
Rebecca Lopez-Valdez 0
Arunkumar Arumugam 0
Sushmita Nandy 0
Thiyagarajan Boopalan 0
Rajkumar Lakshmanaswamy 0
Lucia R. Languino, Thomas Jefferson University, United States of America
0 Center of Excellence in Cancer Research, Department of Biomedical Sciences MSB1, Texas Tech University Health Sciences Center, Paul L. Foster School of Medicine , El Paso, Texas , United States of America
Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor b. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer.
Funding: The study was supported by Texas Tech University Health Sciences Center Paul L. Foster School of Medicine funds. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Pancreatic cancer is the fourth leading cause of cancer related
death even though it is only the thirteenth most common
malignancy in the world . The 5 year survival rate for patients
with pancreatic cancer is the lowest reported for any cancer, which
is less than 1% . This is mainly because pancreatic cancer is
difficult to detect at early stages due to lack of early warning signs
or symptoms . Pancreatic ductal adenocarcinoma (PDAC),
which is the most common form of this extremely aggressive
cancer, is highly invasive and metastatic and is highly resistant to
all forms of existing therapies . Patients who are diagnosed with
PDAC at advanced stages have little hope of effective surgical
resection  and other treatments like radiation, chemotherapy
(Gemcitabine, 5-flurouracil, cisplatin, paclitaxel, docetaxel, etc.) or
targeted therapies (Erlotinib-Tarceva)  do not currently offer
much benefit either. The asymptomatic nature of the disease in its
early stages has ensured that PDAC still remains a deadly and
nearly untreatable cancer despite multiple attempts to find better
treatment strategies . According to the most recent report,
approximately 280,000 new pancreatic cancer cases are diagnosed
globally and the incidence of pancreatic cancer continues to
increase [6,7]. Increasing incidence and mortality indicates that
there is still much lacking in detection and management of the
disease. Therefore, it is absolutely necessary to find better
diagnostic and therapeutic strategies for treating this disease.
Several growth factor receptors such as insulinlike growth
factor 1 receptor (IGF-1R), epidermal growth factor receptor
(EGFR), etc., are aberrantly expressed in many types of cancer
including pancreatic cancer . Increased IGF-1R expression
levels are associated with higher risk of developing various
neoplasms . The IGF-1R signaling axis is highly activated
during the early stages of lung carcinogenesis, where a role for
IGF-1R signaling was demonstrated not only in primary tumor
formation but also in progression to more aggressive lung
adenocarcinoma . Jie Tang et al., 2013 reported high
expression levels of IGF-1R in tumor tissue samples from 25 of
36 patients with epithelial ovarian cancer. They also reported
consistently higher levels of IGF-1 in primary cancer cell cultures
(230 ng/ml) compared to normal ovarian tissue cell cultures
(101.9 ng/ml) . IGF-1R signaling activates intracellular
signaling cascades that include phosphatidyl inositol 3-kinase
(PI3K), AKT, Rac and mitogen-activated protein kinase (MAPK)
[12,13]. These pathways regulate key genes involved in various
cellular functions such as proliferation, survival, differentiation,
transformation and apoptosis . Moreover, IGF-1R targets 70
to 100% of the core metabolic pathways that are often altered in
PDAC pathogenesis . Targeting IGF-1R has already been
shown to enhance the therapeutic effects of mTOR inhibitors in
metastatic renal cell carcinoma, . Likewise, in human
epithelial ovarian cancer, targeting IGF-1R by antisense
nucleotide reduced proliferation by 70% and clonogenicity by 10 fold
. In both in vitro and in vivo systems, an IGF-1R antagonist
(monoclonal antibody MK-0646) was shown to significantly down
regulate X-linked inhibitor of apoptosis (XIAP) protein, which has
been shown to be involved in cell survival and inhibition of cell
death in colorectal cancer . Moreover, IGF-1R targeted
therapies have already moved forward into phase I clinical trials
for prostate, breast, colorectal, liver, synovial sarcoma, etc.,
[12,1719] with promising initial results. However, the potential
benefit of using IGF-1R targeted therapy in pancreatic cancer is
not fully explored. Further, there is still a lack of detailed
knowledge regarding the exact molecular mechanisms by which
IGF-1R regulates pancreatic carcinogenesis.
Therefore, based on mounting evidence for the efficacy of
targeting IGF-1R in a broad spectrum of cancers, we have
determined the effects of targeting IGF-1R in pancreatic cancer in
this study. The ultimate goal of this study is to identify whether
IGF-1R signaling is an effective therapeutic target for pancreatic
cancer with the potential to translate rapidly into clinical use. Here
we clearly demonstrate that blocking IGF-1R expression enhances
apoptosis and suppresses cell invasion, migration and metastasis
via modulation of PI3K/AKT, MAPK and JAK/STAT signaling
pathways in pancreatic cancer cells.
Materials and Methods
All the experiments performed were approved by and
performed following the guidelines of the Institutional Biosafety
Committee of Texas Tech University Health Sciences Center.
Cell Lines and Reagents
Human pancreatic ductal adenocarcinoma cell lines, PANC-1
(epithelioid carcinoma), MIA PaCa-2 (carcinoma) and HPAC
(adenocarcinoma) were purchased from the American Type
Culture Collection, and were maintained in RPMI-1640 medium
supplemented with 10% FBS, 100 Units/mL of penicillin, and
100 mg/mL of streptomycin. Cells were maintained at 37uC in a
humidified atmosphere containing 5% carbon dioxide.
TransIT- siQUEST transfection reagent was purchased from
Mirus Bio (Madison, WI, USA). The 6.5 mm Transwell with
8.0 mm pore polycarbonate membrane inserts was obtained from
Corning Incorporated (Corning, NY, USA). BD Matrigel
(Bedford, MA, USA) and BD Pharmingen Annexin V-FITC Apoptosis
Detection Kit I (San Diego, CA, USA) was obtained from BD
Biosciences. BSA was purchased from Sigma-Aldrich Corporation
(St Louis, MO, USA). siRNA targeting IGF-1R was purchased
from Origene (Rockville, MD,USA). MTS reagent [3-(4,
5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was obtained from Promega (Madison, WI,
USA). Mammalian protein extraction reagent (mPER) was
purchased from Thermo Scientific (Rockford, IL, USA).
The following primary antibodies were used in this study:
pAKT(sc-101629), AKT(5298), Bcl-2(sc-783), pERK, (sc-101760),
ERK (sc-94) and STAT3(H-190)(sc-7179) (Santa Cruz,CA,USA);
IGF-1R(3027), Notch 2 (4530P), Snail (3879), E-cadherin (3195),
N-cadherin (4061), Zeb (3396), Vimentin (5741), Slug (9585), Bax
(2772), Caspase3 (9661), PARP (9542), pPI3K p85(4228), PI3K
p85(4292), IR-b (3024), pIRS-1(2388), IRS-1(2382), pSTAT3
(ser727) (4113), COX-2 (4842), pPTEN (9549), pmTOR (2974),
mTOR(4517), p-p70s6kinase (9206) and p70s6kinase (9202) (Cell
Signaling Technology, (Boston, MA); Caspase8 (ab 25901)
(Abcam, Cambridge, MA, USA); b-actin (Sigma Aldrich,
(St.Louis, MO, USA). Appropriate secondary antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Pancreas Adenocarcinoma Tissue Array
Pancreas adenocarcinoma tissue microarray (TMA) was
obtained from US Biomax, Inc, Rockville, MD The TMA
containing formalin-fixed paraffin embedded samples of
pancreatic adenocarcinoma and normal pancreatic tissues was subjected
to immunohistochemistry (IHC) to determine IGF-1R expression
levels. Institutional Review Board approval was not required for
IHC for IGF-1R antigen was performed using the pancreas
adenocarcionma TMA. TMAs were first incubated in an oven at
58uC for 2 h to enhance tissue adhesion to the charged glass slides.
Deparaffinization was then carried out to remove embedded
medium using xylene incubation for 20-minute. TMAs were
gradually rehydrated in serial alcohol baths (100, 95, 70, 50 and
30%) followed by a distilled water wash for 5 min. Heat induced
epitope retrieval with trilogy (Cell Marque, Rocklin, CA) was then
performed to unmask the antigenic sites within the tissue sections.
TMAs were blocked in TBS containing 1% fetal calf serum and
1% bovine serum albumin for 15 min. Perox-free blocking reagent
(Cell Marque, Rocklin, CA) was also added for 10 min to block
non-specific antibody binding. TMAs were incubated with
IGF1R antibody (1:50 dilution) overnight at 4uC. Slides were then
washed three times in PBS for 5 min and incubated with Ultra
Marque polyscan HRP Label (Cell Marque, Rocklin, CA) for 1 h
at room temperature. TMAs were then washed three times in PBS
and stained with chromogen solution (Cell Marque, Rocklin, CA)
for 20 min. Chromogen staining reaction was stopped by rinsing
with distilled water. Cell nuclei counterstaining was performed
with hematoxylin incubation for 40 sec. TMAs were rinsed with
distilled water and dehydrated with serial ethanol baths (30, 50,
70, 95 and 100%) followed by a xylene bath. Finally, TMAs were
coverslipped with the mounting media (Surgipath Medical
Industries, Richmond, IL) and digital images were captured using
a Nikon Microscope- ECLIPSE 50i.
Silencing of IGF-1R in PANC-1 and HPAC Cells
siRNA targeting IGF-1R were transiently transfected into
PANC-1 and HPAC cells using MIrus bio TransIT siQUEST
transfection reagent. Scrambled siRNA (non-silencing sequence)
was used as a control. Briefly, cells were seeded in 6-well plates at a
density of 2.56105 cells/well. Cells were transfected with different
concentrations and different subtypes (A, B and C) of siRNA
ranging from 10 to 50 nM for 48 h or 72 h, using Mirus
siQUEST Transfection Reagent. The ratio of siRNA to
Transfection reagent was maintained as 1:0.5 for efficient silencing
without toxicity according to the manufacturers protocol. The
final concentrations of siRNA were chosen based on dose
response studies. Forty-eight hours after the transfection, cells were
used for protein isolation or clonogenicity, invasion, and
migration, studies. Apoptosis was studied at 48 and 72 hours after
silencing of IGF-1R.
Cell Viability Assay
PANC-1 and HPAC cells were seeded in 96-well plates at a
density of 0.36104 cells/well and 0.56104 cells/well, respectively,
and transfected with IGF-1R at a final concentration of 30 nM of
subtype B and 50 nM of subtype A for 48 h along with
scrambled controls. After 48 h of transfection, cell viability was
measured using the MTS assay. The absorbance was read at
490 nm in order to calculate the percentage viable cells.
Clonogenicity/Colony Formation Assay in Soft Agar
Colony formation assay was performed in order to measure the
in vitro survival ability of a single cell to grow into a colony in an
anchorageindependent growth environment. Briefly, after
transfecting PANC-1 and HPAC cells with IGF-1R siRNA or
scrambled control for 48 h, these transfected cells were seeded
in complete media at a density of 26104 cells in 60-mm dishes
containing a top layer of 0.7% agar and a bottom layer of 1%
agar. The plates were incubated at 37uC for 3 to 4 weeks and then
stained with 0.2% Crystal violet. Colonies of greater than 20 cells
were counted manually.
Wound Healing Assay
Cell migrating ability of IGF-1R silenced pancreatic cancer cells
were measured using the scratch assay. Briefly, cells were seeded in
6-well plates at a density of 3.56105 cells/well for IGF-1R
transfection. At this density, PANC-1 and HPAC cells reached
monolayer confluency after 486h. A straight wound or scratch
was then gently created in the cell monolayers with a sterile pipette
tip. Cells detached by the scratch were washed twice with PBS and
cultures were then supplemented with fresh medium and
monitored for 96 h at 37uC using the Biostation CT (Nikon
Instruments Inc. Melville, NY, USA) for continuous observation.
The Biostation was automatically programmed to capture
photographs at 2 h intervals up to 96 h. Migration images were
captured and documented at different time points using
NISElement AR software.
Migration and Invasion Assay
The effect of IGF-1R siRNA on invasive properties of
pancreatic cancer cells was evaluated using transwell migration
and invasion assays. Forty-eight hours after transfection, PANC-1
cells and HPAC cells were trypsinized and resuspended in
FBSfree RPMI-1640 medium. For the migration assay, a total of
56103 cells were plated in the top chamber of the transwell with a
noncoated polycarbonate membrane (6.5 mm diameter insert, 8.0
`m pore size; Corning Incorporated). For the invasion assay,
26104 cells were plated in the top chamber of the transwell with a
matrigel-coated (1 mg/mL) polycarbonate membrane.
RPMI1640 medium with 10% FBS was added to the lower chamber as a
chemoattractant. After incubation for 48 h at 37uC with 5% CO2,
cells on the lower surface of the membrane were fixed with 5%
formalin and stained with 0.2% crystal violet. The non-migrated
cells on the upper side of the insert were wiped off with a cotton
swab. Images of the cells which migrated to the undersurface of
the membrane were captured in a blinded manner at 5 different
microscopic fields with 206 magnification. The number of
migrated or invaded cells was counted from five or six randomly
selected fields in a blinded manner. Experiments were done in
triplicates for statistical significance.
Analysis of Cell Death
The effect of IGF-1R silencing on apoptosis was measured using
the Annexin V-FITC Apoptosis Detection Kit I. Cells were
harvested 48 h and 72 h post-transfection and then stained with
Annexin V-FITC and propidium iodide according to the
manufacturers instructions. The percentage of cell death or
apoptosis was quantified using a flow cytometer (FACS Accuri C6)
Western Blotting Analysis
PANC-1 and HPAC cells were transfected with IGF-1R siRNA
for 48 h. After the incubation, protein was extracted from
transfected cells by lysing the cell membrane using mammalian
Protein Extraction Reagent (mPER) according to the
manufacturers protocol. The protein concentration was quantitated using
BSA standard methodology. Equal amounts of protein were
loaded and separated by SDS-polyacrylamide gels, and transferred
onto PVDF membranes. The membranes were blocked with 5%
BSA in 1 X TBST for 1 h and probed with a panel of primary
antibodies against pAKT, AKT, Bcl-2, pERK, ERK, STAT3,
IGF-1R, Notch 2, Snail, E-cadherin, N-cadherin, Zeb, Vimentin,
Slug, Bax, Caspase3, PARP, pPI3K p85, PI3K p85, IR-b, pIRS-1,
IRS-1, pSTAT3, COX-2, pPTEN, pmTOR, mTOR,
pp70s6kinase, p70s6kinase, Caspase8 or b-actin. After washing with
TBST, the membrane was incubated with secondary horseradish
peroxidase-coupled antibodies and then positive bands were
visualized using enhanced chemiluminescence.
The differences between groups were analyzed by unpaired
students t-test. The GraphPad Prism 5 software package version
5.03 was used to do all the statistical calculations. Probability
values ,0.05 were considered to be statistically significant.
IGF-1R is Highly Expressed in Pancreatic Cancers
We examined the expression levels of IGF-1R in pancreatic
ductal adenocarcinoma cell lines (HPAC, MIA PaCa-2 and
PANC-1) using western blot. All three cell lines had a high level of
expression of IGF-1R compared to normal pancreas. HPAC had
the highest expression of IGF-1R among the three pancreatic cell
lines we analyzed while PANC1 had the least expression. So we
chose to use HPAC and PANC1 cells for all our experiments
based on their IGF-1R expression (Figure 1A).
Immunohistochemical analysis of IGF-1R in pancreas adenocarcinoma tissue
microarray (TMA) was done to further confirm the
pathophysiological role of IGF-1R in PDAC pathogenesis. PDAC tumor
tissues at various stages showed increased expression of IGF-1R
compared with normal pancreas tissues (Figure 1B).
Silencing IGF-1R using siRNAs in Pancreatic Cancer Cell
To silence IGF-1R expression, we used 3 different siRNAs at
concentrations ranging from 10 to 50 nM. Scrambled siRNA
which does not target any gene was used as a control. siRNA
transfection efficiency varied based on the subtype and
concentration of siRNA for both cell lines. Accordingly, IGF-1R
expression levels were significantly reduced at a concentration of
30 nM B and 50 nM A for PANC-1 and HPAC cancer cells,
respectively (Figure 1C). Therefore, these two siRNA doses were
selected for all subsequent studies.
Silencing IGF-1R Induces Anti-proliferative Effect in
Pancreatic Cancer Cell Lines
Since IGF-1R is known to stimulate cell proliferation, we
examined the effect of silencing IGF-1R on proliferation of
pancreatic adenocarcinoma cells. Silencing IGF-1R decreased the
viability of pancreatic cancer cells at 48 h post-transfection
compared with scrambled siRNA transfected control cells. Only
45.24% & 47.28% cells were viable upon IGF-1R inhibition in
PANC1 and HPAC cells, respectively (Figure 1D). The
antiproliferative effect of targeting IGF-1R is highly significant in both
of the highly aggressive pancreatic cancer cell lines. These results
suggest a pivotal role for IGF-1R in the proliferation of aggressive
pancreatic cancer cells.
Silencing IGF-1R Inhibits Anchorage-independent
Growth of Pancreatic Cancer Cells
Anchorage-independent growth potential of cancer cells is one
of the important and well-known characteristic features of
transformed cells. Soft agar assays in PANC-1 and HPAC cells
were performed to assess whether IGF-1R knockdown influenced
colony forming potential of these cells. Silencing IGF-1R
dramatically reduced the colony forming capacity in both
pancreatic cancer cell lines (Figure 1E). Results from three
independent experiments were quantified, which reveals .85%
inhibition of colony forming capability in IGF-1R silenced
pancreatic cancer cells (Figure 1F).
(i) Wound healing assay. Reduced colony forming ability is
generally associated with a corresponding loss of invading
capabilities of cancer cells . The classic wound healing assay
was performed to test the role of IGF-1R in regulating the
migratory ability of pancreatic cancer cells. Cell monolayers were
scratched to create a wound to monitor the migrating ability of
both control and IGF-1R suppressed pancreatic cancer cells.
IGF1R suppressed PANC-1 and HPAC cells exhibited reduced
migratory capabilities compared with scrambled controls. HPAC
scrambled control cells reformed a complete monolayer within
48 h and PANC-1 scrambled control cells reformed a complete
monolayer within 96 h. In IGF-1R suppressed PANC-1 and
HPAC cells, a complete monolayer was not reformed even after
96 h (Figure 2A, B & C).
(ii) Transwell migration assay. Suppressed migration of
PANC-1 and HPAC cells achieved by blocking IGF-1R expression
was further confirmed using transwell migration assays. Once
again, compared to controls, IGF-1R siRNA transfected PANC-1
and HPAC cells showed a significant decrease in the number of
cells that migrated (Figure 2D & E). This strongly indicates that
blocking IGF-1R expression suppresses the migrating abilities of
both aggressive pancreatic cancer cell lines.
Knockdown of IGF-1R Inhibits Cell Invasion
Cell invasion is one of the critical steps involved in cancer cell
metastasis. Escape of cancer cells from primary site of origin to
distant sites occurs when the cells acquire the ability to penetrate
the tumor basement membrane and invade into surrounding tissue
and eventually into the blood and lymphatic systems. In vitro
matrigel invasion assays using transwell Boyden chambers which
mimic the internal basement membrane of the tumor
microenvironment were used to investigate the invading potential of
IGF1R siRNA transfected pancreatic cancer cells. Consistent with
reduced migratory ability, cells treated with IGF-1R siRNA
demonstrated a significant reduction in cell invasion ability of
more than 85% in PANC-1and HPAC cells compared with
scrambled siRNA-treated cells (Figure 3A & B). Taken together,
Figure 2. IGF1R silencing suppressed cell migration in pancreatic cancer cell lines. (A) Wound healing assay was performed to evaluate the
migration of PANC-1 and HPAC cells after silencing IGF-1R. Forty eight hours after siRNA transfection, wound healing capacity of cells were monitored
with automated Nikon Biostation CT at 2 h intervals up to 96 h. (B & C): Cell migration was determined by the rate of cells moving towards the
scratched area. The percentage migration was calculated by the NIS-Element AR software. Similar results were obtained in three independent
experiments. (D) Silencing IGF-1R expression inhibits migration of PANC-1 and HPAC cells. Cell migratory abilities were determined using uncoated
transwell Boyden chambers. Post transfection PANC-1 and HPAC cells were allowed to migrate through pores to the bottom surface of transwell.
Migrated cells were fixed and stained with 0.2% crystal violet in 5% formalin. Data are representative of five random microscopic field images taken at
20X magnification (E) Percentage migration for transwell assays is shown for IGF-1R silenced PANC-1 and HPAC cells from the results of three
Figure 3. IGF-1R silencing inhibits invading ability and epithelial-mesenchymal transition of pancreatic cancer cells. (A) PANC-1 and
HPAC cell invasion was assessed in transwell chambers coated with matrigel. Cells that invaded the matrigel-coated insert were fixed, stained and
captured at 206magnification. (B) Number of invaded cells were counted and expressed as percentage invasion. Experiments were done in triplicate
(C) IGF-1R silencing inhibits expression of several epithelial-mesenchymal transition markers. Total protein lysates from scrambled control and IGF-1R
silenced PANC-1 and HPAC cells were analyzed for expression of Notch-2, Snail, E-cadherin, N-Cadherin, Zeb, Vimentin, and Slug along with internal
control b-actin. (D) Densitometic values of EMT markers are shown as % expression. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC
Scrambled, HI-HPAC IGF-1R silenced.
these results indicate that silencing IGF-1R not only decreases
migration ability, but also the invasive properties of pancreatic
ductal adenocarcinoma cells.
Silencing IGF-1R Blocks Epithelial-mesenchymal
Transition (EMT) in Pancreatic Cancer Cells
The process of cancer cell invasion is enabled by EMT which is
the initiator of the metastatic cascade . Cells which undergo
EMT will attain stem cell-like properties that increase cell
proliferation, metastasis, etc. . The EMT-related factors such
as Notch-2, Snail, N-cadherin, Zeb, Vimentin and Slug were
significantly reduced upon silencing IGF-1R in PANC-1 and
HPAC cells (Figure 3C & D). Interestingly, western blot analysis in
IGF-1R suppressed cells showed increased expression of
Ecadherin; loss of this cell-cell adhesion molecule is thought to
promote invasion and metastasis . These data clearly indicate
that silencing IGF-1R in pancreatic cancer cells results in
inhibition of proteins favoring pancreatic cancer EMT.
Silencing IGF-1R Induces Apoptosis
Regulation of apoptosis in pancreatic cancer cells was analysed
upon IGF-1R silencing using Annexin V-FITC Apoptosis
Detection Kit I. Flow cytometric analysis showed a pronounced
induction of apoptosis/cell death by IGF-1R silencing in
pancreatic cancer cells (45.4%, 55.4% in PANC-1 and 47.7%,
59.5% in HPAC was observed after 48 h and 72 h of post
transfection, respectively) (Figure 4A & B).
To identify the molecular mechanism involved in this induction
of apoptosis, we investigated the expression pattern of several
apoptotic signaling molecules in pancreatic cancer cells. Cells
transfected with IGF-1R siRNA had significantly increased
expression of Bax, Caspase 8, Caspase3 and cleaved PARP in
comparison with cells treated with scrambled siRNA (Figure 4C &
D). Silencing IGF-1R also decreased the anti-apoptotic protein
Bcl-2 in both PANC-1 and HPAC cells (Figure 4C & D). These
data reveal that IGF-1R siRNA induces apoptosis via both death
receptor and mitochondrial mediated pathways of apoptosis.
Silencing IGF-1R Alters Key Signaling Molecules
Inhibiting only a single member of one particular signaling
pathway or targeting only one particular cellular function may be
insufficient to treat complex diseases like cancer. Therefore,
targeting molecules with the most impact on multiple oncogenic
signaling pathways will have greater translational potential for
clinical PDAC treatment. In this regard, we chose to perform an
even more comprehensive analysis of the effects of IGF-1R
Figure 4. The effect of IGF-1R siRNA on apoptosis of pancreatic cancer cells. (A) IGF1R inhibition induces apoptosis in PANC1 & HPAC cells.
Post transfection cells were stained with Annexin-V-FITC and propidium iodide followed by flow cytometry. The percentage of early apoptotic
(bottom right quadrant), apoptotic (top right quadrant), late apoptotic and necrotic cells (top left quadrant), and live healthy cells (bottom left
quadrant) are shown. (B) Percentage apoptosis and cell death is summarized for three independent experiments in PANC-1 and HPAC cells. (C) IGF-1R
inhibition induces death receptor and mitochondrial mediated apoptosis in PANC1 & HPAC cells. Bax, Bcl-2, caspase 8, caspase 3 and cleaved PARP
and b-actin expression was assayed using Western blot in IGF-1R siRNA-transfected PANC-1 and HPAC cells. (D) Representative densitometry analysis
shows significant potentiation of apoptosis via intrinsic and extrinsic pathways. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC
Scrambled, HI-HPAC IGF-1R silenced.
silencing in PANC-1 and HPAC cells and have verified that
IGF1R does in fact coordinate the regulation of multiple cellular
pathways involved in survival, proliferation, metastasis, EMT,
apoptosis and cell cycle signaling (Figure 5).
AKT is one of the most constitutively expressed molecules in
many cancers and has been shown to enhance malignant cell
proliferation . We performed Western blot analysis in IGF-1R
siRNA transfected cells in order to measure the expression levels of
the AKT/PI3K signaling cascade, including active downstream
and upstream effectors. We first measured the expression of active
and total forms of AKT and found that the active form of AKT
(pAKT) was significantly downregulated in IGF-1R silenced
PANC-1 and HPAC cells while total AKT levels were unchanged
(Figure 5A & B). A major upstream regulator of AKT is PI3K and
IGF-1R suppression also inhibited the expression of pPI3K
compared with scrambled control (Figure 5C & D).
Activation of P13K induces the phosphorylation of AKT which
in turn activates mTOR and its downstream effector molecule
pp70s6kinase. Once again, IGF-1R silencing resulted in the
suppression of the active forms of mTOR and p70s6k, while the
total forms of the proteins remained unaltered (Figure 5E & F).
Phosphatase and tensin homolog (PTEN), a potent inhibitor or
negative regulator of PI3K, is mutated in most cancers at high
frequency and associated with aggressive metastasis . The loss
of PTEN causes the dysregulation of PI3K, which could favor
pancreatic carcinogenesis; however the comprehensive mechanism
by which this occurs is not fully understood. PTEN was highly
activated upon IGF-1R silencing in PANC-1 and HPAC cells
(Figure 5E & F). Therefore, IGF-1R silencing induces PTEN
expression and inhibits phosphorylation of AKT, PI3K, mTOR
and p-70s6kinase in pancreatic cancer cells. These data suggest
that silencing IGF-1R inhibits cell proliferation and enhances
apoptosis by regulating activation status of PI3K/AKT pathway
ERK or Ras/Raf/MAP kinase signaling is one of the frequently
up regulated pathways in most cancers including PDAC. This
signaling pathway is known to regulate most cellular functions of
the body such as survival, proliferation, mitosis, motility,
differentiation, and apoptosis . ERK is a highly attractive
target for the treatment and development of anticancer drugs for
human cancer. We therefore examined the effects of IGF-1R
silencing on the expression of ERK. IGF-1R silencing resulted in
effective inhibition of p-ERK, without any alterations in total
ERK levels (Figure 6A & B). These results indicate that the ERK
pathway also plays an important role downstream of IGF-1R in
the proliferative and metastatic properties of pancreatic cancer.
The Janus kinase (JAK)/signal transducers and activators of
transcription (STAT) pathway is aberrantly activated in malignant
Figure 5. Suppression of IGF-1R alters key signaling molecules in PANC-1 and HPAC cells. (A, C & E): The effect of IGF-1R suppression on
AKT/PI3K signaling was examined in pancreatic cancer cells. PANC-1 and HPAC cells were treated with IGF-1R siRNA for 48 h. The cells were harvested
and the expression of phospho-AKT, AKT, phospho-PI3K, PI3K, phospho-PTEN, phospho-mTOR, mTOR, phospho-p70s6kinase, p70s6kinase and the
internal control b-actin was measured by Western blotting. (B, D & F): Densitometric analysis is also shown to the right of each representative image.
cancers. This signaling pathway is widely known for its role in
proliferation, immune response and hematopoiesis in response to
various growth factors and cytokines . IGF-1R silencing
profoundly decreased the levels of phosphoSTAT3,
pro-inflammatory cytokine cyclo-oxygenease-2 (COX-2) and phospho-insulin
receptor substrate-1(pIRS) (Figure 6C & D). Non-phospho specific
forms of STAT3 and IRS remained unchanged in both scrambled
control and IGF-1R silenced cells. Interestingly, targeting IGF-1R
also inhibited the expression of Insulin receptor-b (IR-b) (Figure 6C
All together, the results presented here demonstrate that
knockdown of IGF-1R expression inhibits metastasis via inhibition
of EMT. IGF-1R silencing also induces apoptosis and blocks cell
proliferation via inhibition of PI3K/AKT, MEK/ERK, MAPK
and JAK/STAT signaling. Our findings suggest that targeted
therapy against IGF-1R would be effective and beneficial for
treatment of patients with aggressive metastatic pancreatic ductal
Our present study focuses on defining the role of IGF-1R in
pancreatic cancer and lays a strong foundation for the deeper and
broader understanding of the molecular mechanisms by which
IGF-1R contributes to PDAC pathogenesis. This preliminary
study is a springboard for discovering predictive biomarkers useful
for PDAC diagnosis and therapy. IGF-1R is upregulated in a great
proportion of cancer cells [27,28]. We also found aberrantly over
expressed IGF-1R in pancreatic cancer cell lines (PANC-1 and
HPAC) and in human pancreatic adenocarcinoma tissues.
Decreased proliferation and anchorage independent growth was
observed upon effective IGF-1R silencing which indicates that
there is a possibility of extending the life expectancy of PDAC
patients through IGF-1R targeted therapies.
Epithelial-mesenchymal transition (EMT) is well-known as a
crucial event during cancer invasion and metastasis and was also
recently associated with poor disease prognosis in PDAC patients
. In a recent study, IGF-1R was shown to be a critical and
important driver for EMT related events in lung cancer . In
fact, overexpression of IGF1R was found to be associated with
increased mortality in most cancer patients due to enhanced
Figure 6. Silencing IGF-1R alters ERK and STAT signaling in PANC-1 and HPAC cells. (A & C): The effect of IGF-1R suppression on ERK and
STAT signaling was examined in pancreatic cancer cells. Whole cell lysates were separated by SDS-PAGE and analyzed by Western blot for expression
levels of phospho-ERK, ERK, IR-b, phospho-IRS-1, IRS, phospho-STAT3, STAT3, COX-2 and b-actin. (B & D): Representative blots are presented and
corresponding densitometric analysis is shown to the right of each image. PS-PANC-1 Scrambled, PI-PANC-1 IGF-1R silenced, HS-HPAC Scrambled,
HIHPAC IGF-1R silenced.
potential for metastasis . Similar to these prior results, when
we knocked down IGF-1R, we observed reduced motility and
migratory capabilities of PDAC cells. Thus the invasive properties
of PDAC are significantly inhibited when IGF-1R is silenced.
Loss of E-cadherin, which is a hallmark of increased EMT, is
sufficient to confer metastatic properties on breast cancer cells
. Our results demonstrate that silencing of IGF-1R inhibits
metastasis of pancreatic cancer cells by enhancing the expression
of E-cadherin. Our data further demonstrates that IGF-1R
knockdown leads to the suppression of other key regulators of
EMT such as notch-2, snail, zeb, slug, and the mesenchymal
proteins N-cadherin and vimentin. These molecular alterations in
response to IGF-1R knockdown could contribute significantly to
the inhibition of EMT in pancreatic cancer cells. Thus inhibition
of EMT is likely a primary factor contributing to reduced invasion
and metastatic potential of pancreatic cells where IGF-1R is
silenced. Targeting a master molecule like IGF-1R is therefore
much more beneficial for a disease like PDAC than targeting only
one or two components at a time.
Elevated levels of IGF-1 are known to play a pivotal role in
regulating cell proliferation, differentiation and apoptosis via
IGFR signaling . In cells highly dependent on IGF-1R signaling,
apoptosis is highly induced simply by inhibiting IGF-1R
expression . However the role of IGF-1R signaling is not yet well
studied in pancreatic cancer cells. Here, we observed the induction
of apoptosis at a high frequency in IGF-1R silenced pancreatic
cancer cells with increasing time of transfection. We found that
inhibition of the IGF-1R signaling axis reduced the expression of
the anti-apoptotic protein Bcl-2 and increased expression of pro
apoptotic Bax. The classical initiator and effector molecules of
apoptosis such as caspase8, caspase3 and cleaved PARP were
upregulated in IGF-1R silenced cells indicating that apoptosis
occurs via both death receptor and mitochondrial mediated
pathways. Therefore, both intrinsic and extrinsic apoptotic
pathways are effectively activated by IGF-1R silencing.
Interestingly, IGF1R inhibition by siRNA simultaneously
reduced the levels of insulin receptor (IR) in both PDAC cell
lines. Dual inhibition of IR and IGF-1R may confer an enhanced
antitumor effect because IR signaling is also implicated in the
pathogenesis of various tumors [33,34]. IGF-1R-targeted
inhibition is known to have certain metabolic consequences, which
include elevation of blood glucose and insulin levels through
feedback inhibition of the GH-IGF-1 signaling axis [32,35].
IGF1R blocking therapy, however, is reported to be well-tolerated in
clinical phase I and II trials with only 3 to 25% of participants
developing hyperglycemia [32,35].
Signal transduction cascades downstream of both IGF-1R and
IR were effectively inhibited by IGF-1R knockdown, including
PI3K/AKT, MEK/ERK and JAK/STAT pathways.
Hyperactivated AKT and PTEN loss have been reported in several cancers
including pancreatic cancer and are associated with increased
proliferation, metastasis, angiogenesis, cell growth and resistance
to apoptosis . IGF-1R silencing increases PTEN
expression and negatively regulates the PI3K/AKT tyrosine kinase
activities to enhance the anticancer effect in PDAC. Tumor
suppressor PTEN was also reported to suppress the MAP kinase
signaling via Shc phosphorylation  and IRS phosphorylation.
Similarly, IGF-1R suppression inactivated the phospho IRS-1
expression levels thereby suppressing MAP kinase and PI3K/AKT
signaling in PDAC cells. Extracellular signal-regulated kinase
(ERK), the effector molecule of MAPK signaling, was strongly
inhibited by silencing IGF-1R and this could have contributed to
the decreased proliferation of PDAC cells. Currently mTOR is
considered as one of the potential alternative targets for PDAC
treatment and is also considered as a master regulator similar to
what we show here for IGF-1R in pancreatic cancer . It is
therefore very encouraging that IGF-1R silencing strongly blocked
the phosphorylation of mTOR and its downstream target
p70S6Kinase through PI3K/AKT inhibition. Taken together,
our findings support the notion that silencing IGF-1R signaling
inhibits the downstream PI3K/AKT/MAPK pathways which also
affects pathways even further downstream, such as mTOR/
Cytokines like TNF-a are the mediators that link inflammation
and cancer [43,44]. Activation of STAT3 and key inflammatory
molecules such as nuclear factor kappa-B (NF-kB) induces COX-2
expression which in turn produces prostaglandins, resulting in
upregulation of proinflammatory processes that enhance breast
carcinogenesis [45,46]. Previous studies also show that PI3K
inhibitors blocked the phosphorylation of AKT and COX-2
expression to induce apoptosis in PTEN mutated human
endometrial cancer cells . Similar to this, we also found that
AKT and ERK activation was blocked by IGF-1R siRNA in
pancreatic cells leading to significant inhibition of the
proinflammatory molecule COX-2. In a transgenic mouse model
fed with high fat diet, the KRAS oncogene was activated
downstream of COX-2 which mediated pancreatic inflammation
that ultimately led to PDAC progression . In our study,
reduced COX-2 and pSTAT3 expression achieved by IGF-1R
inhibition indicates the potential role of IGF-1R in inflammation
mediated carcinogenesis of pancreatic cancer. In this regard, we
believe there is a primary role for IGF-1R in inflammatory related
In summary, our study demonstrates that silencing IGF-1R
strongly inhibits proliferation, colony forming capability,
migration, and invasive/metastatic potential of pancreatic ductal
adenocarcinoma cells. Further, we show that this occurs through
induction of apoptosis and inhibition of EMT. Key molecular
pathways affected by IGF-1R silencing in PDAC included PI3K/
AKT, MAPK/ERK and JAK/STAT signaling cascades. We
conclude that IGF-IR-targeting drugs hold much promise for the
treatment of PDAC due to the nearly universal effects of IGF-1R
in regulating various pathways involved in PDAC tumorigenesis.
However, it remains important to ensure that the beneficial effects
for PDAC treatment will not be outweighed by potentially harmful
off-target physiologic effects. Therefore, our specific aim in our
future work is to identify a molecular target that would be more
specific for pancreatic cancer cells compared to normal cells which
would still effectively target most of the protumorigenic functions
of IGF-1R in PDAC.
Conceived and designed the experiments: RS RL. Performed the
experiments: RS. Analyzed the data: RS RL. Contributed reagents/
materials/analysis tools: RL. Wrote the paper: RS. Helped in various
experimental techniques: AA SN TB. Edited the manuscript: RS RLV RL.
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