Dual inhibition of epidermal growth factor receptor and insulin-like growth factor receptor I: reduction of angiogenesis and tumor growth in cutaneous squamous cell carcinoma.
ABSTRACT Cutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancer. Most of the approximately 250,000 cases occurring annually in the United States are small, nonaggressive, and cured by excision alone. However, a subset of these tumors which are defined by poorly differentiated histology, large tumor size, invasion of adjacent structures, and/or regional metastases can prove resistant to treatment despite adjuvant radiotherapy and can have an increased risk of recurrence and nodal metastasis. Novel therapeutic approaches are necessary to improve the outcomes for patients with aggressive CSCC.
We analyzed the effect of targeted therapy on the growth and survival of CSCC cell lines using an anti-insulin-like growth factor-I receptor (IGF-IR) antibody, A12, alone or in combination with an anti-epidermal growth factor receptor (EGFR) antibody, cetuximab, both in vitro and in vivo in an athymic nude mouse model of CSCC.
Treatment with A12 and cetuximab inhibited the signaling pathways of IGF-IR and EGFR and inhibited proliferation and induced apoptosis of squamous cell carcinoma (SCC) cell lines in vitro. Immunohistochemical staining revealed decreased proliferating cell nuclear antigen (PCNA), microvessel density, and increased apoptosis within the treated tumor xenografts. In addition, the administration of A12, alone or in combination with cetuximab inhibited the growth of tumors by 51% and 92%, respectively, and significantly enhanced survival in the nude mouse model of CSCC (p = .044 and p < .001, respectively).
These data suggest that dual treatment with monoclonal antibodies to the EGFR and IGF-IR may be therapeutically useful in the treatment of CSCC.
- SourceAvailable from: Giulia Spallone[Show abstract] [Hide abstract]
ABSTRACT: Nonmelanoma skin cancer (NMSC) is the most prevalent cancer in light-skinned populations, and includes mainly Basal Cell Carcinomas (BCC), representing around 75% of NMSC and Squamous Cell Carcinomas (SCC). The incidence of these tumors is continuously growing. It was found that the overall number of procedures for NMSC in US rose by 76%, from 1,158,298 in 1992 to 2,048,517 in 2006. Although mortality from NMSC tends to be very low, clearly the morbidity related to these skin cancers is very high. Treatment options for NMSC include both surgical and nonsurgical interventions. Surgery was considered the gold standard therapy, however, advancements in the knowledge of pathogenic mechanisms of NMSCs led to the identification of key targets for drug intervention and to the consequent development of several targeted therapies. These represent the future in treatment of these common forms of cancer ensuring a high cure rate, preservation of the maximal amount of normal surrounding tissue and optimal cosmetic outcome. Here, we will review recent advancements in NMSC targeted therapies focusing on BCC and SCC.Cancers. 01/2011; 3(2):2255-73.
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ABSTRACT: Objective Heightened levels of sEcad are found in the serum of cancer patients and correlates with an unfavorable prognosis and later-stages of disease. In this study, we explored whether sEcad is elevated in human OPSCC specimens and FaDu cells. Additionally, we investigated sEcad-human EGFR and sEcad-IGF1R interactions and performed a functional analysis of sEcad in OPSCC cancers. Materials and methodssEcad, EGFR and IGF-1R levels were examined in human OPSCC specimens and cells by immunoblotting. sEcad-EGFR and sEcad-IGF-1R interactions were examined by immunoprecipitation and immunoblot assays. Levels of sEcad on EGFR and IGF-1R pathway components were evaluated by IB. The effects of sEcad on OPSCC proliferation, migration and invasion were assessed using standard cellular assays. ResultsStatistical analysis demonstrated that sEcad levels were significantly higher in OPSCC primary tumors and cells compared to normal controls. IP studies indicated that sEcad associated with EGFR and IGF-1R, and addition of sEcad resulted in a statistically significant increase in downstream signaling. Finally, cell based assays demonstrated enhanced sEcad-induced proliferation, migration and invasion, which was blocked by EGFR and IGF-1R inhibitors. Conclusions These findings suggest that sEcad may play an important role in OPSCC oncogenicity via its interaction and activation of EGFR and IGF-1R.This article is protected by copyright. All rights reserved.Oral Diseases 03/2014; · 2.38 Impact Factor
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ABSTRACT: A 77-year old male patient presented with an ulcerated exophytic tumor (T2, N0, M0) with three macroscopically visible satellite metastases in the right temporo-occipital region. Mohs surgery could not control the disease due to lymphangiosis carcinomatosa and perineural infiltration, and recurrence of satellite skin metastases. Re-staging demonstrated a T2, N1, M0 profile (stage III, AJCC). Chemotherapy was limited by the patient's co-morbidities. Therefore, we used targeted therapy with monoclonal anti-epidermal growth factor receptor antibody cetuximab in combination with volumetric modulated arc- radiotherapy (VMAT). Cetuximab was well tolerated except for the loading dose when the patient developed fever chills. To verify the correct application of VMAT, it was applied to a 3-dimensional measuring phantom prior to the patient's first treatment session. To minimize these tolerances, patient set-up was checked and corrected by orthogonal fluoroscopic images recorded daily by the on-board imager used in our Varian accelerator. The average daily beam time was 6 min (6 arcs, 767 monitor units); the total treatment time including patient set-up and set-up correction was less than 20 min. Combined therapy was well tolerated and complete remission was achieved.Dermatology Reports. 12/2011; 3:e57.
DUAL INHIBITION OF EPIDERMAL GROWTH FACTOR
RECEPTOR AND INSULIN-LIKE GROWTH FACTOR
RECEPTOR I: REDUCTION OF ANGIOGENESIS AND TUMOR
GROWTH IN CUTANEOUS SQUAMOUS CELL CARCINOMA
Chad E. Galer, MD,1Christina L. Corey, MD,2Zhuoying Wang, MD, PhD,3
Maher N. Younes, MD, PhD,1Fernando Gomez-Rivera, MD,1Samar A. Jasser, BS,1
DaleL.Ludwig,PhD,6Adel K. El-Naggar, MD, PhD,5Randal S. Weber, MD,1Jeffrey N.Myers, MD, PhD1,4
1Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston,
Texas. E-mail: firstname.lastname@example.org
2Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
3Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
4Bobby R. Alford Department of Otolaryngology–Head and Neck Surgery, Baylor College of Medicine, Houston, Texas
5Department of Head and Neck Surgery, Cancer Hospital of Fudan University, Shanghai, China
6ImClone Systems Incorporated, New York, New York
Accepted 5 February 2010
Published online 19 May 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/hed.21419
noma (CSCC) is the second most common nonmelanoma skin
cancer. Most of the approximately 250,000 cases occurring
annually in the United States are small, nonaggressive, and
cured by excision alone. However, a subset of these tumors
which are defined by poorly differentiated histology, large tu-
mor size, invasion of adjacent structures, and/or regional me-
tastases can prove resistant to treatment despite adjuvant
radiotherapy and can have an increased risk of recurrence
and nodal metastasis. Novel therapeutic approaches are nec-
essary to improve the outcomes for patients with aggressive
Methods. We analyzed the effect of targeted therapy on
the growth and survival of CSCC cell lines using an anti-insu-
lin-like growth factor-I receptor (IGF-IR) antibody, A12, alone or
in combination with an anti-epidermal growth factor receptor
(EGFR) antibody, cetuximab, both in vitro and in vivo in an
athymic nude mouse model of CSCC.
Results. Treatment with A12 and cetuximab inhibited the sig-
naling pathways of IGF-IR and EGFR and inhibited proliferation
and induced apoptosis of squamous cell carcinoma (SCC) cell
lines in vitro. Immunohistochemical staining revealed decreased
proliferating cell nuclear antigen (PCNA), microvessel density, and
increased apoptosis within the treated tumor xenografts. In addi-
tion, the administration of A12, alone or in combination with cetuxi-
mab inhibited the growth of tumors by 51% and 92%, respectively,
Background. Cutaneous squamous cell carci-
and significantly enhanced survival in the nude mouse model of
CSCC (p ¼ .044 and p < .001, respectively).
Conclusion. These data suggest that dual treatment with
monoclonal antibodies to the EGFR and IGF-IR may be thera-
peutically useful in the treatment of CSCC.
C 2010 Wiley Periodicals, Inc. Head Neck 33: 189–198, 2011
epidermal growth factor receptor (EGFR); squamous cell
carcinoma; orthotopic model; preclinical study
Cutaneous squamous cell carcinoma (CSCC) is the
second most common type of nonmelanoma skin can-
cer. The annual incidence is estimated at 1 case per
1000 individuals, and approximately 250,000 cases of
CSCC occurred in the year 2001 alone.1The cause-
specific mortality rate due to CSCC is less than 1%
overall, but rises to 30% at 3 years for those patients
whose tumors have aggressive features.2CSCC inci-
dence has risen dramatically over the past 2 decades
because of lifestyle changes leading to increased vol-
untary exposure to sunlight.3The standard of care
for the management of CSCC is surgical excision.
However, even adequate initial treatment of poorly
differentiated CSCC can be complicated by a recur-
rence rate of up to 25%. Recent successes involving
the therapeutic use of antibodies and small molecule
inhibitors against tyrosine kinases have generated
considerable interest in research aimed at targeting
these receptors in a wide variety of malignancies. In
an attempt to improve the treatment of CSCC, we
explored the effect of inhibition of 2 of these receptors
on cutaneous tumor growth in vitro and in vivo.
The insulin-like growth factor-I receptor (IGF-IR)
Correspondence to: J.N.Myers
Contract grant sponsor: This work was supported by The M. D.
Anderson Cancer Center Specialized Program in Research Excellence
in Head and Neck Cancer; contract grant number: P50 (CA097007);
contract grant sponsor: National Institutes of Health Cancer Center
Support; contract grant number: CA016672; contract grant sponsor: the
PANTHEON Program awarded to Jeffrey N. Myers.
Dale L. Ludwig is an employee of ImClone Systems which is a wholly
owned subsidiary of Eli Lilly and company.
C 2010 Wiley Periodicals, Inc.
Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell CarcinomaHEAD & NECK—DOI 10.1002/hedFebruary 2011 189
composed of 2 extracellular alpha subunits and 2 in-
tracellular beta subunits.4–6Ligand binding (IGF1
or IGF2) to the extracellular alpha subunits trigger
conformational changes in the beta subunits activat-
ing the receptors tyrosine kinase activity, which in
including the phosphatidylinositol 3-kinase/AKT and
pathways.6–11Numerous human tumors have been
shown to overexpress IGF-IR or have increased IGF-
IR kinase activity resulting in enhanced prolifera-
tion, protection from apoptosis, stimulation of migra-
tion and invasion, and stimulation of angiogenesis.
factor (IGF) binding proteins, human monoclonal
antibodies, and small-molecule tyrosine kinase inhib-
itors against IGF-IR, have been developed and show
promise for therapeutic use in both in vitro and in
vivo experiments.12–16A12, a high-affinity human
monoclonal antibody to IGF-IR, has been shown to
induce apoptosis and inhibit tumor growth by com-
petitively binding to the receptor and inducing IGF-
IR internalization and downregulation. Experimen-
tally, A12 has been shown to inhibit the growth of
breast, pancreatic, colon, and renal tumors, both in
vitro and in vivo with little toxicity or weight loss
in nude mouse models.13
The epidermal growth factor receptor (EGFR), a
member of the ErbB tyrosine kinase receptor family,
is a transmembrane glycoprotein receptor. Activation
of EGFR stimulates phosphorylation of downstream
signaling cascades that ultimately regulate cell prolif-
eration, migration, adhesion, differentiation, and sur-
mucosal squamous cell carcinoma (SCC) and is associ-
ated with malignant phenotype and poor progno-
sis.20,21Less is known about the expression of EGFR
in CSCC. Several small studies have shown that 43%
to 80% of CSCCs express membranous EGFR, but
this increases to 100% for metastatic CSCC. In pri-
mary tumors, one group demonstrated baseline acti-
vation of EGFR in 5 of 9 specimens with detectable
EGFR expression. While cetuximab has been well-
studied for the treatment of mucosal SCCs, the bene-
fit for CSCC is not well understood.22–25Barnes et
al21have shown in vitro efficacy of an EGFR inhibi-
tor, Iressa on CSCC, and several case reports have
examined the efficacy of various EGFR inhibitors and
have suggested the benefit of combination therapy
with a second agent.
EGFR and IGF-IR are logical targets for molecu-
lar therapy of cancer based on their frequent overex-
pression and established roles in the pathogenesis
and progression of numerous cancers.18,19,26Recently,
dual inhibition of receptor tyrosine kinases has
emerged as a method to improve the efficacy of tar-
geted therapy. Previous studies of single agents have
shown that tumors often have complex regulation
involving multiple protein tyrosine kinases and may
use these pathways as escape mechanisms when a
single receptor is targeted.25,27
In this study, we analyzed the effects of targeted
therapy against IGF-IR and EGF-R on CSCC cell
lines. We hypothesize that targeted therapy against
IGF-IR (A12) and EGFR (cetuximab) will inhibit
CSCC tumor growth in vitro and in an athymic nude
MATERIALS AND METHODS
Cell Lines and Culture Conditions.
lines Colo16, SRB1, and SRB12 were grown in Dul-
becco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), sodium
pyruvate, L-glutamine, vitamins, non-essential amino
acids (all from Life Technologies, Rockville, MD), and
penicillin-streptomycin (Flow Laboratories, Rockville,
MD). Adherent monolayer cultures were maintained
on plastic and incubated at 37?C in an atmosphere of
5% carbon dioxide and 95% air. The cultures were
maintained no longer than 12 weeks after recovery
from frozen stocks. These 3 cell lines were genotyped
using short tandem repeat analysis and have been
found to be unique and distinct from other cell lines
in the American Type Culture Collection and our
The CSCC cell
mice, age 8 to 12 weeks, were purchased from the
National Cancer Institute–Frederick Cancer Research
and Development Center (Frederick, MD). The mice
were housed and maintained in laminar flow cabinets
under specific pathogen-free conditions in facilities
approved by the Association for Assessment and Ac-
creditation of Laboratory Animal Care. The mice
were used in accordance with the Animal Care and
Use Guidelines of The University of Texas M. D.
Anderson Cancer Center (Houston, TX) under a pro-
tocol approved by the Institutional Animal Care and
Male athymic nude
burg, NJ) was diluted in phosphate-buffered saline
(PBS) to the appropriate concentrations for in vitro
studies and at a concentration of 5 mg/mL for intra-
peritoneal injections in the animal study. A12 was
generously provided by ImClone Systems. For in vitro
administration, A12 was dissolved in PBS to a con-
centration of 10 mg/mL and further diluted to appro-
priate final concentration in RPMI 1640 medium with
or without 2% FBS, as described below. For in vivo
testing, A12 was dissolved in PBS to a concentration
of 4 mg/mL. Both cetuximab and A12 solutions were
prepared immediately before administration to the
The following antibodies were used: anti-IGF-IRb
(C-20) and anti-EGFR (Santa Cruz Biotechnology,
Cetuximab (ImClone Systems, Branch-
190Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell CarcinomaHEAD & NECK—DOI 10.1002/hedFebruary 2011
Santa Cruz, CA); anti-phosphorylated IGF-IR (Tyr
1131)/IR (Tyr 1146), anti-phosphorylated EGFR (Tyr
1068), anti-AKT, anti-phosphorylated AKT (Ser 473),
anti-MAPK mouse (p42), anti-phosphorylated MAPK
(Tyr 42/44; Cell Signaling Technology, Beverly, MA);
anti-b-actin (Sigma, St Louis, MO); mouse anti-prolif-
erating cell nuclear antigen (PCNA) clone PC-10
(DAKO A/S, Copenhagen, Denmark); rat anti-mouse
and rat anti-mouse CD31 peroxidase-conjugated rat
anti-mouse IgG1 (PharMingen, San Diego, CA); per-
oxidase-conjugated goat anti-rabbit IgG and peroxi-
ImmunoResearch Laboratories, West Grove, PA); per-
oxidase-conjugated rat anti-mouse IgG2a (Serotec,
Harlan Bioproducts for Science, Indianapolis, IN);
Hoechst Dye 3342 MW 615.9 (Hoechst, Warrington,
PA), and Alexa Fluor 594-conjugated goat anti-rat
IgG and Alexa Fluor 488-conjugated goat anti-rabbit
IgG (Molecular Probes, Eugene, OR). The terminal
deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL) assay was done using a commercial
apoptosis detection kit (Promega, Madison, WI).
expression and phosphorylation in CSCC lines, total
cell protein extracts were obtained from Colo16,
SRB1, and SRB12 cells. The cells were grown to sub-
confluence in DMEM supplemented with 10% FBS,
washed with PBS, and scraped with lysis buffer, as
previously described.28The proteins (50 lg) were
resolved by polyacrylamide gel electrophoresis and
membranes. The membranes were blocked with 1%
bovine serum albumin
Tween 20 (v/v) in Tris-buffered saline solution for 30
minutes, then probed overnight with the desired pri-
mary antibodies. After incubation with the secondary
antibody, signals were visualized using enhanced
chemiluminescence (Pierce, Rockford, IL).
We performed Western blotting to demonstrate
that cetuximab and A12 are able to specifically inhibit
the phosphorylation EGFR and IGF-IR in vitro, West-
ern immunoblotting was performed. Cells from all 3
lines were incubated in serum-free medium for 24
hours. Then, the cells were incubated with either A12
(50 nM), cetuximab (1 lg/mL) or a combination of
A12 and cetuximab for 2 hours. Untreated cells were
used as a control. The cells were then stimulated
with both IGF-I (10 nM; R&D Systems, Minneapolis,
MN) and EGF (10 nM; Upstate Biotechnology, Lake
Placid, NY) for 15 minutes. Cells were then processed
and Western blotting analysis was performed, as
Colo16 cells were further used to test the ability of
cetuximab and A12 to inhibit the EGFR and IGF-IR
signaling pathways. After serum starvation overnight,
the cells were incubated with no cetuximab, A12, or
To measure EGFR and IGF-IR
the combination; untreated cells were used as control.
The cells were stimulated with both IGF-I and EGF for
15 minutes. Cells were then processed and Western
blot analysis was performed, as described above.
Measurement of Cell Proliferation.
mide (MTT) assay to test the ability of cetuximab and
A12 to inhibit the proliferation of all 3 CSCC lines in
vitro. Two thousand cells per well were grown in
DMEM medium supplemented with 10% FBS in 96-
well tissue culture plates. After 24 hours, the cells
were treated with various concentrations of A12 (0.1–
100 nM) or cetuximab (0.01–50 ug/mL) diluted in
DMEM medium supplemented with 2% FBS. To mea-
sure the number of metabolically active cells after a 3-
day incubation period, we used an MTT and quantified
the results at an optical density of 570 nm using a 96-
well microtiter plate reader (MR-5000; Dynatech Labo-
ratories, Chantilly, VA).
We used a 3-(4,
plated at a density of 2 ? 105cells per well in 38-mm2
6-well plates (Costar, Cambridge, MA) and maintained
for 24 hours. Then, A12 was added in various concen-
trations (0–100 nM) with or without cetuximab (5 lg/
mL) in DMEM medium supplemented with 2% FBS.
After 48 hours, the extent of cell death was determined
by propidium iodide (PI) staining of hypodiploid DNA.
For PI staining, the treated cells were resuspended in
a Nicoletti buffer (50 lg/mL PI [Sigma–Aldrich], 0.1%
sodium citrate, and 0.1% Triton X-100) for 20 minutes
at 4?C. Cell cycle data were then analyzed by flow
cytometry, and the sub-G0/G1 fraction was measured
using the Multicycle System (Phoenix Flow System,
San Diego, CA).
Effects of A12 and Cetuximab on the Growth of
Cutaneous Squamous Cell Carcinoma Xenografts in
CSCC xenografts in nude mice were
established as described previously.28,29Briefly, 1 ?
106cells in an injection volume of 100 lL were
injected subcutaneously into the right side of the face
of each mouse using a 30-gauge needle. The tumors
were allowed to develop for 7 days. The mice were
then randomized into 4 groups (10 mice per group),
and the drugs were administered as follows: (1) A12,
40 mg/kg twice per week by IP injection; (2) cetuxi-
mab, 50 mg/kg twice per week IP; (3) both A12, 40
mg/kg, and cetuximab, 50 mg/kg, IP, twice per week;
and (4) 250 lL of PBS administered IP twice per
week as placebo.
The mice were treated for 18 days and weighed
twice per week. Animals would have been humanely
killed if they lost >20% of their body weight or if they
became moribund. However, all the animals main-
tained their weight and none had to be humanely
killed due to the above criteria before the end of the
Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell Carcinoma HEAD & NECK—DOI 10.1002/hedFebruary 2011191
treatment period. At the end of the 18-day treatment
period, the mice were humanely killed by CO2
asphyxiation and necropsy was performed. The cervi-
cal lymph nodes, the lungs, and the skin tumors were
removed, sectioned, stained with hematoxylin-eosin,
and examined for the presence of metastasis. During
necropsy, the tumor size was measured in 2 dimen-
sions and the volume was determined using the for-
mula V ¼ (length; width2) p/6, expressed in cubic
millimeters. Drug treatment continued until the day
of death with the last dose administered 2 hours
before the animal was humanely killed. Tumor inhibi-
tion was calculated as a percentage according to the
formula (1-[T/C]) ? 100 in which T and C represent
the mean tumor volumes of the treatment group and
the control group, respectively.
For immunohistochemical and routine hematoxy-
lin-eosin staining, half of the tumor was fixed in for-
malin and embedded in paraffin. The remainder was
embedded in OCT compound (Miles, Elkhart, IN),
rapidly frozen in liquid nitrogen, and stored at minus
Effects of A12 and Cetuximab on the Survival of
Nude MiceBearing Cutaneous
established in nude mice as described above. Seven
days after the tumor cell injection, the mice were
randomized into 4 groups (5 mice per group): control,
A12, cetuximab, and a combination group. Each
group of mice was treated with PBS, A12, cetuximab,
or both agents, as described in the previous section.
The mice were weighed twice per week and humanely
killed if the animals showed weight loss of >20% or
seemed moribund. The mice were treated for 5 weeks.
For IGF-IRb, EGFR, and PCNA staining,
paraffin-embedded sections were first deparaffinized
in xylene. Excess xylene was removed by washing the
slides in ethanol. The tissue was treated with pepsin
for 20 minutes at 37?C. Immunohistochemistry was
performed as previously described.30
For staining with antibodies against pIGF-IRb,
pEGFR, and CD31/PECAM-1, frozen tumors were
sectioned (8 to 10 m thick), mounted on positively
charged Superfrost slides (Fisher Scientific, Pitts-
burgh, PA), air dried for 30 minutes, and fixed in cold
acetone for 10 minutes. Immunohistochemistry was
performed, as previously described.30
For TUNEL staining, tissues were fixed with 4%
paraformaldehyde (methanol free) for 10 minutes at
room temperature, washed twice with PBS for 5
minutes, and then incubated with 0.2% Triton X-100
for 15 minutes at room temperature. Then the tissue
sections were incubated with reaction buffer contain-
ing 44 lL of equilibration buffer, 5 lL of nucleotide
mix, and 1 lL of terminal deoxynucleotidyl transfer-
ase (Promega kit) at 37?C for 1 hour, avoiding expo-
sure to light.The reaction
immersing the samples in 2? saline-sodium citrate
buffer for 15 minutes. The samples were then washed
3 times for 5 minutes with PBS to remove unincorpo-
Immunofluorescence microscopy was performed
using a Zeiss Axioplan2 microscope (Carl Zeiss, Thorn-
wood, NY) equipped with a 100-W HBO mercury bulb
and filter sets (Chroma, Brattleboro, VT) to individually
capture red and blue fluorescent images. Images were
captured using a C5810 Hamamatsu color chilled 3-
chip charge-coupled device
Hamamatsu City, Japan) and digitized using Optimas
imaging software (Silver Spring, MD). The stained sec-
tions were imaged with a Microphot-FX microscope
(Nikon, Melville, NY) equipped with a 3-chip charged-
couple device color video camera (Model DXC990; Sony
Corp, Tokyo, Japan).
For evaluation of PCNA and CD31 staining, the
mean positive area was quantified in 5 random 0.159
mm2fields (magnification, ?100) per slide from 5 slides
per study group using Image-Pro Plus software pack-
age (Media Cybernetics, Silver Spring, MD). For
TUNEL staining quantification, the labeled cells were
counted in 5 random 0.159 mm2fields (magnification,
?100) per slide from a total of 5 slides per study group.
The photomontages were prepared using Photoshop
software (Adobe Systems, San Jose, CA).
rank-sum test was used to assess differences in mouse
tumor volume between each treatment group and the
control group. We ran a repeated-measures regression
model with treatment, time, and treatment by time
interaction to compare the tumor volumes observed for
the group receiving A12 plus cetuximab with the group
receiving cetuximab alone. This methodology was also
used to compare tumor volumes from the group receiv-
ing A12 plus cetuximab to the group receiving A12
alone. Survival was analyzed with the Kaplan–Meier
method. Differences between treatment and control
groups were compared to the log-rank test. We com-
pared the results from PCNA, CD31, and TUNEL
staining by independent-samples t test. Least-squares
fit curves were generated for the MTT and PI assays.
SPSS 12.0 for Windows software (SPSS, Chicago, IL)
was used for all statistical analysis. A p value of .05
was considered significant.
The nonparametric Wilcoxon
Epidermal Growth Factor Receptor and Insulin-Like
Growth Factor-I Receptor are Expressed by Cutaneous
Squamous Cell Carcinoma Cell Lines.
analysis revealed that both cell lines tested stained
positively for EGFR and IGF-IR (Figure 1). Only the
Colo16 cell line exhibited baseline phosphorylation of
192 Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell CarcinomaHEAD & NECK—DOI 10.1002/hed February 2011
both EGFR and IGFR; the SRB12 cell line showed a
weak positive for pIGF-IR.
A12 and Cetuximab Treatment Leads to Decreased
Cutaneous Squamous Cell Carcinoma Cell Lines.
evaluated the ability of A12 and cetuximab to inhibit
the phosphorylation of EGFR and IGF-IR in the Colo16
and SRB1 cell lines (Figure 1). Under basal conditions
in serum-free medium, there was a low level of EGFR
autophosphorylation, which was enhanced after expo-
sure to EGF (Figure 1). Baseline expression of pIGF-
1R was minimal; however, phosphorylation occurred
upon stimulation with IGF-1. In contrast, there were
low levels of pIGF-1R but not pEGFR at baseline in
SRB1 cells; both receptors were similarly activated
with growth factor stimulation (Figure 1B). Treatment
with A12 blocked phosphorylation of IGF-IR and
resulted in decreased expression of IGF-1R in both cell
lines, but this effect was more pronounced for Colo16.
EGFR phosphorylation was blocked with cetuximab
treatment but not impacted by A12 treatment in both
cell lines. No differences in total EGFR expression lev-
els were noted in either cell line.
Proliferation of Certain Cutaneous Squamous Cell
Carcinoma Cell Lines.
We examined the effect of
A12 treatment on the proliferation of 2 CSCC cell lines
(Colo16 and SRB1) using the MTT assay. The growth of
cells incubated with increasing concentrations (0–100
nM) of A12 in DMEM containing 2% serum was deter-
mined 72 hours after the addition of the drug (Figure
2A). Treatment with various concentrations of A12 did
not significantly inhibit proliferation in any of the cell
lines studied, although a trend toward inhibition of the
Colo16 cells was noted. Next, we performed MTT
FIGURE 2. Combined treatment with A12 and cetuximab inhibits the growth of cutaneous squamous cell carcinoma (CSCC) cell lines
in vitro. (A) MTT assay was performed on each of the 3 SCC cell lines (Colo16, SRB12, and SRB1) in the presence and absence of
A12. No significant difference in growth was identified. (B) Combined treatment with A12 and cetuximab decreased the growth of
Colo16 (p < .001).
FIGURE 1. Effect of A12 and cetuximab on the inhibition of phosphorylation of insulin-like growth factor-I receptor (IGF-IR) and epider-
mal growth factor receptor (EGFR) in vitro. (A) Colo16, EGFR, and IGF-IR phosphorylation were inhibited by C225 and A12, respec-
tively. Total IGF-IR levels decreased with combined treatment. (B) SRB1, EGFR, and IGF-IR phosphorylation were inhibited by C225
and A12, respectively. No change was seen in total protein levels.
Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell CarcinomaHEAD & NECK—DOI 10.1002/hedFebruary 2011193
assays using the Colo16 cell line to compare the effect
of treatment with cetuximab with the A12/cetuximab
combination on cell proliferation. Proliferation of
Colo16 cells was inhibited by treatment with cetuxi-
mab alone, and to a greater extent by the combination
of A12 and cetuximab in a dose-dependent manner (p
< .01; Figure 2B).
Induction of Apoptosis in the Colo16 Cell Line.
examined the effect of dual inhibition of EGFR and
with A12and CetuximabLeads to
IGF-IR on the rate of apoptosis of the Colo16 cell
line using PI and flow cytometry. Treatment with
A12 alone at increasing concentration did not sig-
leads to induction apoptosis (11.9% vs 22.7%; p <
.012; Figure 3).
Cetuximab Combination Inhibits Tumor Growth in a
Nude Mouse Model of Cutaneous Squamous Cell
The Colo16 cell lines was used for all
in vivo experiments based on the strength of inhibi-
tion of intracellular signaling by A12 and cetuximab
that was noted previously (Figure 1). Cells were
injected into the mice as described with visually evi-
dent tumors noted at 1 week after inoculation.
Treatment witheither A12
reduced the growth of Colo16-derived SCC xeno-
grafts in nude mice by 51% (p ¼ .058) and 49% (p ¼
.041), respectively, when compared to the control
group (Figure 4A). Mice treated with A12 in addition
to cetuximab showed a 92% decrease in the mean
estimated tumor volume when compared to the con-
trol group (p < .001) or animals treated with either
cetuximab or A12 alone (p < .05).
withA12, Cetuximab,oran A12/
or cetuximab alone
Treatment with A12 Alone and in Combination with
Cetuximab Improves Survival
Nude Mouse Model of Cutaneous Squamous Cell
A12 and cetuximab were both well tol-
erated by the animals without substantial adverse
in an Orthotopic
FIGURE 3. Combination treatment with A12 and cetuximab
induces apoptosis in cutaneous squamous cell carcinoma
(CSCC) cell lines. There was no change in the rate of apoptosis
for Colo16 cells treated with A12 alone. Combination treatment
increased the percentage of apoptotic cells.
FIGURE 4. A12 and cetuximab decrease the growth of cutaneous squamous cell carcinoma xenografts and improve survival in an
athymic nude mouse model. (A) A12 or cetuximab treatment alone inhibited cutaneous squamous cell carcinoma (CSCC) xenograft
growth by 51% (p ¼ .058) and 49% (p ¼ .041), respectively. Combined treatment with A12 and cetuximab inhibited squamous cell car-
cinoma (SCC) xenograft growth to an even greater extent, reducing mean tumor volume by 92% (p < .001) (B) Treatment with either
A12 or cetuximab alone prolonged survival. The group treated with the combination of A12 and cetuximab demonstrated even greater
survival than either the control group (p < .001) or the group treated with A12 alone (p < .01).
194 Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell Carcinoma HEAD & NECK—DOI 10.1002/hed February 2011
effects. None of the animals had to be humanely
killed due to weight loss or systemic malignancy, but
rather were killed when the local tumor burden
became excessive (defined as >1 cm in the maximum
dimension or significant ulceration). Treatment with
A12 or cetuximab alone resulted in significantly
greater survival as compared to the control group (p
¼ .044 and p ¼ .023, respectively; Figure 4B). The
combination treatment group also demonstrated a sig-
nificantly improved survival rate as compared to the
control group (p < .001) or A12-only group (p < .01).
There was no statistically significant difference in
survival between the combination treatment group
and the group treated with cetuximab alone (p > .1).
Treatment of Cutaneous Squamous Cell Carcinoma
Cell Lines with A12, Cetuximab, and A12/Cetuximab
Combination in an Orthotopic Tumor Model Leads
Proliferation, and Inhibition of Angiogenesis.
effect of A12 and cetuximab on cellular proliferation
was determined by assessing the expression of PCNA
in tumor sections harvested at necropsy from mice in
all treatment groups. Mean positive area analysis of
PCNA staining showed a significant decrease in cell
proliferation in mice treated with either cetuximab (p
¼ .016) or A12 alone (p ¼ .026) as compared to the
control group (Figures 5A and 5B). Moreover, there
was an additional decrease in cell proliferation in
mice treated with A12/cetuximab combination as com-
pared to the control group (p < .001). However, the
effects of combination treatment on cell proliferation
were not significantly different from treatment with
either A12 or cetuximab alone (p > .1).
To assess the degree of intratumoral apoptosis,
the tumor sections were stained for DNA fragmenta-
tion using the TUNEL method (Figures 5A and 5C).
The mean number of apoptotic cells per unit area (?
SD) in the tumors of control mice and the mice
treated with cetuximab were 11.8 ? 7.4 and 18.2 ?
9.0, respectively (p ¼ .102). Treatment with either
A12 alone or A12 in combination with cetuximab
increased the mean number of apoptotic cells per unit
area to 39.7 ? 20.3 and 35.3 ? 13.6, respectively, rep-
resenting a significant increase in intratumoral apo-
ptosis when compared to the control group (p ¼ .001
and p ¼ .001) or the cetuximab-only group (p ¼ .007
and p ¼ .016).
To determine the intratumoral microvessel den-
sity, we stained tumor sections with CD31-specific
antibodies. Treatment with either A12 or cetuximab
alone resulted in a statistically significant inhibition
of tumor-associated angiogenesis as defined by the
mean CD31-positive area when compared to the con-
trol group (p < .001 and p < .001, respectively; Fig-
ures 5A and 5D). Treatment with A12 in combination
with cetuximab resulted in an additional inhibition of
tumor-associated angiogenesis when compared to the
control group (p < .001) or the group treated with
cetuximab only (p ¼ .003).
Our findings indicate that the combination of A12
and cetuximab simultaneously blocks EGFR and IGF-
IR activation and significantly reduces tumor volume
by both direct antitumor and angiogenic effects. We
showed that elevated IGF-IR and EGFR expression is
consistently and concurrently elevated in CSCC cell
lines. In an orthotopic nude mouse model of CSCC,
dual inhibition with A12 and cetuximab reduced the
tumor volume by 92% as compared to approximately
50% with either agent alone. Combination treatment
also significantly improved survival. In vitro studies
demonstrated the inhibition of activation of IGF-IR
by A12 and EGFR by cetuximab, but showed no
cross-inhibition. The combination of A12 and cetuxi-
mab also showed direct growth inhibitory and apopto-
monotherapy with either A12 or cetuximab caused a
significant increase in apoptosis and a decrease in
both cellular proliferation and microvessel density as
compared to the control group, an effect which was
enhanced for combination treatment.
A12 treatment resulted in downregulation of the
total IGF-IR expression levels, as has been demon-
strated by others.18These results are also consistent
with previous studies of EGFR and IGF-IR overex-
pression in CSCC specimens and cell lines.5,31We
also showed that A12 has effects on downstream ki-
nases, such as AKT, confirming similar findings by
Wang et al.32
Both A12 and cetuximab when used as single
agents demonstrated only minor antiproliferative
activity on CSCC cell lines, whereas combination
treatment led to a significant inhibition of growth
(p < .01). This finding is consistent with multiple
studies demonstrating the efficacy of dual inhibi-
tion of EGFR and IGF-IR pathways in inhibiting
tumor cell proliferation as compared to targeting a
single pathway.31In contrast to studies of small
molecule IGF-IR kinase inhibitors, treatment with
A12 alone did not significantly increase apoptosis
of CSCC in vitro. These data suggest that A12
exerts its antiproliferative effect mainly through a
cytostatic, rather than cytotoxic, mechanism. Simi-
lar results have been reported in other studies
using single agent inhibitors including IGF-IR anti-
bodies (EM164, h7C10, and CP-751,871) and inhibi-
tors of EGFR including AG 1478, mAb225, and
Treatment with A12 and cetuximab significantly
inhibited SCC tumor growth in the murine model. Ei-
ther agent alone resulted in approximately a 50%
reduction in tumor volume, whereas treatment with a
combination of the 2 drugs resulted in a greater than
90% reduction in tumor volume. Similarly, longer
Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell CarcinomaHEAD & NECK—DOI 10.1002/hedFebruary 2011195
survival was also found for the combined treatment
group as compared with the control or A12 treatment
groups. Differences in sensitivity to inhibition of ei-
ther EGFR or IGF-IR alone have previously been
found in other tumors.35However, the concurrent
expression of both IGF-IR and EGFR together in
FIGURE 5. Immunohistochemical analysis of Colo16 xenografts. (A) Representative sections of xenografts treated with A12, cetuxi-
mab, or control were stained with hematoxylin-eosin or immunostained for expression of proliferating cell nuclear antigen (PCNA),
TUNEL, or CD31. Quantification of the mean positive stained area are shown for PCNA (B) and CD31 (C). (D) Apoptotic cells per
high powered field are quantified.
196Inhibition of EGFR and IGF-IR in Cutaneous Squamous Cell CarcinomaHEAD & NECK—DOI 10.1002/hedFebruary 2011
CSCC seems to be important for tumor growth and
development, and simultaneous inhibition of these 2
tyrosine kinases results in a significantly greater
reduction of tumor development and growth. Our
findings are consistent with other studies suggesting
that targeted therapy of IGF-IR can be used in combi-
nation with other therapeutic strategies to achieve
maximum antitumor effects26,27,36
bination therapy may be beneficial in preventing
the development of drug resistance, such as that
seen with trastuzumab (Herceptin), erlotinib, and
Immunohistochemical analyses of tumor sections
from mice treated with either A12 or cetuximab alone
revealed a significant decrease in the proliferative
index as measured by PCNA staining and an increase
in intratumoral apoptosis as measured by the TUNEL
assay. This effect was strongest for groups treated
with A12, either alone or in combination, which is in
agreement with previous work showing only moderate
apoptosis in response to cetuximab treatment alone
but synergism with dual agent therapy. The increase
in intratumoral apoptosis and decrease in prolifera-
tion is in contrast to our in vitro findings showing
limited effects of either agent or the combination on
the growth of Colo16 cells. The differences between
the results from the animal model and in vitro experi-
ments can be explained by reports showing the acti-
vation of vascular endothelial growth factor receptor
(VEGFR) signaling by IGFR in cancer.39
EGFR and IGF-IR are abundantly expressed on endo-
thelial cells.5,40–43EGF and IGF produced at high lev-
els by tumors are able to promote the growth,
survival, and migration of tumor cells, and induce the
synthesis of VEGF-A, VEGF-C, and MMP2, which
may enhance the development of the blood supply
essential for the progressive growth of primary malig-
nancies and their metastases.21,44–46Treatment with
either A12 or cetuximab alone resulted in statistically
significant inhibition of tumor-associated angiogene-
sis, whereas the combination treatment with A12 and
cetuximab resulted in an additional inhibition of
angiogenesis (Figures 5A, and 5C). These findings
concur with other studies that have demonstrated
reduction of angiogenesis resulting from inhibition of
IGF-IR, EGFR, or both receptors simultaneously.5,45
Current studies have indicated that the efficacy of
tyrosine kinase inhibition can be enhanced by com-
bining it with other tyrosine kinase inhibitors, chemo-
demonstrable response was achieved using A12 or
cetuximab as single agents, the enhanced response
obtained when these 2 monoclonal antibodies were
used in combination provides further support for the
use of not only multiple tyrosine kinase inhibitors,
but the use of inhibitors of IGF-IR and EGFR in par-
ticular.51–54This agrees with previous works showing
a synergistic effect of the combination of IGF-IR and
and that com-
EGFR inhibition and suggests the presence of ‘‘cross-
talk’’ between the receptors.33
In summary, dual inhibition of the tyrosine ki-
nases EGFR and IGF-IR can decrease skin cancer
growth both in vitro and in vivo. Administration of
the IGF-IR antibody A12 can significantly inhibit the
proliferation of SCC cells in vitro. Treatment with
A12 alone or in combination with cetuximab signifi-
cantly reduces tumor volume and prolongs the sur-
vival time of nude mice implanted with CSCC by
enhancing their respective cytostatic
inducing antiangiogenesis. These data suggest that
dual inhibition of tyrosine kinases, EGFR and IGF-IR
in particular, may be therapeutically useful and pro-
vide a promising strategy for the treatment of
patients with aggressive CSCC.
Fidler (Department of Cancer Biology, The University
of Texas M. D. Anderson Cancer Center) for providing
the Colo16 cell line and Dr. Gary L. Clayman
(Department of Head and Neck Surgery, The Univer-
sity of Texas M. D. Anderson Cancer Center) for pro-
viding the SRB1 and SRB12 cell lines.
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