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Molecular Oncology, Markers, Clinical Correlates |
Departments of Cancer Biology [C. J. B., M. T. H., D. W. D., C. A. P., R. T., D. J. M., R. R.], Surgical Oncology [D. B. E.], and Gastrointestinal Medical Oncology [J. L. A.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and ImClone Systems, Inc., New York, New York 10014 [D. J. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Single agents or combination chemotherapy have little reproducible impact on patient survival or quality of life in advanced pancreatic adenocarcinoma (1, 2, 3, 4 , 6) . Recently, gemcitabine (2',2'-difluorodeoxycytidine), a deoxycytidine analogue (5, 6, 7, 8) , demonstrated modest improvements in response rate, and median survival in patients with advanced pancreatic cancer was similar to those treated with 5-fluorouracil (1, 2, 3, 4, 5 , 7 , 8) . However, despite these results, the median survival for patients with metastatic disease continues to be <6 months. Although the development of alternative gemcitabine schedules and chemotherapy combinations continues (6 , 9) , study of novel targets and strategies are required to build on the results with gemcitabine.
Among the potential targets are angiogenesis and
EGF-R3
-mediated
proliferation, both of which are fundamental to the development and
progression of human pancreatic carcinoma. Each has been independently
evaluated as a target for therapy, but the link between the two,
involving EGF-R effects on angiogenesis, has been identified only
recently (10, 11, 12)
. Human pancreatic cancers overexpress
EGF-R (13
, 14) , and binding of its ligands, EGF, AR, or
TGF-
, produced in an autocrine manner, can activate signal
transduction pathways that regulate cell proliferation and
anchorage-independent growth (14, 15, 16, 17, 18, 19, 20, 21)
. A statistically
significant decrease in survival was observed in pancreatic cancer
patients overexpressing the EGF-R and at least one of its ligands
(18)
. These data indicate that overexpression of the EGF-R
and its ligands contributes to the malignant phenotype in this disease.
A crucial step in the process of pancreatic tumor progression is the
production of neovascularization in and around the tumor
(22, 23, 24)
. Under homeostasis, the microvasculature is
maintained in a quiescent state with acquisition of the angiogenic
phenotype in solid tumors dependent on the net outcome of stimulatory
and inhibitory factors by the tumor and its microenvironment
(22, 23, 24)
. These factors have not been fully elucidated for
pancreatic cancer; however, VEGF, IL-8, and bFGF are expressed by human
pancreatic carcinoma cells (25, 26, 27)
. After vascular
induction by these factors among others, the rate of tumor growth
increases exponentially (22, 23, 24, 25)
. Interestingly, two of
the most potent inducers of VEGF, a major angiogenesis stimulator for
human cancers (28)
, are EGF and TGF-
(29, 30, 31)
. Recent data from our laboratory also suggests
that therapy with either EGF-R protein tyrosine kinase inhibitors
(32)
or C225 (12)
, an anti-EGF-R antibody,
inhibits human bladder carcinoma growth and metastasis by mechanisms
affecting tumor-induced neovascularization secondary to the
down-regulation of tumor cell-produced angiogenic factors including
VEGF. This effect is not specific to one tumor type (10
, 11)
.
The purpose of the present study was to evaluate whether down-regulating EGF-R signaling pathways by the anti-EGF-R antibody C225 inhibits pancreatic tumor growth and metastasis and whether this effect is potentiated when used in combination with gemcitabine. In our study, C225 treatment resulted in growth inhibition, tumor regression, and abrogation of metastasis. C225 treatment suppressed tumor VEGF and IL-8 production, both in cell culture and in tumors growing orthotopically in nude mice. The down-regulation of these angiogenic factors preceded the involution of blood vessels, as shown by double immunofluorescence microscopy for apoptotic endothelial cells, suggesting a cause and effect. Furthermore, this effect is potentiated in combination with gemcitabine.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and Orthotopic Implantation of Tumor Cells.
Male athymic nude mice (NCr-nu) were obtained from the animal
production area of 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 American Association for
Accreditation of Laboratory Animal Care and in accordance with current
regulations and standards of the United States Department of
Agriculture, United States Department of Health and Human Services, and
NIH, and their use in these experiments was approved by the
Institutional Animals Care and Use Committee. The mice were used when
they were 8–10 weeks of age.
To produce tumors, tumor cells were harvested from subconfluent cultures by treatment with 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% FBS, and the cells were washed once in serum-free medium and resuspended in HBSS. Only single-cell suspensions with >90% viability were used for injections. Orthotopic tumor cell injections were performed as described (26) . The mice were killed 5–6 weeks thereafter, and the size and weight of the primary tumors, the incidence of regional (celiac and paraaortal) lymph node metastasis, and the number of liver nodules were determined. Histopathology confirmed the nature of the disease.
Therapy of Established Human Pancreatic Carcinoma Tumors Growing in
the Pancreas of Athymic Nude Mice.
Treatment was initiated 7 days after tumor cell injection, when tumors
were palpable. Five animals sacrificed at this time point had a median
tumor volume of 18 mm3 (range, 16–18
mm3). To evaluate the therapeutic effect of
gemcitabine alone in this model, an initial in vivo
dose-response experiment was performed. Five mice/group were treated
biweekly with 500, 250, 125, 62, 31, 15.5, and 7.5 mg/kg gemcitabine by
i.p. administration, resulting in 65, 58, 55, 46, 36, 31, and 0.2%
growth inhibition of L3.6pl tumors, respectively, compared with
controls. For the studies herein, a dose of 250 mg/kg gemcitabine was
used. Mice were randomly separated into four groups (16 animals/group):
C225, biweekly treatment with 1 mg/injection; gemcitabine, biweekly
treatment 250 mg/kg/injection; C225 plus gemcitabine, C225
administration on Tuesday and Friday and gemcitabine administration on
Wednesday and Saturday; and control, an equivalent volume of 0.9% NaCl
on the same schedule as the combination therapy group. All groups
received treatment by i.p. injection. Treated mice were closely
monitored for any signs of progressive disease and sacrificed if they
became moribund. Mice in all groups were sacrificed 32 days after tumor
cell implantation. To evaluate the temporal effect of these treatments
on different biological parameters, two mice/group were sacrificed 11,
18, and 30 days after initiation of therapy. The experiment was
repeated using gemcitabine (125 mg/kg) alone and in combination with
C225, with similar results (data not shown).
In Vitro Effects of C225 Alone and in Combination
with Gemcitabine.
Tumor cell cytostasis was determined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium assay as described
(12
, 32, 33, 34)
.
ELISA.
To evaluate VEGF and IL-8 protein expression after treatment with C225,
2000 cells/38-mm2 well were plated in 96-well
microtiter plates in 0.2 ml of supplemented DMEM with 5% FBS.
Twenty-four h later, 2.5, 5, or 10 µg/ml of C225 or control human IgG
(10 µg/ml) was added. After 48 h incubation, viable cells were
counted, the supernatants of wells with similar numbers of tumor cells
were collected, and VEGF and IL-8 protein levels were determined using
Quantikine ELISA kits (R&D Systems, Inc., Minneapolis, MN).
Western Blot Analyses of EGF-R Autophosphorylation after Treatment
with C225.
Serum-starved L3.6pl cells were treated with EGF alone or in the
presence of C225 (1, 5, and 10 µg/ml) for 10 min, washed, and scraped
into PBS containing 5 mM EDTA and 1 mM sodium
orthovanadate and centrifuged. The pellet was resuspended in lysis
buffer [20 mM Tris-HCl (pH 8.0), 137 mM NaCl,
10% glycerol (v/v), 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 0.15
unit/ml aprotinin], sonicated, and centrifuged to recover insoluble
protein. Immunoprecipitations were performed using Mab anti-EGF-R
(clone EGFRI; Amersham Life Science, Inc., Arlington Heights, IL) as
described (34
, 35)
. Immunoprecipitates were analyzed on
7.5% SDS-PAGE and transferred onto 0.45 µm nitrocellulose membranes.
The filters were blocked with 3% BSA (w/v) in TBS [20 mM
Tris-HCl (pH 7.5), and 150 mM NaCl], probed with either
polyclonal sheep antihuman EGF-R (1:1000) or monoclonal
anti-phosphotyrosine (Mab 4G10; 1:2000) in TTBS [0.1% Tween 20 (v/v)
in TBS], and incubated with horseradish peroxidase-conjugated donkey
antisheep IgG (1:2000; Sigma Immunochemicals, St. Louis, MO) or sheep
antimouse IgG (1:2000), respectively, in TTBS. Protein bands were
visualized by the ECL detection system (Amersham).
Immunohistochemical Determination of VEGF, IL-8, PCNA,
CD31/PECAM-1, and EGF-R.
Paraffin-embedded tissues were used for identification of VEGF, IL-8,
EGF-R, and PCNA. Four to six-µm-thick sections were mounted on
positively charged Superfrost slides (Fisher Scientific, Co., Houston,
TX) and dried overnight. Sections were deparaffinized in xylene,
followed by treatment with a graded series of alcohol [100, 95, and
80% ethanol/double-distilled H2O (v/v)] and
rehydrated in PBS (pH 7.5). Sections analyzed for PCNA were microwaved
5 min for "antigen retrieval" (36)
. All other
paraffin-embedded tissues were treated with pepsin (Biomeda, Foster
City, CA) for 15 min at 37°C and washed with PBS. Frozen tissues used
for identification of CD31/PECAM-1 and activated EGF-R were sectioned
(8–10 µm), mounted on positively charged Plus slides (Fisher
Scientific), and air-dried for 30 min. Frozen sections were fixed in
cold acetone (5 min), acetone/chloroform (v/v; 5 min), and acetone (5
min), and washed with PBS.
All samples were incubated with 3% hydrogen peroxide in methanol (v/v) for 12 min to block endogenous peroxidase, washed with PBS (pH 7.5), and incubated in protein blocking solution [5% normal human serum/0.5% normal goat serum in PBS (v/v)] for 20 min. Sections analyzed for activated EGF-R were pretreated with goat antimouse IgG F(ab)2 fragment (1:10 dilution in PBS) for 4–6 h. The sections were incubated with the primary antibody in a humidified chamber for 15–18 h at 4°C, rinsed with PBS, and incubated in protein blocking solution for 10 min. Sections were then incubated for 60 min with the corresponding peroxidase-conjugated secondary antibody [1:200 (v/v)] for 1 h at ambient temperature. Positive reaction was visualized by incubating the slides with stable 3,3'-diaminobenzidine for 10–20 min (Research Genetics, Huntsville, AL) or 3-amino-9-ethylcarbazole (Biogenex Laboratories, San Ramon, CA) after CD31 staining. The sections were rinsed with distilled water, counterstained with Gill’s hematoxylin for 1 min (Sigma Chemical Co., St. Louis, MO), and mounted with Universal Mount (Research Genetics). Control samples exposed to secondary antibody alone showed no specific staining. The positive reaction after staining for activated EGF-R was enhanced with osmium tetraoxide [4% aqueous solution (v/v); Electron Microscopy Sciences, Fort Washington, PA) at a 1:1000 dilution in double-distilled H2O after incubation with 3,3'-diaminobenzidine.
Immunofluorescence Double Staining for CD31/PECAM-1 (Endothelial
Cells) and TUNEL (Apoptotic Cells).
Frozen tissue sections fixed and treated as above were incubated for
18 h with rat monoclonal anti-CD31 antibody (1:100) at 4°C,
rinsed with PBS, and incubated with goat antirat IgG conjugated to
Texas Red (1:200; Jackson ImmunoResearch Laboratory, Inc., West Grove,
PA) for 60 min at ambient temperature in the dark. After sections were
washed with PBS containing 0.1% Brij (v/v), TUNEL was performed using
a commercial kit according to the manufacturer’s instructions with the
following modifications (Promega Corp., Madison, WI). Samples were
fixed with 4% paraformaldehyde (methanol-free) for 10 min at ambient
temperature, washed with PBS, and then permeabilized by incubating with
0.2% Triton X-100 in PBS (v/v) for 15 min. The samples were incubated
with equilibration buffer (from the kit); it was drained off, and
reaction buffer containing equilibration buffer, nucleotide mix, and
terminal deoxynucleotidyl transferase was added to the sections and
incubated in a humidified chamber for 1 h at 37°C in the dark.
The reaction was terminated by immersing the samples in 2x SSC (SSC:
30 mM NaCl/3 mM sodium citrate, pH 7.2) for 15
min, followed by three washes to remove unincorporated
fluorescein-dUTP. Background reactivity was determined by processing
slides in the absence of terminal deoxynucleotidyl transferase
(negative control). Maximum reactivity was observed by preincubating
the tissue sections with DNase I and served to confirm the quality of
the specimen.
Nuclei were stained with Hoechst Dye 3342 (Mr 615.9; 300 µg/ml). Fluorescent bleaching was minimized by treating slides with an enhancing reagent (Prolong; Molecular Probes, Eugene, OR). Immunofluorescence microscopy was performed using a x40 objective (Zeiss Plan-Neofluar) on an epifluorescence microscope equipped with narrow bandpass excitation filters mounted in a filter wheel (Ludl Electronic Products, Hawthorne, NY) to individually select for green, red, and blue fluorescence. Images were captured using a cooled CCD camera (Photometrics, Tucson, AZ) and SmartCapture software (Digital Scientific, Cambridge, United Kingdom) on a Macintosh computer. Images were further processed using Adobe Photoshop software (Adobe Systems, Mountain View, CA). Endothelial cells were identified by red fluorescence, and DNA fragmentation was detected by localized green and yellow fluorescence within the nucleus of apoptotic cells. Apoptotic endothelial cells were quantitated and expressed as an average of the ratio of apoptotic endothelial cells to the total number of endothelial cells in 5–10 random 0.011-mm2 fields at x400. For total TUNEL expression, apoptotic events were quantified in 10 random 0.159-mm2 fields at x100 and divided by the total number of cells/field.
Quantification of MVD, PCNA, TUNEL, and Absorbance.
For the quantification of MVD, 10 random
0.159-mm2 fields at x100 were captured for each
tumor using a Sony three-chip camera (Sony Corporation of America,
Montvale, NJ) mounted on a Zeiss universal microscope (Carl Zeiss,
Thornwood, NY) and Optimas Image Analysis software (Bioscan, Edmond,
WA) installed in a Compaq computer with Pentium chip, a frame grabber,
an optical disc storage system, and a Sony color printer. Microvessels
were quantified as described (12
, 37)
.
For the quantification of the immunohistochemical reaction intensity, the absorbance of 100 VEGF- and IL-8-positive tumor cells in 10 random 0.039-mm2 fields at x200 of the differently treated tumor tissues was measured using the Optimas Image Analysis software (12 , 26 , 27) . The samples were not counterstained; therefore, the absorbance was attributable solely to the product of the immunohistochemical reaction. VEGF and IL-8 cytoplasmic immunoreactivity was evaluated by computer-assisted image analysis and was expressed as a ratio of tumor cell expression to normal pancreatic gland expression relative to the control (26 , 27) . For the quantification of PCNA expression, the number of positive tumor cells was quantitated in 10 random 0.159-mm2 fields at x100 and divided by the total number of cells/field.
Reagents.
All antibodies were purchased from commercial sources as listed: rabbit
anti-VEGF/VPF (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA),
polyclonal rabbit antihuman IL-8 (1:25; Biosource International,
Camarillo CA), rat antimouse CD31/PECAM-1 (1:400), and
peroxidase-conjugated rat antimouse IgG1 (PharMingen, San Diego, CA),
mouse anti-PCNA clone PC 10 (1:50; Dako A/S, Copenhagen, Denmark),
mouse antihuman EGF-R IgG1 (activated form; 1:25; Chemicon, Temecula,
CA), which reacts specifically with the activated and phosphorylated
human EGF-R and does not react with other phosphorylated proteins
(32
, 35
, 38, 39, 40)
, monoclonal mouse antihuman IgG1 EGF-R
clone 30 (1:80; Biogenex, San Ramon, CA), peroxidase-conjugated
F(ab')2 goat antirabbit IgG F(ab')2,
peroxidase-conjugated goat antimouse IgG F(ab)2
fragment, Affinipure Fab fragment goat antimouse IgG (H+L),
peroxidase-conjugated goat antirat IgG (H+L), and Texas
Red-conjugated goat antirat IgG (1:200; Jackson Research
Laboratories, West Grove, CA), peroxidase-conjugated rat antimouse
IgG2a (1:75; Serotec, Harlan Bioproducts for Science, Inc.,
Indianapolis, IN), monoclonal anti-phosphotyrosine MAb 4G10 (1:2000)
and polyclonal sheep antihuman EGF-R (1:1000; Upstate Biotechnology,
Lake Placid, NY), peroxidase-conjugated donkey antisheep IgG and human
IgG (Sigma Immunochemicals), and Hoechst Dye 3342
Mr 615.9 (300 µg/ml; Hoechst,
Warrington, PA). Chimeric anti-EGF-R Mab C225 was provided by ImClone
Systems, Inc. Gemcitabine (HCl, 2',2'-difluorodeoxycytidine) was
purchased from Eli Lilly & Company (Indianapolis, IN).
Statistical Analysis.
Pancreatic tumor weights, expression intensities of VEGF and IL-8, and
quantification of the number of PCNA, TUNEL, CD31, and apoptotic tumor
and endothelial cells were compared by unpaired Student’s t
test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Pretreatment of the cells with C225,
specific for the EGF-R, for 1 h followed by treatment with EGF (15
min) abrogated this effect in a dose-dependent manner (1–10 µg/ml;
Fig. 1
). The Mr 170,000 band was
confirmed as EGF-R by Western blot analysis using independent
anti-EGF-R antisera (Fig. 1)
. Northern blot analyses demonstrated high
levels EGF-R-specific transcripts in L3.6pl cells that did not vary in
response to treatment with either EGF or C225 treatment (data not
shown).
|
. The maximum inhibition
observed was 20% after 72 h of continuous exposure of 12.5
µg/ml in either 1 or 5% serum-containing medium. The combination of
C225 (2.5 µg/ml) and gemcitabine (0–64 ng/ml) produced an additive
cytotoxic effect at gemcitabine concentrations below
IC80 (Fig. 2B)
. No synergistic effects
were observed in vitro.
|
. The incidence of
macroscopically visible pancreatic tumors was 5 of 10 and 0 of 9 in the
C225 and C225 plus gemcitabine treatment groups, respectively, compared
with 10 of 10 in the gemcitabine-treated and control groups (Table 1)
.
Upon histological examination, three of nine and three of five
macroscopic tumor-free animals in the C225 plus gemcitabine and C225
treatment groups, respectively, showed microscopic disease (Fig. 3
; see H&E). All mice in the gemcitabine
and control treatment groups showed progressive disease. Independent
experiments using gemcitabine at 125 mg/kg demonstrated similar results
(see "Materials and Methods"). The observed differences in
pancreatic tumor weights and median tumor volume in the C225 and C225
plus gemcitabine groups suggest regression because the treated tumors
were smaller than those when therapy was initiated (see "Materials
and Methods").
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. No macroscopic visible liver
metastases were observed in the C225 plus gemcitabine treatment group
(Table 1)
.
Analysis of Total EGF-R and Activated EGF-R in L3.6pl Pancreatic
Tumor Sections of Treated versus Control Mice.
We next determined the EGF-R content and level of activated EGF-R
(tyrosine phosphorylated form) in L3.6pl tumors growing in the pancreas
of nude mice after therapy with saline (control), gemcitabine, C225, or
C225 plus gemcitabine. Immunohistochemical analysis for the activated
and tyrosine phosphorylated EGF-R (32
, 35
, 38, 39, 40)
in
L3.6pl tumors growing in the pancreas of nude mice demonstrated
specific immunoreactivity both peripherally and centrally in tumors
from control and gemcitabine treated mice, whereas immunoreactivity was
significantly reduced in C225 and C225 plus gemcitabine-treated tumors
(Fig. 3, act. EGF-R)
. In contrast, the identical tumors
showed similar levels of immunoreactivity for total EGF-R (does not
distinguish activated from inactivated) regardless of treatment group
(Fig. 3, EGF-R)
. A431 epidermoid carcinoma, known not only
to overexpress EGF-R but also to exhibit a high degree of EGF-R
autophosphorylation (32
, 35)
, demonstrated high
immunoreactivity for both total and activated EGF-R (data not shown).
These results suggest C225 or combination treatments inhibit EGF-R
function.
In Vivo Inhibition of Proliferation (PCNA) and
Increase of Apoptosis (TUNEL) after C225 Blockade of EGF-R.
We next analyzed the effect of C225 treatment alone and in combination
with gemcitabine on tumor cell proliferation in vivo.
Tumor cell proliferation was reduced as measured by the percentage of
PCNA positive versus total cells (per microscopic field) in
each tumor section 11, 18, and 30 days after initiation of C225 or C225
plus gemcitabine treatments (Fig. 4
;
Table 2
). A significant difference was
also observed in tumors (18 days after therapy) from mice treated with
C225 (34%) or C225 plus gemcitabine (7%) compared with gemcitabine
alone or control tumors (69 or 94%, respectively; Fig. 4
; Table 2
).
These results indicate an antiproliferative effect of C225 treatment
alone that is further augmented in combination with gemcitabine
(P < 0.008).
|
|
; Table 2
). The results
suggest that treatment with C225 results in not only cytostasis but
also cytotoxicity in vivo, correlating with inhibition of
tumor growth and regression.
Down-Regulation of VEGF and IL-8 in L3.6pl Human Pancreatic Cancer
Cells Growing in Vitro after Treatment with C225.
Because C225 treatment in vitro resulted in a 20% maximal
cytostatic effect and treatment in vivo of established
tumors not only decreased tumor cell proliferation but also increased
apoptosis, we investigated the mechanism for this differential effect.
Recent studies have demonstrated that an indirect effect in which tumor
cell production of angiogenic factors is inhibited, thereby affecting
tumor-induced angiogenesis, resulting in tumor regression
(10, 11, 12)
. ELISA detected a dose-dependent down-regulation
of VEGF (48%; P < 0.04) and IL-8 (20%;
P < 0.05) protein production in conditioned media of
subconfluent L3.6pl pancreatic carcinoma cells after continuous
exposure to C225 for 48 h (Table 3)
.
No effect was observed on cell-associated bFGF protein production (data
not shown). Northern blot analysis directly correlated with the ELISA
results, demonstrating a 40 and 20% decrease in steady-state mRNA
transcripts for VEGF and IL-8, respectively, after treatment with C225
(5 µg/ml for 48 h) compared with controls (data not shown).
|
; Table 4
). Furthermore, no significant reduction
in bFGF protein expression was seen after treatments with either C225
or C225 plus gemcitabine in comparison to gemcitabine-treated or
control tumors (data not shown).
|
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; Table 4
). MVD was significantly
reduced in tumors treated for 18 and 30 days with C225 and C225 plus
gemcitabine. No significant difference in MVD was observed in tumors
from gemcitabine-treated and control animals (Fig. 5
; Table 4
). The
down-regulation of VEGF and IL-8 protein production observed as early
as 11 days after therapy preceded the reduction in MVD (Table 4)
. This
temporal sequence suggests that the reduction in the levels of VEGF and
IL-8 within the tumor contribute to the involution of neovessels
observed after treatment.
Endothelial Cell Apoptosis.
Double-staining immunofluorescence was used to evaluate the
percentage of apoptotic endothelial cells (per microscopic section) in
tumors from animals receiving the different treatments for 11, 18, and
30 days (Fig. 6)
. The highest significant
difference was observed 18 days after treatment with C225 (69% median
apoptotic endothelial cells; P < 0.00004) or C225 plus
gemcitabine (62% median apoptotic endothelial cells; P < 0.0004) compared with control tumors (0% median apoptotic
endothelial cells; Fig. 6
; Table 4
). Gemcitabine treatment did not
enhance endothelial cell apoptosis compared with controls (Fig. 6
;
Table 4
). These data directly correlate with the observed reduction in
MVD (Table 4)
. The down-regulation of VEGF and IL-8 protein production
by tumor cells, therefore, preceded the reduction in MVD, suggesting
that the decline in VEGF and IL-8 directly contributed to the reduction
in neovascularity as a result of endothelial cell apoptosis. These data
also indicate that therapy with C225 and C225 plus gemcitabine, but not
gemcitabine alone, caused not merely an inhibition but an actual
involution of the neovasculature, leading to tumor cell apoptosis and
regression of established tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EGF-R and its ligands (including EGF, AR, and TGF-) are commonly
overexpressed in pancreatic cancer, and their expression is associated
with decreased survival duration (13
, 18
, 19)
. This
coexpression of both receptor and ligand and the fact that EGF, AR, and
TGF- are mitogens for pancreatic cancer cell lines have led to the
hypothesis that an EGF-R-dependent autocrine loop contributes to the
malignant phenotype (14
, 17
, 20
, 21)
. Its importance in
the biology of pancreatic cancer is supported by in vitro
experiments targeting either EGF-R or its ligands
(41, 42, 43)
. In addition, Schmiegel et al.
(43)
demonstrated a longer median survival in unresectable
pancreatic cancer patients treated with the anti-EGF-R Mab 425. One
complete response was observed in a patient who remained in an
unmaintained remission for 3 years (43)
. These results
suggest that the EGF-R is a potential therapeutic target in pancreatic
carcinoma.
In the present study, we describe a new therapeutic approach for pancreatic cancer, the combination of anti-EGF-R antibody C225 and gemcitabine. Whereas gemcitabine inhibits DNA synthesis by cells in S-phase and by blocking cell cycle progression through G1-S, blockade of EGF-R signaling pathways results in cell cycle arrest at the G1-S checkpoint. The induction of G1 arrest by C225 is associated with the inhibition of cyclin-dependent kinase 2 activity and increased levels of cyclin-dependent kinase inhibitor p27Kip1 (44 , 45) . In fact, C225 has been shown recently to augment the antitumor activity of several chemotherapeutic agents and irradiation in different mouse xenograft models (10 , 14 , 45, 46, 47, 48, 49) . The molecular pathways for this effect are unclear but may be mediated by several different mechanisms, including those affecting DNA repair (41 , 46 , 48 , 50 , 51) , multidrug resistance (52 , 53) , cell cycle checkpoint control (45) , or angiogenesis (10, 11, 12 , 58) . For example, C225, but not EGF, triggered a specific physical interaction between the internalized EGF-R and DNA-PK, implicated in the repair of DNA double strand breaks, in a variety of cell types (51) . This association significantly reduced the level and activity of DNA-PK in the nucleus and concomitantly increased DNA-PK levels in the cytosol, suggesting that EGF-R blockade and down-regulation by C225 may impair DNA repair by reducing the nuclear level of DNA-PK (51) .
The capacity of C225 to modulate tumor cell cycle distribution may also play a central role in regulating the increased sensitivity to chemotherapeutic agents and irradiation. This may involve cell cycle checkpoint control as an activator of cell death (45) . EGF-R blockade results in cellular arrest at the G1 restriction point, and cells damaged by chemotherapy or radiation typically arrest in G2-M to repair DNA alterations. Mendelsohn (45) has hypothesized that tumor cells die if they consecutively ignore two checkpoint signals (e.g., activated by EGF-R blockade and cytotoxic drug or radiation treatment). Interestingly, nonmalignant epithelial cells, which obey checkpoint control signals, may be less susceptible to the cytotoxic effects of these combination treatments. These data indicate that inhibition of EGF-R signaling may act through several potential mechanisms to sensitize tumor cells to cytotoxic agents or radiation.
Our data indicate a significant regression of established human pancreatic carcinoma growing orthotopically in the pancreas of nude mice after administration of C225, which could not be explained by the modest 20% cytostatic effect observed after in vitro treatment of the same highly metastatic L3.6pl human pancreatic cancer cells. The in vivo treatment effect was also significantly potentiated when C225 was combined with gemcitabine. This discrepancy may be explained, at least in part, by inhibition of the host angiogenic response. Indeed, our in vivo and in vitro data confirm a down-regulation of tumor cell-produced VEGF and IL-8 (but not bFGF) after treatment with C225. This down-regulation preceded the reduction in MVD, suggesting that the decline in VEGF and IL-8 directly contributed to the reduction in neovascularity as a direct result of endothelial cell apoptosis. These data also indicate that therapy with C225 and C225 plus gemcitabine, but not gemcitabine alone, caused not merely an inhibition but in fact an involution of the neovasculature, leading to tumor cell apoptosis and regression of established tumors. This is the first report to our knowledge demonstrating that therapy with C225 leads to apoptosis of endothelial cells within pancreatic tumors as determined by a double-labeling immunofluorescence procedure for CD31 and TUNEL.
This premise is further strengthened by the recent studies of Koch et al. (55) and Benjamin et al. (54 , 58) using a genetic system designed for conditional VEGF expression in either a xenografted glioma or well-established, slow-growing human prostate cancers. Their data demonstrate the dependence of immature vessels on VEGF for survival and prove that this can be exploited to an existing tumor vasculature and hence the dependent tumor mass (54 , 55 , 56) . Antiangiogenic therapy therefore capitalizes on the concept that the proangiogenic processes in a tumor are more exaggerated in intensity and localized to a more limited anatomical site than are normal physiological processes (22, 23, 24 , 57) . A shift to an antiangiogenic phenotype can result from a suppression of proangiogenic mechanisms and/or enhancement of the opposing antiangiogenic processes.
The mechanisms by which EGF-R signaling pathways regulate VEGF and IL-8
are unclear. However, it is established that activation of EGF-R
signaling pathways by EGF and TGF- up-regulate VEGF (31
, 58)
. Furthermore, it is known that after stimulation of EGF-R
signaling pathways, ras and raf are activated,
resulting in phosphorylation of c-fos and c-jun,
which leads to increased AP-1 transcriptional activity (54
, 58, 59, 60, 61)
. Increased AP-1 activity leads to transcription of genes
with AP-1 binding sites in their promoter including VEGF and IL-8
(58
, 60
, 62)
. Experiments in progress are analyzing the
effect of EGF-R blockade on the specific transcription factors involved
in VEGF and IL-8 regulation in the human L3.6pl pancreatic carcinoma
cells.
In summary, our experiments indicate that therapeutic strategies targeting EGF-R signaling have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice and the effect is mediated by inhibition of tumor-induced angiogenesis, which in turn leads to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine. These experiments indicate that normalization of angiogenesis related biomarkers such as VEGF, IL-8, or MVD may be clinically useful (in addition to other markers including EGF-R activation status, PCNA, and TUNEL for tumor or endothelial cells) to demonstrate regression or eradication of cancer after EGF-R-directed therapy. Analysis of these biomarkers should be included in clinical trials for evaluating this form of therapy. Finally, combination of this approach with gemcitabine may provide increased benefit in pancreatic carcinoma patients.
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ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by a grant (to R. R.) from The
Marc Lustgarten Foundation for Pancreatic Cancer Research, Grants
CA67952 (to R. R.), CA 69676 (to D. J. M.), and Cancer Center Core
Grant CA16672 from the NIH, National Cancer Institute, ImClone Systems,
Inc., and Habilitationsstipendium of the "Lise-Meitner-Programm" of
the Ministerium fuer Wissenschaft und Forschung, Nord-Rhine-Westphalia,
Germany (to C. J. B.). 
2 To whom requests for reprints should be
addressed, at Department of Cancer Biology, The University of Texas
M. D. Anderson Cancer Center, Box 173, 1515 Holcombe Boulevard,
Houston, TX 77030. Phone: (713) 792-8643; Fax: (713) 792-8747;
E-mail: rradinsk{at}mdanderson.org
3 The abbreviations used are:
EGF-R, epidermal growth factor receptor; AR, amphiregulin; TGF,
transforming growth factor; VEGF, vascular endothelial growth factor;
IL, interleukin; bFGF, basic fibroblast growth factor; FBS, fetal
bovine serum; Mab, monoclonal antibody; MVD, microvessel density; PCNA,
proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling; PECAM, platelet/endothelial
cell adhesion molecule; DNA-PK, DNA-dependent protein kinase.
Received 2/ 8/00; revised 2/ 9/00; accepted 2/11/00.
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T. Karashima, P. Sweeney, J. W. Slaton, S. J. Kim, D. Kedar, J. I. Izawa, Z. Fan, C. Pettaway, D. J. Hicklin, T. Shuin, et al. Inhibition of Angiogenesis by the Antiepidermal Growth Factor Receptor Antibody ImClone C225 in Androgen-independent Prostate Cancer Growing Orthotopically in Nude Mice Clin. Cancer Res., May 1, 2002; 8(5): 1253 - 1264. [Abstract] [Full Text] [PDF]
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J. D. Wayne, E. K. Abdalla, R. A. Wolff, C. H. Crane, P. W.T. Pisters, and D. B. Evans Localized Adenocarcinoma of the Pancreas: The Rationale for Preoperative Chemoradiation Oncologist, February 1, 2002; 7(1): 34 - 45. [Abstract] [Full Text] [PDF]
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E. M. Gibson, E. S. Henson, N. Haney, J. Villanueva, and S. B. Gibson Epidermal Growth Factor Protects Epithelial-derived Cells from Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis by Inhibiting Cytochrome c Release Cancer Res., January 1, 2002; 62(2): 488 - 496. [Abstract] [Full Text] [PDF]
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C. Mundhenke, J. P. Thomas, G. Wilding, F. T. Lee, F. Kelzc, R. Chappell, R. Neider, L. A. Sebree, and A. Friedl Tissue Examination to Monitor Antiangiogenic Therapy: A Phase I Clinical Trial with Endostatin Clin. Cancer Res., November 1, 2001; 7(11): 3366 - 3374. [Abstract] [Full Text] [PDF]
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F. Ciardiello and G. Tortora A Novel Approach in the Treatment of Cancer: Targeting the Epidermal Growth Factor Receptor Clin. Cancer Res., October 1, 2001; 7(10): 2958 - 2970. [Abstract] [Full Text] [PDF]
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A. Viloria-Petit, T. Crombet, S. Jothy, D. Hicklin, P. Bohlen, J. M. Schlaeppi, J. Rak, and R. S. Kerbel Acquired Resistance to the Antitumor Effect of Epidermal Growth Factor Receptor-blocking Antibodies in Vivo: A Role for Altered Tumor Angiogenesis Cancer Res., July 1, 2001; 61(13): 5090 - 5101. [Abstract] [Full Text] [PDF]
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F. Ciardiello, R. Caputo, R. Bianco, V. Damiano, G. Fontanini, S. Cuccato, S. De Placido, A. R. Bianco, and G. Tortora Inhibition of Growth Factor Production and Angiogenesis in Human Cancer Cells by ZD1839 (Iressa), a Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Clin. Cancer Res., May 1, 2001; 7(5): 1459 - 1465. [Abstract] [Full Text]
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P. D. Ryan and B. A. Chabner On Receptor Inhibitors and Chemotherapy Clin. Cancer Res., December 1, 2000; 6(12): 4607 - 4609. [Full Text]
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