From the Gastroenterology Division,
** Department of Genetics, § Abramson Cancer
Center and Family Cancer Research Institute,
Wistar Institute,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, September 6, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Epidermal growth factor receptor
(EGFR) overexpression is observed in a number of malignancies,
especially those of esophageal squamous cell origin. However, little is
known about the biological functions of EGFR in primary esophageal
squamous epithelial cells. Using newly established primary human
esophageal squamous epithelial cells as a platform, we overexpressed
EGFR through retroviral transduction and established novel
three-dimensional organotypic cultures. Additionally, EGFR was targeted
in a cell type- and tissue-specific fashion to the esophageal
epithelium in transgenic mice. EGFR overexpression in primary
esophageal keratinocytes resulted in the biochemical activation of Akt
and STAT pathways and induced enhanced cell migration and cell
aggregation. When established in organotypic culture,
EGFR-overexpressing cells had evidence of epithelial cell
hyperproliferation and hyperplasia. These effects were also observed in
EGFR-overexpressing transgenic mice and the esophageal cell lines
established thereof. In particular, EGFR-induced effects upon
aggregation appear to be mediated through the relocalization of p120
from the cytoplasm to the membrane and increased interaction with
E-cadherin. EGFR modulates cell migration through the up-regulation of
matrix metalloproteinase 1. Taken together, the functional effects of
EGFR overexpression help to explain its role in the initiating steps of
esophageal squamous carcinogenesis.
Epidermal growth factor receptor
(EGFR)1 is a transmembrane
protein receptor with tyrosine kinase activity that triggers numerous signaling pathways (1-3). Activation of the EGFR tyrosine kinase results in the generation of a number of intracellular signals, which
culminate in not only cell proliferation but also other processes that
are crucial to cancer progression, including angiogenesis, metastatic
spread, and the inhibition of apoptosis. These events are mediated
through various downstream targets of EGFR (e.g. the
serine/threonine kinase Raf and mitogen-activated protein/extracellular signal-regulated kinase 1/2). In addition, Ras activation by
EGFR is required for a vast array of cellular functions, foremost of which is the regulation of cellular proliferation. Activation of EGFR
also results in the activation of the lipid kinase phosphatidylinositol 3-kinase, generating the second messenger phosphatidylinositol 3,4,5-trisphosphate, which in turn activates Akt. We have previously demonstrated that there is differential activation of the Akt isoforms
by EGFR in esophageal cancer cells (4). Apart from the
mitogen-activated protein kinase and phosphatidylinositol 3-kinase
pathways, EGFR also activates other pathways such as phospholipase-C
and its downstream protein kinase cascades, small GTPases such as Rho,
and multiple signal transducer and activator of transcription (STAT) isoforms.
EGFR activation is not only important in normal cellular processes, but
it is frequently altered or overexpressed in many malignancies,
especially those of squamous cell origin. Mechanisms that mediate EGFR
overexpression include gene amplification, truncation of the carboxyl
terminus, transcriptional activation, and posttranslational modifications. EGFR overexpression is a frequent genetic alteration in
premalignant esophageal squamous dysplastic lesions and the early
stages of esophageal squamous cell cancer (5-8). This has led us to
focus upon the role of EGFR-mediated signaling in esophageal epithelial
cell biology and examine its role in models of esophageal squamous cell carcinogenesis.
We describe herein novel organotypic esophageal cell culture and
esophageal specific transgenic mouse models as the basis for
examination of EGFR-mediated biological effects in physiologic environments that are often not possible in cancer-derived cell lines.
We observe that EGFR overexpression results in hyperproliferation both
in vitro and in vivo but is not sufficient to
induce cancer, explaining its frequent association with premalignant
stages. Furthermore, EGFR activation results in increased cell
migration. Mechanistically, the increase in cell migration induced by
EGFR is mediated through up-regulation of matrix metalloproteinase-1 (MMP-1). Whereas EGFR overexpression did not influence the assembly of
adherens junctions and desmosomes, it did induce a translocation of
p120 catenin from the cytosol to the cell membrane that mediates the
increased cell aggregation. We believe that these effects help to
explain how EGFR modulates the persistence of proliferative basal cells
into the suprabasal compartment of the squamous epithelium and that
EGFR is critical for the initiating events in squamous carcinogenesis.
Cell Lines--
Primary esophageal keratinocytes, designated as
EPC1 and EPC2, from normal human esophagus were established. Surgical
specimens from normal esophagi were promptly removed and transported
aseptically in Hanks' solution (Invitrogen) with 100 units/ml
penicillin, and 100 µg/ml streptomycin (Invitrogen) and 5 µg/ml
gentamicin (Invitrogen). The tissue specimen was incubated with 1.5 units/ml dispase (Roche Molecular Biochemicals) at 4 °C overnight,
and the epithelium was dissected away and incubated with trypsin
(Invitrogen). The reaction was stopped with soybean trypsin inhibitor
(Sigma) and centrifuged. The pellet was resuspended in keratinocyte-SFM medium (KSFM) (Invitrogen) supplemented with 40 µg/ml bovine
pituitary extract (Invitrogen), 1.0 ng/ml EGF (Invitrogen), 100 units/ml penicillin, 100 µg/ml streptomycin (Invitrogen), 5 µg/ml
gentamycin, and 100 units/ml nyastatin (Invitrogen). EPC cells were
grown at 37 °C and 5% CO2 with KSFM, with 40 µg/ml
bovine pituitary extract, 1.0 ng/ml EGF, 100 units/ml penicillin, and
100 µg/ml streptomycin.
When cells were starved, the medium was KBM supplemented with
0.5 µg/ml hydrocortisone (Bio Whittaker Inc., Walkersville, MD) and
0.09 mM calcium chloride (Bio Whittaker). Cells were
starved for 48 h without EGF and then stimulated with 10 or 100 ng/ml EGF for the indicated periods at 37 °C, washed three times
with ice-cold phosphate-buffered saline, lysed in buffer, and
centrifuged for 15 min at 4 °C. A431 cells conditioned in KSFM
served as a positive control.
The growth curves of EPC cells and those transduced with green
fluorescent protein (GFP) or EGFR (see below) were generated by plating
cells in six-well plates (1.0 × 104 cells/plate) and
grown in KSFM supplemented with 40 µg/ml bovine pituitary extract.
Cells were harvested at indicated periods and counted with a
spectrophotometric quantitation method (9). All experiments were
carried out at least three times in triplicate with generation of S.D. values.
Retroviral Vectors and Infection--
pFB-neo retroviral vectors
(Stratagene, La Jolla, CA) were used to infect EPC1 and EPC2. In
addition, we subcloned into the pFB-neo vector the entire coding
sequence for the human EGFR or green fluorescent protein (GFP). The
inserted region in the resulting construct was sequenced, and the
plasmid was transfected into Phoenix-Ampho cells by the
calcium-phosphate precipitation method (Calphos;
Clontech, Palo Alto, CA) according to the
manufacturer's instructions. In brief, culture supernatants from
individual Phoenix-Ampho cells were used to infect EPC1 and EPC2 cells.
These cells were infected with filtered (0.45-µm pore size)
supernatant from an overnight culture of Phoenix-Ampho cells, producing
the pFB-neo retroviruses encoding EGFR or GFP. Cells were passaged
48 h after infection and selected in 300 µg/ml G418 for 14 days.
Organotypic Cell Culture--
To grow human esophageal
epithelial cells (keratinocytes), 5 × 105 cells were
seeded on to the collagen matrix, containing 1× minimal essential
medium with Earle's salts (Bio Whittaker), 1.68 mM
L-glutamine (Cellgro, Herndon, VA), 10% fetal bovine serum
(Hyclone, Logan, UT), 0.15% sodium bicarbonate (Bio Whittaker), 76.7%
bovine tendon acid-extracted collagen (Organogenesis, Canton, MA), and
7.5 × 104 human skin fibroblast cells. Cells were fed
with Epidermalization I medium for 2 days, which is Dulbecco's
modified Eagle's medium (JRH Biosciences, Lenexa, KS)/Ham's F-12
(Invitrogen) (3:1) supplemented with 4 mM
L-glutamine, 0.5 µg/ml hydrocortisone, 0.1 mM
O-phosphorylethanolamine, 20 pM
triiodothyronine, 0.18 mM adenine, 1.88 mM
CaCl2, 4 pM progesterone (Sigma); 10 µg/ml
insulin, 10 µg/ml transferrin, 10 mM ethanolamine, 10 ng/ml selenium (ITES) (Bio Whittaker); and 0.1% chelated newborn calf
serum (Hyclone). For the following 2 days, cells were fed with
Epidermalization II medium, which is identical to Epidermalization I
except that it contains 0.1% unchelated newborn calf serum. Then cells were raised to the air-liquid interface and cultured in
Epidermalization III medium for 6 days containing the same growth
supplements as Epidermalization II except 2% newborn calf serum. Cells were fixed with 10% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin.
Generation of EGFR Transgenic Mice and Cell Lines of Mouse
Esophageal Keratinocyte Origin--
We employed the Epstein-Barr virus
ED-L2 promoter and fused it to the human EGFR cDNA to create a
transgene from which founder lines were generated and maintained in the
B6SJL-F1 gene background. The EGFR transgene is expressed in a
tissue-specific fashion with targeting to the tongue, esophagus, and
forestomach (10). The EGFR transgenic mice and age-matched wild-type
mice were sacrificed at age 6 months for histology and
immunohistochemistry. The cell lines were established in the same
fashion same as described for EPC1 and EPC2.
Antibodies--
Antibodies against EGFR were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against
anti-phospho-EGFR (Y1173) and Akt1 were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Antibodies specific for focal
adhesion kinase were from BD Biosciences (San Diego, CA), Antibodies
specific for Akt phosphorylated at Ser-473, phosphofocal adhesion
kinase, phospho-STAT1, phosphoextracellular signal-regulated kinase 1/2 and total extracellular signal-regulated kinase 1/2 were obtained from
Cell Signaling (Beverly, MA). Antibodies specific against desmoglein 1 and 2 (clone DG3.10) were purchased from Biodesign (Kennebunk, ME), and
antibodies against Desmoplakin were from Serotec.
E-cadherin-neutralizing antibody was purchased from Sigma. Antibodies
against E-cadherin, p120, and Immunoprecipitation--
Preconfluent cells starved in KBM
medium and stimulated with 10 ng/ml EGF for 15 min as well as
unstimulated cells were washed with phosphate-buffered saline without
calcium and magnesium (PBS) and incubated with 700 µl of lysis
buffer (1% Triton X-100, 1% Nonidet P-40, 50 mM Tris, pH
8, and proteinase inhibitors 2 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 10 mM NaF, 2 mM Na3VO4, 5 mM sodium pyrophosphate;
less stringent buffer does not contain Nonidet P-40) for 30 min on ice.
70 µl of 4% bovine serum albumin and 140 µl of 1.5 M
NaCl were added to the extracts, which were then preabsorbed with 10 µl of recombinant Protein G-agarose (Invitrogen) for 1 h
at 4 °C. Preabsorbed extracts were incubated with antibodies against
EGFR, plakoglobin, p120, and Western Blotting--
Subconfluent cells were lysed in lysis
buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl,
1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 2 mM sodium orthovanadate, and a
protease inhibitor mixture tablet (Roche Molecular Biochemicals)).
Protein concentration was determined by the BCA protein assay (Pierce). The solution was subsequently solubilized in NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA) containing 50 mM dithiothreitol. Total protein samples (10 µg) were
separated on a 4-12% SDS-PAGE and transferred to a polyvinylidene
difluoride membrane (Immobilon-P; Millipore Corp., Bedford, MA). The
membrane was blocked in 5% nonfat milk (Bio-Rad) in TBST (10 mM Tris, 150 mM NaCl, pH 8.0, and 0.1% Tween
20) for 1 h at room temperature. Membranes were probed with
primary antibody diluted 1:1000 in 5% TBST milk overnight at 4 °C,
washed three times in TBST, incubated with anti-mouse or anti-rabbit
horseradish peroxidase antibody diluted 1:3000 in TBST for 1 h at
room temperature and then washed three times in TBST. The signal was
visualized by an enhanced chemiluminescence solution (ECL Plus;
Amersham Pharmacia Biotech) and was exposed to Eastman Kodak Co. X-Omat
LS film.
Immunohistochemistry and
Immunofluorescence--
Immunohistochemistry for Ki67 was performed
with the Vecta Elite kit (Vector Laboratories, Burlingame, CA)
following the manufacturer's protocol. In brief, paraffin sections
were pretreated with xylene and then placed in a microwave in 10 mM citric acid buffer. Endogenous peroxidases were quenched
using hydrogen peroxide before sections were blocked in avidin D
blocking reagent and biotin blocking reagent. Sections were incubated
with primary and secondary antibody, and then signal was developed
using the DAB substrate kit for peroxidase.
Reconstructs were embedded in ornithine transcarbamylase and
frozen at RT-PCR and SYBR Green Real Time PCR--
RNA was isolated from
EPC2-GFP and EPC2-EGFR cells using Trizol reagent (Invitrogen), and
cDNA was synthesized using the Superscript first strand synthesis
system for RT-PCR (Invitrogen) according to the manufacturer's
instructions. Primer for PCR and SYBR green real time PCR were designed
using the TaqMan probe software and synthesized by Invitrogen (primer
sequences: MMP-1 (5'-TCC GGT TTT TCA AAG GGA ATA; 3'-CCT CAG AAA GAG
CAG CAT CGA), MMP-2 (5'-AGA CCG CCA TGT CCA CTG TT; 3'-TGG TCG CAC ACC
ACA TCT TT), GAPDH (5'-CAC CCA CTC CTC CAC CTT T; 3'-TCC ACC ACC CTG
TTG CTG TAG)). SYBR green real time PCR was performed and analyzed
using the ABI 6000 (Applied Biosystems, Foster City, CA) with reagents
from the SYBR green PCR kit (QIAgen).
Cell Migration and Invasion Assay--
Haptotactic cell
migration assays were performed using 24-well inserts (Falcon cell
culture inserts (8-µm pore size); BD Biosciences) with or without
matrigel Biocoat (BD Biosciences) according to the manufacturer's
instructions. In brief, the lower chamber was filled with 0.6 ml of KBM
containing 0.5 µg/ml hydrocortisone with 10 ng/ml EGF, and 0.5-ml
cell suspension in KBM under serum-starving conditions were plated in
the upper chamber in duplicate or triplicate wells and incubated at
37 °C for 12 h. Then cells attached to the upper side of the
membrane were removed gently with a cotton swab and rinsed. Cells that
migrated through the membrane and attached to the bottom of the
membrane were fixed and stained with reagents from the Diff Quik
staining set (Dade Behring, Newark, DE). Membranes were cut out and
photographed, so migrated cells could be counted on the pictures taken.
All experiments were performed at least three times in triplicate. The
specific inhibitor of EGFR tyrosine kinase, AG1478, was purchased from
Calbiochem.
Cell Aggregation Assay--
Cell aggregation assays were based
upon previously described methods (11). Single cell suspensions,
starved for 48 h in KBM and stimulated with 10 ng/ml EGF, and
those that were unstimulated were obtained with 1 mM EDTA
in 10 mM Hepes-buffered calcium- and magnesium-free Hanks'
solution. Cells were then washed twice in Hepes-buffered Hanks'
solution containing 10 ng/ml DNase I. Cells were resuspended, and
3 × 105 cells were incubated on a rotary shaker for
1 h in six-well plates coated with 2% bovine serum albumin.
AG1478 was added to the cell suspension during the 1-h incubation
period on the shaker. Plates were incubated for another 1 h
without shaking, so cell aggregates settled for analysis using the NIH
Image software.
EGFR Overexpression and Activation by Retroviral Transduction in
Normal Human Esophageal Epithelial Cells--
Human primary esophageal
cells, designated as EPC1 and EPC2, were established and had
morphologic, cytogenetic, and biochemical properties of normal cells as
illustrated by normal diploid status, expression of cytokeratins 5 and
14 found in basal cells, absence of p53 mutation, and the ability to be
differentiated in a postconfluent state or with high calcium
concentration (1.0-1.2 mM) (data not shown). These cells
also reach senesence after 40-44 population doublings.
The cells were transduced with retroviral vectors containing either
wild-type human EGFR or GFP. Western blotting indicated EGFR
overexpression in EPC1 and EPC2 compared with parental EPC or
cells transduced with GFP (Fig.
1). In fact, EGFR expression was
comparable with EGFR expression found in A431 cells, a vulvar squamous cancer cell line, and a prototypic cancer cell line for EGFR
investigation. 125I-EGF binding experiments and Scatchard
analysis were performed to determine EGFR number: EPC2-GFP,
Kd = 0.9 nM, r = 0.9 × 106/cell; EPC2-EGFR: Kd = 1.2 nM, r = 3.4 × 106/cell. All subsequent experiments are depicted for EPC2
cells but were identical in EPC1 cells as well.
Functionally, phosphorylation levels of EGFR were also increased in
EPC2-EGFR cells compared with parental cells and EPC2-GFP cells (Fig.
1). These results indicated that EGFR can be overexpressed in primary
human esophageal epithelial cells with functional activation of the
EGFR by EGF ligand.
EGFR Overexpression Results in the Activation of Key Downstream
Targets--
Extracts of cells serum-starved overnight and then
stimulated with EGF ligand were analyzed by Western blot for the
activation of downstream targets of EGFR. EGF stimulation induced
increased phosphorylation of Akt-1 and STAT1 in EPC2-EGFR cells (Fig.
2) compared with control cells. Akt1
phosphorylation was induced at early time periods but sustained for a
longer phase in EPC2-EGFR cells. STAT1 phosphorylation was dramatically
increased in EPC2-EGFR cells 15 min after stimulation compared with
EPC2-GFP cells. In addition, EGFR activation induced phosphorylation of
extracellular signal-regulated kinase 1/2 (data not shown).
EGFR Induces Esophageal Epithelial Cell Hyperproliferation in
Organotypic Cell Culture and Transgenic Mice--
To analyze the
behavior of GFP- and EGFR-transfected cells in a more physiologic
environment, we established and examined organotypic cultures of these
cells. Parental cells as well as GFP and EGFR-EPC2 cells were grown on
a collagen gel containing human skin fibroblasts submerged in the
tissue culture medium for 4 days followed by cultivation at the
air-liquid interface for 7 days to induce terminal differentiation.
These epithelial reconstructs were evaluated by histology,
immunohistochemistry for EGFR and proliferating cell nuclear antigen,
and the terminal deoxynucleotidyltransferase-mediated dUTP nick end
labeling method for apoptosis. Compared with normal and GFP-transduced
primary human esophageal epithelial cells, EGFR overexpression resulted in a thicker epithelium and basal cell hyperplasia (Fig.
3A). EGFR was expressed in the
basal and suprabasal cell layers and showed not only membrane staining
but also diffuse cytoplasmic staining, whereas reconstructs of
parental cells showed only faint staining in the basal cells (Fig.
3B). Proliferating cell nuclear antigen staining revealed
that EGFR-overexpressing cells were found in the basal and suprabasal
cell layers and not restricted to the basal cell layer in parental
and GFP-derived cells (Fig. 3C). Furthermore, there was no
change in the scant apoptosis that was confined to the basal cell
compartment between these control cells and EGFR-overexpressing cells
(data not shown). By adding the EGFR tyrosine kinase inhibitor AG1478
to the medium of the organotypic culture, the hyperplasia
induced by EGFR was dramatically diminished such that the epithelium
was significantly thinner in a dose-dependent fashion (Fig.
3D).
In order to address the effects of EGFR overexpression in
vivo, we generated transgenic mice in which the Epstein-Barr virus ED-L2 promoter was fused to the human EGFR cDNA. This promoter has
been previously demonstrated to direct transgene expression specifically to the oral and esophageal squamous epithelia (10). Transgene expression in two different founder lines was confirmed by
RT-PCR, Northern blotting, Western blotting, and immunohistochemistry (data not shown). Furthermore, there was increased tyrosine
phosphorylation of the EGFR transgene product in transgenic mice when
compared with age-matched littermates (data not shown). Compared with
age-matched (6 months) littermate wild-type mice, EGFR transgenic mice
revealed evidence of increased proliferation in the basal and
suprabasal cells (Fig. 4), consistent
with what was observed in organotypic culture.
EGFR Overexpression Increases Cell Migration and MMP-1 Expression
Level--
Activation of EGFR is associated with cell migration and
invasion. To determine whether overexpression of EGFR in primary esophageal keratinocytes induces cell migration, we performed cell
migration assays. Migration of EPC2-EGFR cells was increased compared
with control cells (Fig. 5). We could
demonstrate that the observed enhanced migration is
EGFR-dependent by use of the EGFR-specific inhibitor,
AG1478 (Fig. 5).
In order to assess how EGFR mediates the increased cell migration, we
determined whether matrix metalloproteinases may be up-regulated.
Indeed, based upon RT-PCR data, we could show an up-regulation of MMP-1
in EPC2-EGFR cells. Levels for MMP-2 and MMP-9 were the same in both
EPC2-GFP and EPC2-EGFR cells (Fig. 6A). After the addition of
AG1478 to the cell culture medium, the up-regulation of MMP-1 was
reversed (Fig. 6B). We confirmed the up-regulation of MMP-1
with SYBR Green real time PCR in EPC2-EGFR cells compared with EPC2-GFP
cells, whereas MMP-2 and MMP-9 levels remained unchanged (data not
shown).
Western blot analysis revealed increased MMP-1 in conditioned
media of EPC2-EGFR cells compared with EPC2-GFP control cells. Furthermore, the AG1478 inhibitor abolished MMP-1 secretion (Fig. 6B).
EGFR Overexpression Increases E-cadherin-dependent Cell
Aggregation--
After staining of EPC2-GFP and EPC2-EGFR cells in
migration assays, we observed clustering of EPC2-EGFR cells that was
not present in EPC2-GFP cells (Fig. 5B). We investigated
whether EGFR overexpression and stimulation with EGF have effects on
cell-cell adhesion using a cell aggregation assay. EPC2-EGFR cells
demonstrated stronger aggregation than EPC2-GFP cells without EGF
stimulation (Fig. 7). After stimulation
with EGF, cell aggregation is further enhanced, and the differences in
aggregation between EPC2-GFP and EPC2-EGFR cells are accentuated. The
addition of AG1478 nearly abolishes cell-cell aggregation, suggesting
that aggregation is EGFR-dependent (Fig. 7). To prove that
the observed aggregation is E-cadherin-dependent, we
performed aggregation assays adding neutralizing E-cadherin antibody
(DECMA-1) to the cells in suspension during the 1-h incubation period.
Samples that were incubated in the presence of DECMA-1 were unable to
aggregate (Fig. 7).
To examine whether EGFR overexpression induces changes in
the assembly of adherens junctions and desmosomes, we performed immunoprecipitations and Western blot on cell extracts before and after
EGF stimulation. Expression levels of components of adherens junctions
and desmosomes (E-cadherin,
Sequential detergent extraction followed by immunoprecipitation with an
antibody against E-cadherin confirmed the strong interaction between
E-cadherin and p120 in the Triton X-100 soluble pool (membrane fraction), whereas p120 but not E-cadherin could be immunoprecipitated from the saponin pool (cytoplasmic fraction) (data not shown).
p120 Translocation from Cytoplasm to the Membrane Correlates with
Strong Adhesion--
To determine the localization of p120 in
organotypic culture (Fig. 8C) and monolayer cell culture
(Fig. 8D), we performed immunofluorescence staining in
EPC2-GFP and EPC2-EGFR cells. Confocal microscopy of organotypic
culture shows that EGFR overexpression in the basal and suprabasal
layers correlates with strong p120 staining. Merged scans demonstrate
partial co-localization of EGFR with p120 at the cell membrane of
EGFR-positive cells and co-localization of p120 with E-cadherin (Fig.
8C), whereas controls stained with antibody against
desmoplakin, a component of the desmosome, did not show co-localization
with p120. In order to analyze the localization of p120 in more detail,
EPC2-GFP and EPC2-EGFR cells were stained with an antibody against
p120, which demonstrates there is a large cytoplasmic pool of p120 in
EPC2-GFP cells. In EPC2-EGFR cells, the distribution of p120 is shifted toward the cell membrane with little cytoplasmic staining (Fig. 8D). Furthermore, EPC2-EGFR cells display more sites of
cell-cell contact in culture that stain positive for p120 than EPC2-GFP cells. In summary, EGFR induces a shift in the distribution of p120
from the cytoplasm to the cell membrane that coincides with a
co-localization with E-cadherin and co-distribution of EGFR and p120 in
organotypic culture. Interaction of p120 with E-cadherin is believed to
promote adhesion and helps to explain the stronger aggregation we
observe in EPC2-EGFR cells.
EGFR-mediated activation of diverse signal
transduction pathways results in protean cellular manifestations
(1-3). Furthermore, EGFR overexpression is important in the
premalignant stages of carcinogenesis. In order to address the
biological roles of EGFR-mediated effects in physiological settings, we
have developed and characterized primary human esophageal squamous
epithelial cells that can recapitulate the stratified squamous
epithelium in organotypic cell culture. In addition, we generated and
characterized transgenic mice where EGFR is specifically targeted to
the esophageal squamous epithelium. Notably, EGFR overexpression
induces a hyperproliferative state, whereby proliferating basal cells
persist into the normally differentiated suprabasal compartment,
perhaps reflecting a combination of enhanced cell migration and cell
aggregation. Furthermore, EGFR transgenic mouse-derived esophageal
epithelial cells also reveal evidence of increased cell proliferation
in the basal and suprabasal compartments.
Cell migration is a highly coordinated process involving the precise
regulation of cell adhesion to and dissociation from extracellular
matrix (ECM) proteins (12). In our experimental systems, EGFR
overexpression and activation result in increased migration of human
and mouse esophageal epithelial cells. This is reversed by use of an
inhibitor of EGFR tyrosine kinase, indicating a direct effect of EGFR
upon cell migration. Our results are consistent with the importance of
EGFR-mediated cell migration in developmental and spatial paradigms.
EGFR can substitute for fibroblast growth factor in modulating cell
migration in Drosophila (13). It has also been observed that
autocrine EGF signaling stimulates directionally persistent mammary
epithelial cell migration (14).
EGFR-mediated Cell Migration and MMP-1--
Many proteinases are
capable of degrading ECM components, but one family of enzymes that
appears to be particularly important for matrix degradation is the
MMPs. Currently, the MMPs comprise a large family of over 20 secreted
or transmembrane proteins that together can degrade all known
components of the ECM and basement membrane (15, 16). MMP family
members share functional and structural characteristics and can be
categorized into the collagenase, gelatinase, stromelysin, and
membrane-type MMP subfamilies. We find that EGFR-mediated cell
migration appears to be coordinated specifically with increased
secretion of MMP-1. Egfr
Originally, MMPs were considered to be most important almost
exclusively in invasion and metastasis. However, recent studies document that MMPs are involved in several steps of cancer development. MMPs can regulate cell growth in different ways (e.g. the
release of membrane-bound growth factors like tumor growth factor-
Up-regulation of MMP-1 in malignant tumors compared with normal tissue
has been described for cancer cells, stromal cells, and fibroblast in
squamous carcinogenesis, but these observations have been only
correlative in nature (24-28). Our findings support a direct and novel
role of EGFR signaling and MMP-1 transcriptional regulation.
E-cadherin and p120 Modulate EGFR Effects upon Cell
Adhesion--
Through the promotion of increased migration, EGFR is
thought to decrease cell adhesion by way of its interaction with
E-cadherin associates with p120, but this is not necessary for its link
to the cytoskeleton (31), although it is important for cell adhesion
and may serve as a scaffold for recruitment of other small RhoGTPases
(32, 33). Furthermore, p120 appears to be an important regulator of
adhesion through its interactions with RhoGTPase family members that
modulate transitions between migration and adhesion (34-36). We find
that the p120 cytosolic pool is decreased in EGFR-overexpressing
esophageal epithelial cells, which may contribute to the increased cell
aggregation by virtue of the fact that more p120 binds E-cadherin.
Functionally, the cell aggregation assay is supportive of this notion.
Furthermore, our confocal microscopy data show that p120 has
predominantly a cytoplasmic distribution in EPC2-GFP cells, which
shifts to a membranous localization in EPC2-EGFR cells. Furthermore,
the localization of p120 in organotypic culture correlates with EGFR overexpression.
The clustering potential of E-cadherin is attributable to the
juxtamembrane domain, which seems to be activated by the homophilic interaction between cadherin extracellular domains (37). Mutational analysis of the juxtamembrane domain has demonstrated the importance of
this region for the regulation of adhesion mediated by p120 (38) as
uncoupling of E-cadherin from p120 results in weaker adhesion.
In summary, EGFR overexpression leads to a hyperproliferative state
with induction of enhanced cell migration. This permits cells in the
basal compartment to migrate to the suprabasal compartment. The
migration may be related to an increased secretion of MMP-1. We believe
that there is activation of cell-cell aggregation once cells migrate to
the suprabasal compartment. This appears to be modulated by the
availability of p120 to bind E-cadherin, thus permitting a homeostatic
balance between proliferation in the basal compartment and
differentiation in the suprabasal compartment. Our studies allow for
novel insights into EGFR-mediated effects on cellular processes in the
normal esophageal epithelium and may help to explain the initiating
events in squamous carcinogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin were obtained from
Transduction Laboratories (Lexington, KY), and antibodies against MMP-1
were purchased from Neomarkers (clone X2A; Fremont, CA). Secondary
anti-mouse and anti-rabbit horseradish peroxidase antibodies were
purchased from Amersham Biosciences.
-catenin. After a 1-h incubation at
4 °C, the antigen-antibody complex was incubated with 10 µl of
recombinant Protein G-agarose for 1 h at 4 °C. The
precipitates were washed three times with 1 ml of wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100,
0.5% deoxycholate, 0.1% SDS; less stringent wash in PBS only) and
boiled with 100 µl of lithium dodecyl sulfate buffer containing dithiothreitol for 10 min. Supernatants were used for Western blotting as described above.
80 °C. Sections were cut and fixed in acetone for 10 min
at
20 °C. Cells in culture were seeded into chamber slides (Nalge
Nunc, Naperville, IL) and fixed in 1:1 methanol/acetone for 8 min.
After fixation, objects were treated with 0.1% Triton X-100 in
PBS for 5 min. Objects were washed in PBS and blocked with 1%
bovine serum albumin (Sigma) for 1 h. Incubation of primary antibodies was for 1 h at room temperature or overnight at
4 °C. After washing with PBS, objects were incubated with Texas Red- conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR) or
fluorescein isothiocyanate-conjugated secondary antibody (Roche
Molecular Biochemicals) for 1 h. Stained objects were examined with a Nikon Microphot microscope and imaged with a digital
camera at magnifications as indicated. Confocal microscopy was
performed using the Radiance2100 confocal and multiphoton imaging
systems and documented with Laser Sharp software (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Retroviral transduction of EGFR results in
overexpression and tyrosine kinase activation. Western blotting
shows EGFR expression levels in EPC2, EPC2-GFP, and EPC2-EGFR cells.
Phospho-EGFR levels are increased in EPC2-EGFR cells after stimulation
with EGF. -Actin serves as a loading control.
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Fig. 2.
Western blotting of EGFR and its
downstream target molecules demonstrates EGFR-mediated activation of
intracellular signaling pathways. A, Akt1 phosphorylation is
increased in EPC2-EGFR cells compared with EPC2-GFP cells before and
after stimulation with EGF. B, STAT1 phosphorylation peaks
after 15 min of EGF stimulation in EPC2-EGFR cells, whereas STAT1 is
not phosphorylated in EPC2-GFP cells. -Actin serves as a loading
control (data not shown).
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Fig. 3.
Organotypic cell culture demonstrates
hyperplasia and hyperproliferation due to EGFR overexpression.
A, Hematoxylin and Eosin (H&E) staining
of EPC2-, EPC2-GFP-, and EPC2-EGFR-overexpressing cells reveals
hyperplasia as a function of EGFR overexpression (magnification,
×400). B, immunofluorescence staining (IF)
reveals expression of EGFR in basal and suprabasal layers
(EGFR-positive cells) in EPC2-EGFR cells, whereas EPC2 and EPC2-GFP
cells only express EGFR in the basal layer (magnification, ×400).
Staining in EPC2 and EPC2-GFP in the keratinized layer is nonspecific.
C, the hyperplasia correlates with proliferating cell
nuclear antigen (PCNA)-positive cells in the basal and
suprabasal layers of EPC2-EGFR cells compared with only proliferating
cell nuclear antigen-positive basal cells in EPC2 and EPC2-GFP as
revealed by immunohistochemistry (IHC) (magnification,
×400). D, the addition of AG1478, an EGFR-specific tyrosine
kinase inhibitor, leads to a dose-dependent decrease in the
thickness of the epithelium in EPC2-EGFR cells.
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Fig. 4.
The esophageal epithelium of
EGFR-overexpressing mice shows increased proliferation by Ki-67
immunohistochemistry. A, Ki-67-positive cells are confined
to the esophageal basal cell layer from wild-type mice (WT)
(magnification, ×400). B, transgenic mice overexpressing
EGFR under the control of the Epstein-Barr virus ED-L2 promoter
(L2-EGFR) show increased proliferation in the esophageal basal and
suprabasal layers based upon Ki-67 staining (magnification,
×400).
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Fig. 5.
EGFR overexpression results in increased cell
migration. A, cell migration was measured in 8-µm pore
size cell culture inserts and shows increased migration in EPC2-EGFR
cells (black bar) compared with EPC2-GFP cells
(white bar). Migration was inhibited by the
addition of an EGFR-specific tyrosine kinase inhibitor, AG1478.
B, staining of the cells migrated through the PET membrane
shows single EPC2-GFP cells (arrows, left
panel) compared with clusters of migrated EPC2-EGFR cells
(arrows, right panel).
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[in a new window]
Fig. 6.
EGFR overexpression up-regulates MMP-1
mRNA and secreted protein levels. A, RT-PCR reveals
increased amplification of MMP-1 levels in EPC2-EGFR cells compared
with EPC2-GFP cells, whereas levels for MMP-2 and MMP-9 were unchanged.
The addition of EGFR inhibitor AG1478 reversed the up-regulation of
MMP-1. B, Western blot of conditioned media revealed
an increase of MMP-1 secretion in EPC2-EGFR cells compared with
EPC2-GFP cells, which was inhibited by the addition of EGFR inhibitor
AG1478 (Coomassie Blue stain of the gel serves as loading
control).
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Fig. 7.
Stimulation of EGFR leads to increased
cell-cell aggregation that is abolished by EGFR inhibitor AG1478.
A, cell aggregation assays show enhanced aggregation in
EPC2-EGFR cells that is abolished by EGFR inhibitor AG1478. Cell
aggregation assays were performed in six-well plates with capture and
analysis after cells settled with the NIH image software. B,
the addition of neutralizing anti-E-cadherin antibody abolishes
aggregation, thereby demonstrating that aggregation is
E-cadherin-dependent. Rat IgG serves as a control.
-catenin,
-catenin, plakoglobin,
desmoplakin, and desmoglein) remained unchanged (data not shown).
Furthermore, immunoprecipitations and Western blots of E-cadherin did
not reveal changes in interaction with its cytoplasmic partners that
link it to the cytoskeleton (data not shown). However (and
importantly), analysis of its interaction with another member of the
catenin family, p120, demonstrated that p120 had stronger interaction
with E-cadherin in EPC2-EGFR cells, whereas p120 was predominantly in
the cytosol in EPC2-GFP cells (Fig.
8).
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Fig. 8.
EGFR overexpression induces a shift in p120
from the cytoplasm to the membrane. A, immunoprecipitation
of p120 shows increased p120 levels in EPC2-EGFR and interaction of
p120 with membrane-bound E-cadherin. IB, immunoblotting;
IP, immunoprecipitation. B, immunoprecipitation
using anti-p120 antibody after cell lysis under less stringent
conditions shows less cytoplasmic p120 in EGFR-overexpressing cells
than in GFP-expressing cells. C, confocal microscopy of
organotypic culture of EGFR-overexpressing EPC2 cells demonstrates
strong membranous staining of p120 (red) in cells that
overexpress EGFR (green). The merged picture shows partial
co-localization of p120 and EGFR (yellow-white
arrows, far right upper
panel). p120 localizes to the adherens junction as
demonstrated in double staining with E-cadherin (green) and
p120 (red), whereas the control experiment with
desmoplakin reveals that localization of p120 to adherens
junction can be distinguished from the desmosomal junction
(far right lower panel)
(magnification, ×60). D, immunofluorescence staining using
p120 antibody shows a shift in the localization of p120 from cytoplasm
(round-stemmed arrows) to membrane
(arrows) in EGFR-overexpressing cells compared with
GFP-expressing cells. EGFR-overexpressing cells adhere closer and
provide more cell-cell contact (arrows) sites than
GFP-expressing cells. Confocal microscopy (four
smaller pictures to the right) allows
one to follow localization of p120 from top to the bottom of the cells.
Cytoplasmic localization in EPC2-GFP is marked by round-stemmed
arrows, and localization in adherens junctions is marked with
arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice show low MMP expression in early
lung development (17). Stimulation with EGF could up-regulate MMP in
wild-type mice but not so in Egfr
/
mice, which suggests a direct
link between EGFR and MMPs. MMP-9 has been shown to be up-regulated in
ovarian cancer cell lines that harbor EGFR overexpression (18), and
this may be mediated through phosphatidylinositol 3-kinase activity
(19). Additionally, MMP-9 is up-regulated in head and neck squamous carcinoma cell lines with EGFR amplification (20).
)
(21). ECM-bound growth factors also become more easily available after ECM degradation (22, 23). During metastasis, cancer cells must cross
several ECM barriers. They cross the epithelial basement membrane,
invade the surrounding stroma, and enter blood vessels or lymphatics.
One of the first steps in invasion is migration.
-catenin, which after tyrosine phosphorylation can no longer mediate
the connection of the cadherin-catenin complex with the
actin-cytoskeleton (29). Whereas we confirmed binding of EGFR to
-catenin, it did not result in changes of the phosphorylation status
of
-catenin, since
-catenin is phosphorylated even in EPC2-GFP
cells. It also has been reported that EGFR-mediated phosphorylation of
plakoglobin after EGF stimulation leads to a loss of adhesion (30). We
could not confirm this in our experimental conditions. By contrast, our
results show that EGFR overexpression and activation do not induce
changes in the assembly of adherens junctions and desmosomes but that
EGFR mediates increased cell aggregation through relocalization of
p120, a component of adherens junctions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grants P01 DE12467 and P01 CA098101 (to A. K. R.), a grant from the Leonard and Madlyn Abramson Family Cancer Research Institute at the University of Pennsylvania Cancer Center (to A. K. R.), the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases (Grant P30 DK50306), and its Morphology, Molecular Biology, Mouse, and Cell Culture Core Facilities, and NIH Grants CA 80999 and CA 25874 (both to M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These three authors contributed equally to this work.
To whom correspondence should be addressed: 600 CRB, University
of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104. Tel.:
215-898-0154; Fax: 215-573-5412; E-mail:
anil2@mail.med.upenn.edu.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M209148200
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor receptor; STAT, signal transducers and activators of transcription; MMP, matrix metalloproteinase; EGF, epidermal growth factor; GFP, green fluorescent protein; PBS, phosphate-buffered saline; ECM, extracellular matrix; KSFM, keratinocyte-SFM medium; RT, reverse transcription.
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